The selectivity of ion channels in nerve and muscle

0306.4522:82;071335-32sO3.oOiO Pergamon Press Ltd 0 1982 IBRO

Nrr,rerti<~rr<<~Vol. 7. No. 6, pp. 1335 lo 1366. 19X2 Prmted in Great Britain

COMMENTARY THE SELECTIVITY OF ION CHANNELS NERVE AND MUSCLE

IN

C. EDWARDS NeurobiologyResearch Center and Department of Biological Sciences, SUNY Albany, 1400 Washington Avenue, Albany, NY 12222, U.S.A. CONTENTS Introduction Some practical considerations Chloride channels Nerve Muscle Other cells and systems Sodium channels Nerve Muscle Egg cells Potassium channels Nerve Muscle Egg cells and other systems Calcium channels Channels blocked by manganese and/or cobalt Channels permeable to manganese Ion effects on the calcium channel Channels permeable to zinc Channels possibly permeable to magnesium Larger cation channels Excitatory postsynaptic potentials Mechanoreceptor potentials Photoreceptor potentials Voltage dependent channels Ion movements across membranes at rest Afterword

INTRODUCTION There is much evidence that ions cross the membranes of nerve and muscle through selective channels. This commentary will focus on experimental studies of selectivity. The data covered are mostly from nerve and muscle cells, although some relevant data from other cells and systems are included when appropriate. The concentrations of ions inside living cells differ dramatically from those in the fluids surrounding the cells. These differences are due largely to the properties of the lipid membrane enclosing the cell. which is impermeable to a number of substances within the cell and in the bathing fluid. A complete

Ahhrrviarions: ACh, acetylchoiine; EPP, endplate potential; EPSP, excitatory post-synaptic potential; E,_, membrane potential as which current is zero; GABA, r-aminobutyrate: IPSP, inhibitory post synaptic potential; Pj, membrane permeability of jth ion; Sj, activity of jth ion; i, inside a cell: o, outside a cell; TTX, tetrodotoxin.

estimate of the internal contents of rat skeletal muscle has been given by Conway.54 There are several mechanisms by which ions may cross membranes. Some ions are moved against electrochemical gradients, e.g. the sodium/potassium pump, and this requires metabolic energy. In some other cases, the influxes and effluxes of ions are coupled so that there is no requirement for metabolic energy; e.g. the electrochemical energy yielded by the movement of Na+ into the cell is apparently used to move Ca’” ions out of the cell against an electrochemical gradient.226 In addition, ions cross membranes down electrochemical gradients; these movements are mostly, if not entirely, through a variety of selective channels. One argument for the existence of selective channels in the membrane is based on the properties of lipid membranes. The resistance of a pure lipid bilayer membrane in saline solution is typically 106-lOa ohm cm2.1g3*1ggThe resistance of the mem-

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brane of frog muscle is about 4 x 10J ohmcm2,s3 which is several orders of magnitude lower. The addition of polypeptide ionophores, such as gramicidin, to hilayers decreases the resistance to 3 x IO2 to 1030hmcm’.‘93-‘“9 i.e. to values similar to those of muscle membrane. In this regard it is interesting to note that the best current model of the cell membrane assumes it to be a lipid bilayer containing approximately equal amounts by weight of lipids and proteins and that some of the protein molecules probably span the membrane (see review of Singer & NichofsoPf ). A stronger argument for the existence of channels derives from the recent measurement of the currents through single membrane channels by means of the extracellular patch clamp developed by Neher, Sakmann & Steinbach.zO” This technique has been used to resolve the currents through the individual open channels controlled by acetylcholine at the frog neuromuscular junctian,2”5 those controlled by glutamate at the locust neuromuscular junction,“’ those responsibte for the Ca”’ dependent outward currents in the membrane of N&Y neurons,“” the voltagedependent K + channels in squid axonss3 and the vofGore-de~ndent Na” channels in rat muscle cells in culture2”” and in the membrane of the tunicate egg.“” In all cases. square wave-like current pulses are found; the widths of the pulses vary stochastically. Similar fluctuations in the conductance of lipid &layers with incorporated proteins were reported by Bean, Shepherd, Chan & Eichner.24 The estimated rate of charge transfer through the acetylcholine activatcd channel at the endplate. 5 x lo7 ions/s.‘43 is significantly greater than the maximum rate found so far for a carrier. 3 x 104 ionsis.‘70 In the following discussion, it wiIl be assumed that ions cross the cell membranes through protein channels. Most, if not all, of the channels discussed here have at least two conformations, one that allows the passage of ions through the channels and one that does not. When the channels are open, a region of the channel. usually called the selectivity filter, limits the ions that pass through the channel on the basis of charge. size and ability to form hydrogen bonds. Operationally. most channels pass either cations or anions. The size and other properties of the filters have been analyzed by measuring the permeabihties of the channels to ions of various sizes. The selectivity is presumably determined by the geometry of the liIiter, as well as the nature of the chemical groups that interact with the ions. The properties of a number of channels in a variety of cells have now been examined and the available evidence suggests that only a limited number of selectivity filters exist in nature. In a discussion of the ion selectivity of channels, Mull~ns2~~ has pointed out that ‘-. . . (a) high ion selectivity necessarily involves the total dehydration of an ion and its enclosure in a very close fitting structure which effectively solvates the ion. (b) moderate ion selectivity means that the ion involved is partially

hydrated but with not more than a single layer of water molecules between the ion and the structure providing solvation, and (c)a system with tow or negligible ion selectivity will take an ion into a structure with more than one Iayer of aqueous hydration. . ,‘_. The data given below on channel size and selectivity are quite consistent with Mullins’ views. Several properties of some of the inorganic ions of interest are given in Table 1 including the crystal radii, the estimated radii of ions with a primary single hydration shell, the limiting ionic conductances and the heats of hydration. The properties of some of the nitrogenous cations used to probe cation channels have been discussed by Moreno & Diamond.“”

The ideal way to examine the selectivity of a channel is to measure the voltage dependence of the current in a single channel and the changes in the currents when the solutions on both sides of the membrane are altered. It is assumed. of course, that the properties of the membrane remain constant. However. most experiments have been conducted under less than ideal conditions. The recent development of techniques to perfuse the interior of cetls,‘h3~“2 to incorporate functional acetylcholine activated channels into bilayer m~mbranes20h*23s and to record the currents from single channels in small patches of membrane excised from celfs’3” increases the probability that one day it may be possible to do experiments under ideal conditions. In most previous studies of channel selectivity, the composition of the external solution was altered and it was usually assumed that the ionic composition within the ceil was constant. This assumption may not always be valid. The removai of Na’ from the bath solution will increase the cytoplasmic free Ca’+ in many cells; this happens because the exchange of external Na+ for internal Ca2 + is one mechanism for keeping the internal Ca” level Iow.*~~ In some cells, including the squid giant axon. Li “ and presumably other cations. cannot replace Na * in this role.30~32 unless the intracellular Ca ‘* level is
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The selectivity of ion channels in nerve and muscle Table I. Some properties of inorganic ions

Ion Li’ Na+ K’ Rb’ CS’ TIC Be” Mgz’ Ca’+ Sr2 + Ba’ ’ Zn2+ Cd2+ MnZ+ Fe2+ c02+ NiZ+ FClBrI-

Crystal Radius’ (pm) 90 116 152 166 181 164 59 86 114 132 149 88 109 97 92 88.5 83 119 167 182 206

Radius of ion and its primary hydration shell’ (pm)

Limiting ionic conductance3 (cm’ intR- ’ equiv- ‘)

218 208 218 224 232

38.7 50.4 73.5 77.8 78.5 76.0 44 53 59.6 59.8 63.6 54 53.9 51.8 54 52 50.5 54.7 76.4 78.2 76.6

214 224 216 222

231 241 238 255

Heat of hydration (Kcal/mole)“ -130 - 104 -83 -78 -70 -85 - 608 -473 - 395 -359 - 325 - 502 - 446 -455 -473 - 505 -517 -98 -80 -72 -62

1Shannon.236 * Goldman & Bates”“; estimates not based on Shannon radii, but on older data. ALandolt-Bornstein.‘68 4 Noyes2”

This condition probably applies to most nerve cells. Therefore, elevation of the bath K+, without a corresponding reduction in Cl-, is likely to lead to the entry of K’ and Cl-. This will produce cell swelling and will probably alter the concentrations of the internal ions, and complicate the interpretation of the shifts of the various measured potentials. Thallium has been used widely in studies of the selectivity of the K+ channel because the ionic radii are similar (Table 1). TlCl is relatively insoluble; the solubility constant is about 2 x 10-4.240 This means that 2.5 mM TlCl in frog ringer is saturated and so experiments in artificial sea water or even crayfish ringer are precluded. TlN03 is over ten times more soluble and the perchlorate and acetate are still more soluble. A problem with the use of acetate salts is noted below. Another problem arises from the use of weak acids to replace Cl-. Small amounts of the acids are unionized at physiological pH and the uncharged molecules can penetrate the cell membrane. In time, the cell interior can become significantly more acid. In crab muscle fibers, the replacement of some of the bath Cl- by acetate (pK, = 4.75) or propionate (pK,, = 4.87) shifts the internal pH by about 0.5 pH unit; methanesulfonate (pK, = - 1.2) and isethionate (pK, < 1.25) are without effect.237 The substitution of an anion for Cl- may sometimes alter the composition of the solution. Some of the anions used to replace Cl-, such as propionate and acetylglycinate;“’ and isethionate and acet-

ate49,223 bind Cazf. In addition, calcium sulfate is relatively insoluble; to maintain the free Ca2+ level in frog Ringer, the replacement of 75% of the Cl- by sulfate requires that Ca2+ be increased more than three fold.“’ The greater concentrations of Ca’+ and Cl- in sea water restrict still more the choice of substitute anions. Methanesulfonate seems to be one of the better substitute anions, but it may not be impermeant in all cases7’ There are several problems in experiments using divalent cations, Cd ‘+ forms a series of complexes with Cl-; one compound, CdCl+, may carry current through the acetylcholine-controlled channel at the frog neuromuscular junction.* These complexes are even more of a problem in artificial sea water because of the much higher Cl- concentration. Another problem is the di~culty in choosing the activity coefficient. In a IOOmM solution, the mean activity coefficients for MgCl,, CaCl,, SrC12, BaC12, MnCl,, CoCl,, NiClz are between 0.51 and 0.53 (the coefficient for CdCl, is about 0.23, possibly because of the existence of complexes just discussed).230 The estimated single ion activity coefficients are somwhat lower: Mg*+, 0.279; Ca*+, 0.269; Sr2’. 0.266 and Ba*+, 0.259.23,23s Both coefficients have been used in recent work, and the choice remains, perhaps, somewhat arbitrary. The interior composition of a cell can change if the permeability of the membrane is altered for prolonged periods by either changes in membrane potential or exposure to certain transmitters. For example, the entry of CI- into muscle cells during prolonged ex-

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posure to acetylcholine (ACh) is sufficient to produce a depolarization when the ACh is removed.14’ Also, the leakage of ions from a microelectrode into the cell may affect its contents. These sources of error can be minimized by Iimiting the duration of the current pulses and of the exposures to experimental solutions, and by not filling microelectrodes with ions such as Cl- which may affect the currents being studied. The results of ion substitution experiments are dependent upon the way the effects are measured. Perhaps the simplest way to examine the permeability to various test ions is to measure the effects of various test solutions on the voltage changes produced by activation of the channels. The quantitative analysis of data so obtained is dependent upon the assumption that the voltage response is proportionaX to the current flowing through a single channel. There are several reasons why this assumption may not be valid. The measured voltage change depends upon both the current through the channels and the membrane resistance; altering the ionic composition of the bath can change the membrane resistance, and in turn alter the change in membrane voltage independently of any effects in ionic current. The size of the voItage response is dependent on the membrane potential and becomes smaller as the membrane potential appraaches the reversal potential. Finally. the substituted ions may affect the number of channels that are opened by a given stimulus. Some of these problems can he avoided if the membrane current is measured and the voltage is held constant, Tbe currents measured at constant voltage reflect ion selectivity. however, only if the number of open channels is the same in all solutions, and if there are no saturation or channel blocking effects by any of the ions studied.’ I8 Most of these problems can be avoided by measuring the reversal potentiai, i.e. the potential at which the net current flowing through the channel is zero. The reversal potential for an ion is independent of the number of channels that are open. In the presence of univalent ions of the same sign, which pass through identical channels, a permeabiiity ratio can be calcufated from the Goldman-~odgkin-K~~z equation, which states that at the potential at which the current is zero,

Where R, T and F have the usual meanings, Pj is the membrane permeability of the jth ion, and [SJ is the activity of the jth ion, either in the cell (i) or in the external bath (0). The problems involved in the use of this equation have been discussed by HilIe.itl Note that the values of the permeability ratio Pi/p, calculated in this way apply only for the voltage, E,,,, at which they are determined; it cannot be assumed that the ratio is ~ndepende~t of voltage. Further. the analy sis assumes the independence principle, i.e. that the

chance that a given ion crosses given potential is independent of & Huxley;i2’ see also discussion tions between ions which suggest be valid are frequently found, examples wiil be cited below.

the membrane at a other ions (Hodgkin in Hille’ZL. Interacthe principle not to and a number of

CHLORIDE CHARNELS One of the most abundant biologicai anions is Cl-. The seiectivity properties of the channels through which Cl moves will be discussed first. It should be remembered here that the pattern of hydration of Clis quite different from that of the cations to be discussed below (cf. discussion in Robinson & Stokes”‘). This is true regardless of what is assumed about the pattern of hydration of the cations and is due basically to the difference in charge.

The first channel whose ion selectivity was studied is the chloride channel responsible for the inhibitory postsynaptic potential (IPSP) in the motor neuron of the cat spina cord. In the first experiments, it was found that bromide, nitrate and thiocyanate pass through the IPSP channel, whereas bicarbonate, acetate and glutamate do not.j5 The results of a later study with many more anions are shown in Table 2.i3’ The anions are Iisted according to the size of the hydrated ion, as determined by the limiting ionic conductance in water. With two exceptions, the evidence suggests a clear relationship between the size of the hydrate ion and the ability to pass through the channel. The permeability to CIO; and the ;mpermeab~My to PF; suggest that the diameter of the selectivity filter should be between about 1.2 and 1.3 x that of Cl-. HS- should enter the channel, but appears not to; Ito er ~1.‘~~ suggested that this may be a consequence of its conversion to H,S within the cell, as well as its binding to proteins. The permeability of the larger HCO; ion has been attributed to its ellipsoidat shape.i3 In these experiments, the motor neuron, whose function is of great interest physiol~gic~ly was studied in situ and so its external environment could not be altered in a controlled, reproducible manner. During hyperpolarizat~on, the current passing through an intracellular micro&&rode may cause the entry into the cell of sufficient anion to alter its ionic composition If the injected anion can permeate the IPSP channel, the normally hyperpolarizing IPSP becomes depolarizing because of the reduction of the concentration gradient of the permeable anion; the magnitude of the depolarization decreases with time as the ion diffuses from the cell. There is no change in the IPSP following the injection of anions which cannot move through the channel. One problem with the method is the uncertainty about the exact ionic composition within the cefl.

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The selectivity of ion channels in nerve and muscle Table 2. Permeability of several Cl- channels to various anions

Anion BrICINO; NO; N; c10; SCNBF; HSOCNc10; PF, BrO; FHCO; CH,SO; HSO; HCO; Cl acetateacetate10; Br acetateH,PO; propionatemethane sulfonatebutyratebenzoateglutamate CrO; so; c,o; so; HPO; Fe(CNJ citrate E Fe(CN);

Relative hydrated size’

Motor neuron IPSP2

0.98 1.00 1.00 1.06 1.07 1.11 1.13 1.16 1.17 1.18 1.19 1.19 1.30 1.38 1.39 1.40 1.47 1.53 1.72 1.83 1.87 1.89 1.98 2.12 2.13

+ + + + + + + + + + + -

2.15 2.34 2.36 2.82 1.82 1.92 2.02 2.12 2.68 2.19 3.20 2.77

Helix aspersa abdominal ganglion cell IPSP’

Mauthner cell IPSP4

Cat hippocampal pyramidal cell5

Cat spinal ganglion GABA

+

+ + + + + + + + + + + +

+

+ + +

-+

+

+ + +

+

+

+

+

+

+ +

+

-

-

Cerebral cortical neurons IPSP’

Crayfish neuromuscular IPSPB

+ + + + + + + + + + + + +

-

-

-

-

-

i Almost all of the relative size data are from Araki, Ito & 0scarssoni3; the estimates are based on data in various compilations. The values for methyl sulfate and glutamate are from Kelly, Krjevic, Morris & Yimiso and that for methanesulfonate is from McBain, Dye & Johnson. ‘sl The value for methyl sulfate appears to me to be low, judging from the number of major atoms. * Ito, Kostyk & Oshima.“‘s 3 Kerkut & Thomas.‘52 4 Asada14; Diamond, Roper & Yasargile4 ’ Eccles, Nicoll, Oshima & Rubia.‘4 6 Gallagher, Higashi & Nishi.96 ’ Kelly, Krnjevic, Morris & Yim.i5’ s Takeuchi & Takeuchi.2s4

Several other Cl- channels in nerve cells have been investigated by similar techniques (Table 2). In studies of the large Mauthner neuron in the goldfish central nervous system, the neurally-evoked IPSP, as well as the conductance increase induced by ionophoretic application of the putative inhibitory transmitters, glycine and y-aminobutyric acid, were studied. Although fewer anions were examined, the results suggest that the selectivity filter of the channel opened

by either the IPSP or the amino acids is similar in size to that of the cat spinal motorneuron.‘4.64 The channels mediating the IPSP and the ACh response of cells in the abdominal ganglion of Helix aspersa,152 the IPSP of cat hippocampal pyramidal cells74 and both the spontaneous IPSPs and the response of cat spinal ganglion cells to y-aminobutyrate (GABA) also seem to be very similar in size to that in the spinal motor neuron. There are, however, a few dis-

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crepancies. Formate (HCO;) goes through all channels except those in the cat spinal ganglion cell. HS goes through the channel in the cat spinal ganglion, though not that in the spinal motor neuron or the Mauthner cell. While the similar patterns of permeant and impermeant ions might suggest that the selectivity filters of these postsynaptic channels are similar in size, the impermeability of the cat spinal ganglion cell to formate may mean that the channel is indeed different. The release of transmitter from the presynaptic terminal of the crayfish neuromuscular junction by action potentials is inhibited by ;‘-aminobutyric acid.‘j* The effects of anion substitution on this process have been examined. 253 NO; is permeant and methylsulfate, acetate, propionate and glycerophosphate are not. GABA increases the membrane conductance of rat sympathetic ganglion cells.’ The effects on E,,, of Cl- replacement by various anions suggest that Br - is permeant, and that isethionate, glutamate and SOd are impermeant. The selectivity of the channel producing the evoked IPSP and the similar response to GABA at a synapse on the giant motor fiber of the crayfish has been studied by 0chi.209 The reversal potential shifts on the replacement of Cl- by propionate. The incomplete data suggest that the selectivity filters of these channels may be similar to those just discussed. In the neuron of the crayfish stretch receptor. the membrane potential changes produced by stimulation of the inhibitory nerve and by applied GABA are similar.ibh The usually hyperpolarizing IPSP is reversed by replacement of Cl- by glutamate,io7 or methanesulfonate and is almost completely abolished by prolonged exposure to the latter.2’6 The data suggest a Cl- channel smaller than methanesulfonate, which could be similar in size to the channels just discussed. However, additional experiments with smaller anions are necessary to determine whether this is, in fact, the case. The results of studies of the permeability of the Clchannel of cerebral cortical neurons by anion injection were somewhat different.rsO This channel seems to be significantly larger, for it admits anions as large as chloroacetate, propionate and glutamate. It has been estimated that the diameter of the selectivity filter of this channel is 2-3 x that of a Cl- ion. or about 5-8 A. Spreading depression in the brain appears to be accompanied by the entry of Na+ and Cl- into nerve cells.‘64~260 To investigate the permeability of the Clchannel in cells in the rat cerebellum, the entry of anions which replaced the Cl- in the extracellular fluid has been studied with ion selective electrodes.221 SCN-, hexafluorophosphate and hexafluoroarsenate are permeant, while r-naphthalene sulfonate is not. The data suggest the channel size to be similar to that mediating inhibition in cortical neurons,r5’ and to be larger than that in spinal motor neurons’38 and in hippocampal cells.74

The anion permeabilities of the membrane of the cell body of a giant neuron of Aplj+~ have been examined by using ion selective electrodes to measure internal ion activity following changes in the ionic contents of the bath solution. and also by determining the values of the membrane conductance.72 The data suggest the permeability of methanesulfonate to be about half that of Cl-. The selectivity filter should be larger than that in the cat spinal motor neuron and it might be similar in size to that in cortical neurons. In some neurons of Ap/lG, ACh produces a rapid and a slow inhibition.‘“’ The IPSPs produced by stimulation show two similar components. The fast channel appears to be a Cl- channel. Measurements of the change in the reversal potential when the external Cl- is replaced by other anions suggest the channel to be impermeant to SO, and methylsulfate, and slightly permeant to propionate and isethionate. A GABA activated Cl channel in ApIyiu has been reported to behave similarly.“” The anion permeabilities are somewhat inconsistent in that propionate and isethionate are larger than methylsulfate. and the data are different from those of any previously described channel (see Table 2). The anomaly may be a consequence of the binding of Ca2+ by isethionate (K,,, = 2.99. 223) and the acidification of the cell interior by propionate (see discussion above).

At the crustacean neuromuscular junction the IPSP as well as the potential changes initiated by GABA, the putative transmitter. were attributed by Boistel & Fatt3” to an increase in the permeability of the membrane to Cl-. GABA does not alter the permeability of the membrane to large anions, such as pyroglutamate, acetylglycine or propionate.33s224,253 Quantitative measurements of the effects of the replacement of Cl- by various anions on the GABA-induced increase in membrane permeabiiity at the crayfish neuromuscular junction were made by Takeuchi & Takeuchi.254 The sequence found was: Br- > Cl- > SCN- > I- > NO, > HCO; > ClO; > ClO; > BrO;. Note that the sequence based on the relative hydrated size (Table 2) is Br- > II, Cl- > NO; > Cl04 > SCN- > ClO; > BrO; > HCO;, which is quite different. The sequence found in the crayfish does not relate to the sequences set by hydration energies, relative hydrated size, or crystal ionic radii.254 The small, but finite, permeability to BrO; suggests that the size of the selectivity filter of this channel may exceed that of the motor neuron IPSP channel. In later experiments, the measured permeabilities of the anions were found to depend on GABA concentration.256 In higher concentrations of GABA, NO;. and I- are less permeant than Cl-. while in lower concentrations they are more permeant. NO such crossover is found with Br- or SCN-. Thus the sequences are I- > Br- > NO, > Cofor low levels of GABA and Br- > Cl- > I- > NO; for

The selectivity of ion channels in nerve and muscle higher levels. In I- solution the reversal potential for the neurally evoked IPSP is more negative at lower stimulation frequencies than at higher frequencies. It was suggested that GABA might somehow combine with the receptor to alter the electrical charge profile of the membrane. Further, in solutions in which Cl- was partially replaced by these test ions, the changes with ion concentration were not linear, suggesting the presence of some interaction between the anions.255 In the lobster neuromuscular junction, the anion sequence found from measurement of the reversal potential of the IPSP is NO; > SCN- > Br- > Cl- ; the channel is somewhat permeable to acetate and propionate but impermeable to BrO;, isethionate and methylsulfate. ig7 The impermeability of this GABA-controlled channel to BrO; suggests that the size of the selectivity filter may be quite similar to that of the cat spinal motorneuron and the other similar channels discussed above (Table 2). In some of the work cited above, there was evidence suggestive of a small K+ component to the IPSP.55,152 However, the results of direct studies on juncthe crayfish254 and the lobster neuromuscular tion19’ seem to preclude a role for K+ so that the channel admits anions only. Further, it has been stated that in the hyperpolarizing IPSP in cat neurons “ . we have no positive evidence for the involvement of K+ ions.“’ In frog muscle, at rest, the Cl- permeability of the membrane is quite high; indeed it is approximately twice as high as the K+ permeability.iz6 The permeability of this Cl- channel to other anions has been examined and the selectivity filter appears to be smaller than those of the postsynaptic channels discussed above. The membrane resistance of frog muscle is increased by total replacement of Cl- by resistances anions. relative are other The Cl-:Br-:NO; :I- = 1.0:1.5:2.0:2.3. There is an increase with thiocyanate as well; however, the increase is greatest at low concentrations and falls somewhat at higher concentrations. 134 The efflux of tracer Clfrom frog muscles is slowed by replacement of Cl- by other anions; the most potent anion is SCN-, followed by ClOi, I-, NO; and Br- .ii’ If it is assumed that the Cl- conductance is 0.67 of the total membrane conductance, the relative permeabilities of anions these estimated to be may be Cl-:Br-:NO;:I= 1:0.5:0.25:0.16. In rat diaphragm, the relative permeabilities estimated from the membrane conductance and the assumption that methylsulfate is impermeant are Cl-:Br-:I= I.0:0.2:0.15.217 The sequence for the halide ions for frog muscle is that expected from the crystal radii (Table l), and is quite different from that expected from the radii of the hydrated ions, as estimated from the limiting ionic conductances (Br- > I-, Cl- > NO;). Further, the relatively large differences in permeability for these ions, whose hydrated sizes are so similar, are suggestive of a small, relatively tightly-

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fitting selectivity filter such as is found with the Na+ and K+ channels to be described below. Under certain conditions the frog muscle Cl- conductance shows voltage dependent changes. At pH 9.8, a hyperpolarizing current causes the Cl- conductance to decrease, while at pH 5.0, the conductance increases in response to a hyperpolarization.‘35 The permeability of the frog muscle membrane to various anions has been estimated from the membrane conductance by Woodbury & Milesz6’ Trichloroacetate is about 1/6th as permeant as Cl-. The permeabilities of benzenesulfonate, isethionate, methanesulfonate and glutamate are 0.05 times or less than Cl-. The data for a number of other anions, formed by dissociation of weak acids, e.g. benzoate, valerate, butyrate and propionate, may be questioned because of the possibility that the undissociated acids may have entered the cell, altered the internal pH and I thereby changed the membrane conductance. In contrast, the Cl- channel in the membrane of crayfish muscle seems to be much larger. The resting conductance is not significantly altered on replacement of Cl- by methanesulfonate, but is increased 4-5-fold following substitution by isethionate acid (Dekin, personal communication). Dude1 & Ri.idel” have reported the membrane to be permeable to methylsulfate (see also ref. 255) but not to propionate. The problems in interpreting the results of propionate experiments were mentioned above. In the skeletal muscle fibers of the stingray, Tueniura lymma, the Cl- conductance of the membrane at rest is 8-10 times greater than the K’ conductance.‘14 The permeabilities of the Cl- channel have been measured from the potential changes during ion substitution. The permeability sequence is Psc., > P NO, > Pc, = P,, > P, > PcIo,. The measured conductances for partial replacement of Cl- by various anions deviate significantly from those expected from the independence principle, as is found also with the IPSP channel in crayfish muscle.iL4 The impermeance of larger anions, such as F-, acetate, methanesulfonate and SO: ‘I 3 suggests that the selectivity filter may be similar to that at the cat spinal motor neuron. The selectivity of the Cl- channel of the resting muscle fiber of the barnacle has been examined by observing the changes in membrane potential initiated by injection of various K+ salt solutions.103 The channel appears to be permeable to Br- and I-, but not to acetate, methanesulfonate, citrate and sulfate. The data suggest its size may be similar to that of the motor neuron Cl- selectivity filter discussed above. At pH 3.9, the membrane is primarily permeable to anions.‘16 The permeability sequence determined from the change in potential on replacement of Cl- by various anions is SCN- > I- > NO; > Br- > ClO; > Cl- > BrO; > p-toluenesulfonate, methanesulfonate > IO;. Measurements of the conductance of the membrane in the same solutions give a different sequence, Br-, Cl- > Cloy, NO; >

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C. Edrvards

SCN-. The discrepancies between the sequences measured in different ways were attributed to the presence of positively-charged sites in the membrane and competition among the anions for these sites. In somatic muscle of itscar& the resting membrane potential appears to be due largely to the transmembrane Cl- concentration gradient. Replacement of Cl- by various anions hyperpolarizes the membrane potential; the order of effectiveness is NO, > I- > Br- > Cl-.6’ Depolarization of the membrane potential of chick skeletal muscle celis grown in culture produces action potentials lasting tens of seconds. At least part of long plateau is due to a voltage dependent Cl- channel, which is impermeant to acetate.” In Purkinje fibers of sheep heart, a voltage dependent Cl- channei has been described.69 This channel is impermeant to gluconate, mareate, propionate and methyl sulfate, and so its size could be about the same as that giving rise to the IPSP at the crayfish neuromuscular junction. The anion permeability of resting isolated strands of papillary muscle and Purkinje tissue of mammalian hearts has been examined by measuring the membrane resistance.i3” This may possibly be the same channel studied by Dude1 rt u&49 because, if methyl sulfate is assumed to be impermeanf the contribution of the Cl- channel to the resting conductance is less than 30%. In the heart of oyster, the behavior of the reversal potential for the depolarizing response evoked by acetyicholine suggests the channel to be permeable to Cl-. to be impermeable to SO; and to be somewhat permeable to isethionate.”

The anion permeabilities of the dark-adapted photoreceptor of the barnacle have been estimated from the changes in membrane potential following ion substitution4’ The permeability sequence is gluconate 7 I _ > SO: > glutamate > Br- > glucuronate > Cl- r isethionate > methanesuifonate. No other anion channel has been found to be permeable to SO;, and the sequence is not consistent with either crystal radii (Table 1) or relative hydrated size (Table 2). In artificial sea water, the replacement of Cl- by SO,’ reduces the Ca’+ level significantly, and this might be expected to depolarize the membrane potential. However, a hyperpolar~zation was found, and so the results do not fit any previously described channel A large increase in the membrane conductance of a planar phospholipid bilayer system is produced by the addition of membrane vesicles prepared from the electric organ of Torpe& c~~j~r~~~~,2~7Much of the additional conductance is due to a voltage dependent anion channel. The selectivity has been estimated with equation I from the reversal potential of the tail currents seen after a square voltage pulse with Cl- on one side and the test cation on the other side.i9r The

only permeant anions are Cl- and Br-, and PHr/PcI is about 0.68. The relative Ps for SO;, F-, NO; and acetate are less than 0.1. Further, I- and SCN- inhibit the movement of Cl- through the channel. Explanation of the anion data requires a minimum of three channels, a small selective one to fit the frog muscle data, a larger one to fit the data from the cat spinal motorneuron and probably a number of other cells, and a still larger one to fit the data from the cortical neuron. The data from some cells, which are somewhat incomplete, may require additional channels. However. the similarities among the specificities of a number of the channels suggest that the total number may not be too great.

SODIUM CHANNELS tvmz The rising phase of the action potential in the giant axon of the squid, and in many other excitable cells, is due to the entry of Na+ through a voltage-controlled channel.izg In the squid axon, Li is about as permeant as Na”. “* In the myehnated axon of frog, P,ujP,, was found to be 0.3 by Dodgeb6 and Frankenhaeuser & Moore’? showed the K+ permeability of the Na+ channel to be small but measurable. An extensive examination of the selectivity of the Na’ channel in frog nerve has been made by Hilfe.’ Is*’ I9 The permeability ratios, measured from the reversal potential, are Iisted in Table 3. The first I1 cations are measurably permeant; none contains a methyl (-CH,) group. The addition of methyl groups (-CH3) to permeant cations makes them impermeant, e.g. methyl ammonium (CH,-NH;) and hydrazinium (NH,-NH;) are simiiar in size, but the former does not pass through the channel In general, the size of the permeant cations is simiIar to that of a partially hydrated Na’. In the permeant cations, the C, 0 and N atoms are coplanar and the hydrogens are suited for donating hydrogen bonds. The single exception to the latter is the Ii on formamidinium,~~’ The barriers to Na” Row through the channel seem not to be very high.“’ A single Na+ channel can admit an estimated 8 x IO6 ions/s5’. The temperature coefficient is about 1.3 for Na* channelsE6 which is similar to the coefficient for aqueous diffusion.230 Hiilei’9 has proposed a model consistent with the required low barrier. In this model the Na’ channel selectivity fiiter opening is 3 x 5 A, and is lined with oxygen atoms with either a dipole such as a C===O group or a partial negative charge. The selectivity of the filter depends on its size and the abiIity of the groups lining it to hydrate the ion. The permeant cations are partly solvated by the oxygen ligands of the pore, but are also in contact with one 01 more H,O molecules, which must also fit the pore. The numbers of H,O molecules required for the best fit are two for Li”. three for Na’ and four for K’, Rb’ and NH:. Hillel l9 has shown how the permeant ions

The selectivity of ion channels in nerve and muscle Table 3. Permeability ratios for monovalent cations in sodium channel of frog node calculated from reversal potentials1’8~“9 P,JP,, 1.0 0.94 0.93 0.59 0.33 0.16 0.14 0.13 0.12 0.086 0.06 < 0.056 < 0.025 <0.014 <0.013
Ion Sodium Hydroxylammonium Lithium Hydrazinium Thallium Ammonium Formamidinium Guanidmium Hydroxyguanidinium Potassium Aminoguanidinium n-Methylhydroxylammonium Methylhydrazinium Ethanolammonium Triaminoguanidinium Biguanide Methylguanidinium Acetamidinium Imidazolium Tetraethylammonium Choline Dimethylammonium Methylammonium Tris (hydroxymethyl)amino methane Tetramethylammonium

can be made to fit the channel and the importance of hydrogen bonding in the fit. -CH3 and -CH2 groups do not fit, because of their size and their inability to form hydrogen bonds. In squid the the axon, ratios PLi:PNa:PK:PRb:Pca = 1:1.0:0.085:0.085:0.045 were found from E,,, during voltage clamp.195 The ratio PNH4:PNa was measured to be 0.27.29 The value of Pco/P,s has been estimated to be_ about 0.01 (Ca’+ entry measured by aequorin; Baker, Hodgkin & Ridgeway’s), 0.02 (entry of radioactive Ca’+; Rojas 8~ Taylor231), or 0.1 to 0.14 (current during voltage clamp in perfused axons in Na-free, Ca’+ bathing solution; Meves & Voge118*). In internally perfused giant axons, the selectivity sequence estimated from E,,, is Na+ > guanidinium > NHf > K+ > Rb+ 1 CS+.~~ The relative permeabilities estimated = 1.0:1.0:0.083: from E,,, are PLi:PNa:PK:PRb:Pcs 0.025 :0.01646 and PLi:PNa: Pformamidinium: Psuanidinium: 1.12: 1.0:0.20:0.20:0.085: P,,:P methylguanidinium = 0.061.123 In the perfused giant axon of Myxicola, the ratios are P,,:PLi:P, = 1 :0.94:0.076.73 The data are similar to those found for the frog node Na+ channel (Table 3). The permeability to cations of the membrane of the cell body of a giant neuron of Aplysia has been examined by both internal ion-selective microelectrode and conductance measurements.” The results show Tris to be quite permeable; if it moves only through the Na+ channel, the value of Pt,,.JPNa is about 0.84. An effect of the internal K+ concentration on P,JP,, in the perfused squid giant axon was reported

1343

by Chandler & Meves.46 The ratio P,JPNs is increased from 0.078 to 0.29 on reducing the internal K+ from 530mM to 50mM.43 In axons perfused with solutions in which the only cation is NH:, the selectivity of the Na+ channel is dependent on the NH: concentration. The ratio PNHJPNa is decreased by an increase in internal NH:, and alterations in the external NH; level are without effect.26 Further, the addition of Cs* to the perfusate reduces P,/P,,. In the perfused axon of Myxicolu, a decrease in internal Kf also increases PK/P,,.73 These interactions suggest that the independence principle is not obeyed. The squid data have been fit by a three barrier, twosite ionic permeation model.26 The selectivity of the voltage dependent Na’ channel in myelinated nerve may be modified by several pharmacological agents. in the presence of the alkaloid aconitine the ratios (my calculation based on published values for the change in E,,,) are P,,,:P,,:P,,:P,,:P,:P,,:P,, = 1.35:1.0:0.8:0.58: 0.32:0.2.‘98 Batrachotoxin, a steroidal alkaloid found in the skin of a Colombian frog, also changes the relative permeabilities, giving P,,: PNH4:P,,: PLi: P~:Pg”anidi”c:PRb:Pcr:Pc~:Pme,hy,umi”, = 1.53: 1.4: 1.0: 1.0:0.41:0.37:0.26:0.15:0.15:0.1.1s3 In both cases, the loss of selectivity (note the increase in PK/PN.) and the increase in P for larger cations, e.g. Cs+, suggest that the agents enlarged the selectivity filter.153 Muscle The selectivity of the Na+ channel responsible for the action potential of the muscle membrane of several animals has been examined by similar techniques. The results from both the frog, P,,:PLi: P hydroxylammonium: P hydrazinium: PNH4 : Pguanidinium: P,: = 1:0.96:0.94:0.31:0.11:0.093:0.048: ~%;~“ni~~~m the rat PLi : P,, : Phydroxy,ammonium : P hydrszinium. Pguanidinium: PNH~: Ptetraethylammonium: PK = 1.14:1.0:0.9:0.3:0.16:0.15 < 0.012:0.045218 suggest that the channels are very much like that found in myelinated nerve. The selectivity of the voltage dependent Na+ channel of single rat heart cells has been determined from the maximum rate of rise of the action potential and from the reversal potential measured by voltage clamp (N. Akaike, K. Nishi, Y. Oyama, Kuraoka & Y. Tsuda, personal communication). The cells were dispersed by collagen treatment and perfused internally by suction pipette. The sequence found, Na+ > Li+ > hydrazine > formamidine > guanidine > hydroxylamine > methylguanidine > methylamine is similar to that reported for frog nerve, except for hydroxylamine. This compound may have a pharmacological action, because it irreversibly depressed the Na+ current. Egg cells The membrane of the unfertilized egg of the tunicate, Halocynthia roetzi, has a voltage dependent Na+ channel.19* The selectivity of the channel has been

1344

C. Edwards Table 4. Permeability ratios for monovalent cations in potassium channel of frog node calculated from reversal potentials’z”~‘z’ Minimum pore diameter,’ .A

Ion

P,,,;P, t0.018 <0.010 1.00 2.3 0.91 0.13 < 0.029 <0.025 < 0.077 io.021 < 0.020 <0.013

Lithium Sodium Potassium Thallium Rubidium Ammonium Hydrazinium Hydroxylammonium Cesium Methylammonium Formamidinium Guanidinium

1.20 1.90 2.66 2.80 2.96 3.0 3.3 3.3 3.38 3.6 3.6 4.8

’ Diameters measured using CPK models of cations and measuring the diameter of the 5-oxygen pentagonal pore through which the model can just pass. Where appropriate the models were built with hydrogen-bonding hydrogens.

investigated either

by examining

the external

solution

the

effects

or the internal

of changes

of

solution

on

reversal potential in voltage-clamped cells. The selectivity ratios measured with changes in the bath solution are P,,:P,:P,,:P,, = 1.0:0.088:0.045: 0.027.212 The ratios found with changes in the internal solution in internally perfused cells are P,,:P,:P,,:P,, = 1.0:0.14:0.05:0.04.2s1 The values of PK/PNa are close to that found for frog nerve (0.086) and neither Rb+ nor Cs+ are measurably permeable in frog nerve. ‘19 Therefore the selectivity filter of the egg channel seems to be quite similar to that of frog nerve and muscle and the squid axon. The selectivity data for nerve, muscle, heart and the tunicate egg membrane can, by and large, be fitted by a single channel. The ionic interactions found in the membrane of the squid giant axon suggest the presence of multiple barriers and/or sites within the channel. the

POTASSIUM

CHANNELS

The falling phase of the action potential in the giant axon of the squid, and in many other tissues, is due in part to a voltage-dependent increase in the K’ permeability. 12’ For the squid axon K+ channel, Rb+ is permeant,*** NH: is less permeant” and Na+,‘* Cs+ 28,222 and Ca2+ 18 are not permeant. At membrane voltages in the range 2 160 mV, the K+ channel becomes significantly less selective, and Na+ is more permeant.” The permeability of the K’ channel in the myelinated nerve fiber of the frog has been investigated by HilIe.‘*” The permeability ratios, measured from the reversal potential, are given in Table 4, along with the estimated minimum size of a selectivity filter per-

meable to the ions. The diameters for the few permeant ions fall in the range of 2.6 and 3.OA. These ions are smaller than a number of the ions to which the Na+ channel is permeant. Indeed, HilIe’*’ notes that K’ channels discriminate more than do Na’ channels, since there are fewer permeant cations for the K+ channel. Also the ratio P,,/PK for the K’ channel (~0.01) is smaller than PK/PN:, for the Na+ channel (0.086) (see Tables 3 and 4). A model for the K + channel selectivity filter has been suggested by Hille. I*” It is very simi!ar to that for Na+ just discussed, but its size, 3 A in diameter, is somewhat different. The basic mechanism of permeation is the same, but the ions fit differently, as required by the experimental results. A cation in the filter is in contact with only two water molecules, along with the 0 atoms lining the pore. Thus ions are dehydrated in both filters, but with the exception of Li+, the ions in the K’ filter have fewer water molecules than do those in the Na+ filter. A similar model for the selectivity filter for the K+ channel has been proposed by Bezanilla & Armstrong.28 An increase in the intracellular free Ca*+ level produces a hyperpolarization in many neuons, as a consequence of an increase in the potassium conductance.‘h5.‘87 The selectivity of this channel in AplJ1.G neurons has been examined by measuring the reversal potential in solutions of various cations.270~Z7’ The permeability ratios as measured from the reversal potential are: P,:P,,:P,,:P,,,,:P,, = 1:0.98:0.7: 0.12:0.03 (the Na+, Li + and tetramethylammonium permeabilities are too small to measure). Except for Tl+, the results are similar to those found for frog nerve. Molluscan neurons show a delayed, time- and voltage-dependent outward current which is quite similar to the voltage-dependent K’ current in the squid

1345

The selectivity of ion channels in nerve and muscle axon and the myelinated frog nerve.50 For a snail neuron, the relative permeabilities, measured from the reversal potential, are P,,: P,: P,,: Pc,: PNH4:PLi: P,, = 1.29:1.00:0.74:0.18:0.15:0.09:0.07. The sequence differs slightly from that for frog nerve and muscle (NH; and Cs+ are reversed) and the magnitudes are also somewhat different.“’ It is uncertain at this time whether the difference is significant. In some neurons of Aplysia, ACh produces a rapid and a slow inhibition.i4’ The IPSPs produced by stimulation show two similar components. The fast channel is a Cl- channel and was discussed above. The slow channel is a K+ channel; the permeability to Rb+ is about half that to K+, and the permeability to Cs’+ is too small to be measured. The permeability to cations of the membrane of the cell body of a giant neuron of Aplysia has been examined by both intracellular ion selective electrodes and membrane conductance measurements.” The results show Tris to be quite permeable; if it moves only through the Na’ channel, the value of Tris/P,, is about 0.84. However, the membrane is more permeable to K+ than to Na+ (PNa/PK = 0.1 to 0.13), and so Tris could possibly be crossing the membrane through the K+ channel or even by some non-specific cation channel. The membrane of internally-perfused snail neurons shows a TEA-resistant voltage-dependent outward K+ current. The change in the reversal potential on replacement of K+ by Tris gives a value of PTris/PK of about 0.1.16’ This permeability to Tris is much higher than would be expected from the K+ selectivity filter in frog nerve. In the membrane of frog nerve, the so-called leakage current is the residual current after the voltageand time-dependent currents are subtracted from the voltage clamp records. The normal potential for the current is probably between - 70 and - 80 mV. The channel accepts several metal cations and NH:, with a sequence Cs+ = K+ > NH: > Tl+. Hille12’ suggests that.. “Although the evidence is incomplete, leakage channels seem to be potassium-preferring cation channels with less ionic selectivity than potassium channels”. Muscle The selectivity of the voltage-dependent K+ channel responsible for the repolarization phase of the action potential in frog muscle has been examined by voltage clamp. The relative permeabilities, estimated from the reversal potential are Pk: Pit,: Pc,: P,,: P,i = l:0.95:0.11:0.03:0.02,ga which are quite similar to those found for frog nerve. A similar value for P,JPk has been reported by Adrian, Chandler & Hodgkin.4 Egg cells and other systems The membrane of the immature egg of the starfish shows inward rectification, i.e. the membrane conductance for inward currents is much higher than for outward currents. The permeability ratios, calculated

from the slopes of the curves of the relationship between membrane potential and cation concenP,,:P,:P,,:PNH4 = 1.5: 1.0:0.330.4: are tration, 0.03-0.04. The permeabilities of Na+, Cs’ and Tris are too small to measure.‘15 The sequence is the same as that found for the K+ channel in frog nerve but the values are somewhat different (Table 4). In mixtures of K+ and Tl+, the membrane conductance does not change linearly with the molar fraction of Tl+ ; the effect can be explained by assuming the presence of binding sites for cations in the channels so that their permeability properties depend on the nature of the cation bound.iog The properties of a voltage-dependent K+ channel isolated from the sarcoplasmic reticulum of rabbit muscle have been examined by incorporating the channel into a lipid bilayer membrane. The selectivity of the channel was calculated from the potential across the membrane with KC1 on one side and the test cation on the other. The permeability ratios are PNn4:Pk:Ps,:PN,:PLi = 1.27: 1.0: 0.88: 0.51 : 0.13.5s The selectivity profile for this channel is quite unlike those of the other channels discussed above. The selectivity data for nerve, muscle and starfish egg are more or less consistent with the existence of a single selectivity filter; however, there are some discrepancies which may require additional filters. CALCIUM

CHANNELS

A number of cells show voltage-dependent Ca2+ currents. There are several problems connected with studying these currents (discussed extensively by Hagiwara & Byerly’02). The reversal potential cannot be measured because the Ca2+ concentration inside (10m6 M or less) is too low for there to be an outward current. The determination of E,,, by extrapolation is difficult, because of the problem of isolating the Ca2 + current from the other currents present. Careful studies of the current through the Ca2+ channel show that it saturates with increasing concentration, i.e. C Ic, = Ic”,“”___ C + K,,

(2)

where C = Ca2+ concentration, and Kc, is a constant given by the Ca2+ concentration at which Ic, = l/2 IF:“. In this equation, Ic”,a”can be considered to be a mobility factor and Kc, the affinity of a binding site in the channel. The selectivity of the Ca2+ channel is usually determined from the measurements of the currents in solutions of the different divalent cations. However, the equation shows that these currents are determined by the 1:: term only when the concentration of the test ion greatly exceeds Kc,, i.e. at or near saturation. Another problem encountered in characterizing the Ca’+ channel is that passage of divalent ions through the channel seems to require that these cations occupy sites on the membrane. The magnitude of the overshoot of the Ca2+ action potential in barnacle

1346

C. Edwards

muscle is reduced by the presence of other divalent ions or of La3+. The effects have been attributed to the competition between these ions and Ca2+ for the occupancy of membrane sites; the size of the overshoot is determined by the density of Ca2+ on these sites.“’ The sequence of binding as determined from the effect on the rate of rise of the action potential is UO$+ > Zn*+, Co’+, Fe2+ > Mn2’ > La3+, Ni2+ > Ca2+ > Mg2+ > Sr2+. The selectivity of the Ca2+ channel seems to be a function of the size of the ion. The two ions larger than Ca2+, in terms of both crystal radii and of limiting conductance, Sr2+ and Ba*+ (Table l), are usually permeant through Ca2+ channels, as first shown in sequence of the crayfish muscle. *2 The permeability smaller ions varies with the composition of the solution. For the membrane of barnacle muscle when there is only one divalent cation in the bath, the sequence is Ba2+ > Sr2+ = Ca*+; however, in the presence of Co2 +, which blocks the Ca*+ channel. the sequence

becomes

Ca2+

> Sr2’

> Ba2+.‘0’

Table 5. Block of voltage-dependent

Organism

Cal’

channels

Channels blocked by manganese

and/or cobalt

It is widely accepted today that block of a voltagedependent inward current by Mn2+, Co’+ or various other of the transition metal cations is evidence for the presence of a Ca* I channel. Some cells for which this is true are listed in Table 5. A preparation which behaves similarly is that of pinched nerve terminals (synaptosomes) prepared from rat brain. These show an increased uptake of labelled CaZC in the presence of elevated Kf levels,3’ and this increase in uptake is blocked by Mn2’. The membrane of the immature egg cell of the starfish, Mediaster aequalis, shows two voltage-dependent inward currents when studied with voltage clamp.“’ The two currents depend on Cazf. and Sr” and Ba2+ are also permeant. Both currents are blocked by Co’+ and procaine. However, one of the inward currents may be carried by both Na+ and Ca*+. The current increases with both Na’ concentration and Ca2’ concentration. Further, the current is unaffected

and other Ca2+ effects by various

divalent

Blocking ion

Site of effect

ions

Reference

Nerve Cells Aplysiu

Snail

Squid

Leech Lobster

Portunus sanyuinolentus

Frog

Xenogus

Chick

Pigeon

Component of action potential in nerve cell Nerve cell action potential Nerve cell action potential in absence of Na+ Slow inward current in pacemaker neurons Slow inward current in internally perfused neurons Nerve cell action potential Late phase of depolarization induced Ca*+ entry in axon Action potential in presynaptic nerve terminal in stellate ganglion Action potential in Retzius cell in Na-free solution TTX-resistant component of action potential in presynaptic nerve terminals Slow, regenerative, TTX resistant responses in cardiac ganglion cells Slow after hyperpolarization of motorneurons Action potentials in nerve termina! of spindle in TTX and high Ca2+ Action potential in dissociated neurons in culture Action potential in Rohon Beard cells in spinal cord in early stages Component of action potential of dorsal root ganglion cells in culture Dendritic action potentials in cerebellar Purkinje cells

co2+

Geduldig

co*+ co*+

WaId263 Standen“”

co2 +

Eckert

& Junge99

& Lux’~

Cd’+

Kostyuk

& Krishtallb’

Cd Zi CoZ’ C&+ Co”, Mn’+

Akaike,

Lee & Brown5

Cd*+, Mn’+

LlinBs. Steinberg

Co’+.

Kleinhausls4

Mn’”

Baker, Meves & Ridgway”’ & Walton”’

co2+

Kawai

& Niwa’4h

Mn’+

Tazaki

& Cookez5*

& Barrett”

Co’+,

Mn*+

Barrett

Co’+,

Mn”

Ito & Komatsu’37

co’+ Co’+,

Mnzi

coz+

Co’+,

Mnzi

Spitzer & Baccaglinizd5 Baccaglini

Dichter

& Spitzer”

& Fischbachhs

LlinLs & Hess’ ”

The selectivity

of ion channels

in nerve and muscle

1347

Table 5.--continued

Organism Guinea-pig

Mouse

Rat

Blocking ion

Site of effect Action potential in nerve cells in Auerbach’s plexus Burst response in cerebellar Purkinje cells Component of action potential in neuroblastoma cells in culture Component of action potential in dorsal root ganglion cells Component of action potential in dorsal root ganglion cells in culture TTX-resistant action potential in sympathetic neurons

Mn2+

Reference Hirst & Spencelz4

Cd2+ Co2+ Mh2+ co2+

Llinas & Sugimori”* Spector,

co2+

Yoshida,

co2+

Matsuda, zaware6

Kimhi

& Nelson

Matsuda

244

& Samejima2”

Yoshida

&

Co*+, Mn’+

McAfee 8~ Yarowsky’80

Mn2+ Zn2+, Co’+, Fez+, Mn2+, Ni’+, Mg2+ co2+

Hagiwara Hagiwara

Yone-

Muscle Balanus

Muscle

Drosophila

Action potential in larval muscle fibersin presence of TEA Action potential in larval muscle Action potential in larval skeletal muscle Action potential in larval muscle in presence of TEA Action potential in muscle in absence of Na+ and presence of procaine Plateau of action potential of muscle cells in culture

Mealworm Xylotrupes dichotomus

Waxmoth Amphioxus

Chick

action

potential

& Nakajima”’ & Takahashi1r2

Suzuki & Kane’@

co2+

Yamamoto

co2+

Fukuda,

Mn”

Yamamoto

Co2’

Hagiwara

Mn2+ co2+

Kano & Shimada14* Kanor4’

Mn2’

Hagiwara

Mn2’

Rougier, Vassort, Garnier Gargouil & Coraboeufz3’ McDonald, Sacks & DeHaan”’

& Washio274 Furuyama

& Kawa”

& Fukami273 & Kidokororob

Heart Frog

Chick Cat

cow Dog Rabbit

Rat Sheep

Plateau phase of action potential in ventricle cells Component of action potential in atrial cells Spontaneous beating of aggregates of embryonic heart cells in culture Action potential in ventricle Slow current in ventricle trabeculae Plateau of action potential in Purkinje fibers Action potential in ventricle cells Action potential in atrioventricular nodal cells Action potential in ventricle cells Component of action potential in ventricle Slow inward current in Purkinje fibers

Mn” Mn2+ Co’+ Mn2+ ~i;i,+

Mn2+

potential

Balanus

Action neuron

potentials in photo-receptor in presence of TEA

Toad

Regenerative responses in rods in presence of TEA Glucose induced action potentials in pancreatic islet cells

Mascher’ 84 Kolhardt, Bauer. Krause Fleckenstein’56 Carmeliet & Vereecke45

&

Mn2’

Other Action

Stylonychia

& Nakajima”’

Mn2+

Zipes & Mendezzso

Mn2+ Mn2+

Takeya & Reiterz5’ Coraboeuf & VassortSb

Mn2’

Vitek & Trautweinz6’

Mn2+

de Pever & Deitmar63

cells

(ciliate) Co2+ M 2+ M’,2 +g

co2+ Mouse

Cd’+ Co’+ MHZ+ Mn*+

Ross & Stuartza2 Edgington & Stuart” Fain, Gershenfeld & Quandt” Dean & Matthews”

C. Edwards

1348

on replacement of Na+ by Li+, Cs+ or Rbf, but Tris is impermeant. However, it is uncertain if Na+ enters the channel or if its action is to modify the Ca” current, Thus it is unclear whether the selectivity filter controlling this current is or is not the same as one of those already described.

Channels permeable

to manganese

Squid axons perfused with a solution in which the only electrolyte is CsF give action potentials in external solutions in which the only electrolyte is CaCI,. The action potential is still present if the CaCl, in the external solution is replaced by 50mM MnC12.272 This suggests that voltage dependent Ca2+ channels may be present in squid giant axon, and that they are permeable to Mn2’. However, under these perfusion conditions, the action potentials. found in external solutions containing only CaCI,, BaCl,, or SrCI, are blocked by tetrodotoxin,265.2h6 which is a specific blocking agent for the voltage-dependent Na+ channel. Further. in axons perfused with NaF, the action potentials found in solutions containing 100mM CaCl, have components sensitive to tetrodotoxin and to tetraethylammonium ion;‘3h the latter is a specific blocker for the voltage-dependent K+ channel. Therefore there are some problems that remain to be resolved concerning the selectivity of channels in the perfused squid axon. In the clonal nerve cell line, PC12, elevated external K+ increases the uptake of radioactive Ca2’, and this increase is inhibited by Mn2+. However, in the absence of Ca”, elevated K+ produces an uptake of Mn2+.24h Furthermore, the release of dopamine from these cells by high K+ is Ca2+-dependent, but Ca2+ can be replaced by Mn2f.228 Thus, Mn2+ seems to be permeant through the Ca2+ channel in the absence of Ca2+, but also may block Ca2+ entry. The Ca’+ channel of frog skeletal muscle fibers is permeable to Mnzf, and P,, < P,,.* Myoepithelial cells of the marine polychaete worm Syliis spongiphila show overshooting membrane potential responses after transmembrane current pulses or the addition of carbamylcholine.’ The responses are reversibly abolished by removal of Ca 2f The magnitude of the overshoot is increased by the addition of Mn2+ to the bath, either in the presence or in the absence of Ca2+, suggesting that Ca2+ and Mn2’ are similary permeant, The responses are blocked by Co2+, Ni2+ or Zn”, whose crystal radii are smaller than that of Mn2+. In frog atria, 2 mM Mn2+, in the presence of I mM Ca’+, reduces the overshoot of the action potential, while higher levels of Mn2+ (8 mM) increase it. It is proposed that Mn2+ at low levels may block the Ca2+ current and at higher levels may replace ca2+,47.48

The overshoot of the action potential in guinea-pig hearts in Ca-free and Na-free solution increases with the Mn2+ concentration.2’0 The increase over the

range 2 to 20 mM is about 24 mV. which is not too much below the 30mV expected from the Nernst relation. In bath solutions containing 2mM Ca2+, the addition of Mn2+ usually, but not always, reduces the overshoot of the action potentiaL6’ In reduced Ca’+ (0.1 mM), 10mM Mn 2+ increases the overshoot. Here again, the behavior is consistent with the proposal that Mn2+ can inhibit Ca2+ entry into the cell, and that Mn2’ can enter when its concentration is an order of magnitude greater than the Ca2+ level. The Ca2+ channels in several, but not all, oocytes are permeable to Mn ’ + The action potential of the sea urchin oocyte, and the off response following membrane depolarization of the mouse oocyte involve a Ca2+ channel that is permeable to MnZ+.21’ In the egg cells of starfish, there is a small inward Mn2+ current in the absence of Ca2+.io8 In contrast, the voltage-dependent Ca2+ channel in the membrane of the egg cell of the tunicate is not permeable to Mn2+.213 Thus, the demonstration of the permeability to Mn2+ requires, in some cases, that Ca2+ be removed or that the Mn2+ concentration be several times the Ca2+ level. Since in many published experiments, the Mn2+ levels were similar to the Ca2+ levels, there remains some doubt about the permeability to Mn2+ in these situations. However, the membranes of some ceils are impermeable to Mn2+ when tested in this way; for example, crayfish muscle gives no sign of a regenerative response in isotonic MnCI, (160 mM).82

Ion effects on the Ca2+ channel If the Ca2+ level in the external medium is reduced the properties of the Ca2+ channel may change dramatically. In internally dialysed molluscan neurons, the reduction of external Ca2+ by addition of EGTA causes a Na2+ inward current to appear, the kinetics of which are similar to those of the Ca2+ current in the normal medium.‘62 The Na’ current is blocked by agents which block the Ca’+ channel; thus, it is thought that the selectivity of the Ca2+ channel is altered so that it becomes permeable to Na+.159 The muscle fibers of larvae of the mealworm Tenehrio mollitor have a Ca2+ action potential, and when the Ca is removed in some fibers, the channel become permeable to Na’. The Na+ action potential is blocked by Co2+, which usually blocks the Ca2’ action potentials, and is not affected by tetrodotoxin, which usually blocks Na+ action potentials.274 Action potentials are present if Na+ is replaced by Li+. After replacement by choline or guanidine, there are possible voltage-dependent conductance changes, but no action potentials. There are no responses in the presence of NH, or tetraethylammonium ion.275 Thus the profile of permeant and impermeant cations is different from that of the voltage-dependent Na’ channel described above (Table 3) where the relative permeabilities are P,,: PLi: PNH4:Pgudnidine:P.,,,: Pcho, = 1.0:0.91:0.16:0.13: <0.008: <0.007.

The selectivity of ion channels in nerve and muscle Channels permeable

to zinc

The action potential of the muscle fibers of the larva of the beetle Xylotrupes dichotomus in the presence of TEA is due almost solely to Ca*+ entry. The channel also admits Ba*+, Sr*+, Mn*‘, Cd*+, Zn’+ or Be2+; however Co*+, Ni*+ and Mg2+ are not permeant9* The specificity cannot be attributed to either the crystalline or hydrated ionic radii, since the crystal radius of Be 2+ is smaller and its size based on the limiting ionic conductance is greater than that of any of the other ions. In giant neurons of the Japanese land snail, Zn*+, Cd2+ and Mn2+ have been found to be permeant the voltage-dependent Ca*+ channel through (Kawar4’ and personal communication). The Zn*+ action potentials are blocked by Ca2+ channel blockers, such as Co2 + and La2 + . The crystal radii of the permeant cations are 88 pm or larger (Table 1). The restriction on permeability might be attributable to crystal radius, except for the impermeability of Co2+ whose radius is 88.5 pm. Note that the radii given in Table 1 are the best estimates currently available, for they are based on the broadest and most complete study of the data made to date. However, there are other estimates of the radii which give equal values for Co*+ and Zn2+.220 Therefore, there is a problem in interpreting the data in this way. The differences in the energies of hydration of various divalent cations have been used to explain their diverse actions on the Ca2’ action potentials of Syllis spongiphila.” The selectivity of the voltage-dependent Ca*+ channel in the membrane of the snail, Helix, has been examined by voltage clamp in internally-perfused cells.6 The permeability sequence (10 mM) is P,,:P,,:P,,:P,,:Pz, = 2.3:1.5:1.0:0.22:0.18. However, at higher concentrations (25 mM), P,, > P,, Co2+ and Ni*+ are not permeant. Channels possibly permeable

to magnesium

In cat ventricular trabeculae muscles, the similar effects on the slow inward current during voltage clamp on addition of Mg*+, Sr2+ and Ba*’ to low Ca*+ solutions suggest each of the three ions may enter the Ca*+ channel.“’ There is another report that Mg*’ 1s permeant, though weakly so, through the Ca2+ channel in heart muscle (Porteau, cited in Reuter & Scholz225). The Ca’+ channel of frog skeletal muscle shows a small Mg*+ current.8 However, the authors note that it is possible that the Mg2+ current flows “. . through a relatively unselective permeability mechanism.” Strips of arterial smooth muscle in elevated K+ show action potential like responses in sucrose gap recordings which last about a second. In the absence of Na+, both Ca2’ and procaine must be present to give the response; presumably, procaine is necessary to inhibit the high resting K’ conductance and thereby permit the responses to occur. The response is also

1349

found in the presence of either Mn2+ or Mg*+, which suggests that these ions may also enter the channel responsible for the slow inward current.14’ Electrical stimulation of the muscle membrane of mealworm larvae in a solution containing K+, Ca2+ and high Mg*+ (to replace Na+) gives an action potential.*’ The inward current was attributed to *+ but the Ca*+ level (10mM) may have been Mg 1 sufficient for Ca2+ to be the carrier. The details of the selectivity in this preparation remain to be determined. It is a bit difficult to sort out the Ca*+ channel data, as noted above. There appear to be no fewer than three Ca* + selectivity filters, one impermeable to Mm*+, one permeable to Mn*+, and one permeable to Zn*+. There may also be a channel permeable to Mg*+. Although Mn*+ blocks Ca*+ entry in a number of channels, the question of its permeability has not been investigated adequately. The interpretation of additional data, may, of course, require that additional channels be considered. LARGER CATION CHANNELS Excitatory

postsynaptic potential channels

At the frog neuromuscular junction, acetylcholine (ACh) reacts with receptor molecules on the membrane to produce a depolarization called the endplate potential, or EPP. The EPP is due to an increase in the permeability of the muscle membrane to cations.62,252 The EPP is still present after the Na+ in the bathing fluid is replaced by organic cations as large as hydrazinium,“’ methyl ammonium, ethyl ammonium and dimethyl ammonium,94 and trimethylethanol ammonium and dimethyldiethanol ammonium.204 The results of a study of the effects of replacing Na+ by a series of organic cations on the size of the ACh depolarization led to the suggestion that the channel is a pore about 6.4 A in diameter.‘s3 This was based on the finding that dimethyldiethanol ammonium is permeant, while methylethyldiethanol ammonium and diethyldiethanol ammonium are not. The results of a more recent study of the EPP with voltage clamp in which more cations were examined’i are similar (Table 6). Some ions, such as NH:, hydroxylamine, methyl ammonium, hydrazine, guanidine, formamidine, hydroxyguanidine and aminoguanidine are more permeant than Naf ; the permeability ratios, Px/PN, for these ions are 1.3 to 1.9. Cations as large as glucosamine and triethanol ammonium are slightly permeant. It was concluded that the selectivity filter must be at least as large as a square, 6.5 x 6.5 A. Further, for large cations, the selectivity seems to be controlled more by frictional factors than by chemical ones. Similar values for the relative permeabilities, based on voltage clamp measurements of E,,,, have been reported for ammonium, methylamine, hydrazine, formamidine and Lit.2h4 The reduction of the ionophoretically-induced acetylcholine current by guanidine

1350

C. Edwards Table

6. Permeability

ratios for the ACh controlled neuromuscular junction’.”

X

Mol wt

Monovalent (pH = 5.1)

organic

Hydroxylamine Ammonium Guanidine Formamidine Hydroxyguanidine Aminoguanidine Methylamine Hydrazine (pH = 6.6) Acetamidine Ethylamine Imidazole (pH = 6.0) Aminoethanthiol Dimethylamine Isopropylamine Methylguanidine Methylethylamine Ethanolamine n-Propylamine Ethylenediamine (pH = 8.4) Guanylurea Biguanide 4-Aminopyridine Methylethanolamine n-Butylamine Diethylamine Dimethylethanolamine Trimethylamine Trimethylsulfoxonium Trimethylsulfonium Piperazine (pH = 7.0) Triaminoguanidine Isobutylamine t-Butylamine Diethanolamine Dimethylethanolamine Glycine methylester (pH = 5.9) lsopropylethanolamine Tris (pH = 6.8) Isopropylethanolamine Diethylethanolamine Choline Glycine ethylester (pH = 5.7) Dimethyldiethanolammonium Triethylamine Dimethyldiethanolammonium Histidine (pH = 6.0) Glucosamine (pH = 6.3) Ethyldiethanolamine (pH = 6.6) Triethanolamine (pH = 6.6) Arginine Tetrakisethanolammonium Lysine Methylethydiethanolammonium

cations 34.0 IX.5 60. I 45.1 76. I 75.1 32.1 33.1 59. I 46.1 69. I 78.2 46. I 60. I 74.2 60.1 62.1 60. I 61.1 102.1 102.1 95.1 76.1 74.2 14.2 90.1 60.1 93.2 17.2 87.1 105.2 74.2 74.2 106.2 90.1 90.1 104.2 122.1 104.2 118.2 104.2 104.1 134.2 102.2 134.2 156.2 180.2 134.2 150.2 175.2 194.3 147.2 148.2

Divalent organic Ethanediamine (pH = 6.0) Butanediamine (pH = 6.85) Hexanediamine (pH = 5.9) 4-Aminomethylpiperidine (pH = 6.9) Inorganic Ion TI+ CS’ Rb’ K’

channel

Major atoms

2 I

4 3 5 5 2 2 4 3 5 4 3 4 5 4 4 4 4 7 7 7 5 5 5 6 4 5 4 6 7 5 5 7 6 6 1

at the frog

P,:P,,, I .92 1.79 1.59 1.58 1.50 1.37 1.34 1.32 I 20 I.13 0.95 0.94 0.87 0.x2 0.79 0 77 0.72 0.68 0.68 0.60 0.60 0.54 0.44 0.43 0.38 0.38 0.36 0.33 0.30 0.30 0.30 0.29 0.28 0.25 0.23 0.23 0.22 0.18 0.17 0.15 0.13 0.12 0.090 0.090

8 7 8 I 7 9 I 9 I1 I2 9 10 12 13 10 10

< 0.01 0.043 0.034 0.030 0.030 <0.014
cations 62 90 I18 I15

4 6 8 8

0.4 0.4 0.3 0.2

cations Relative hydrated

size

P,P\.,

0.66 0.64 0.65 0.68

2.51 1.42 I.30 I.1 I

1351

The selectivity of ion channels in nerve and muscle Table 6.-eonrinued Ion Na+ Li+ Mg2+ Ca2+ sr2+ Ba* + MnZf co2 + Ni’+ Zn*+ Cd*+

Relative hydrated size

PJPS,

1.00 1.30 0.95 0.84 0.84 0.79 0.97 0.97

1.00 0.87 0.25 0.22 0.18 0.21 0.25 0.23 0.25 0.26 0.13

1.oo 0.93 0.93

The values of E, for the monovalent ions were measured in solutions containing 114mM of the test ion; those for the divalent ions in solutions containing 20-21 mM of the test ion. Relative hydrated size data from Table 1, assuming Na+ = 1.00. and aminoguanidine, and the block by methylguanidine and choline, were interpreted as suggesting the channel to be a rectangle 3.8 x 4.8 A. However, they note that guanidine, aminoguanidine and methylguanidine block the ACh response at low concentrations and choline has been shown to produce a ‘use dependent block’ of the ACh channel.71 The channel size proposed by Watanabe & Narahashi264 is somewhat smaller than the 6.5 A square proposed by Dwyer er al.” based on the data summarized in Table 6. The large amount of data from voltage clamp studies given by Dwyer et af.” strongly supports their model. However, it should be noted that the changes in E,,, produced by changes in Ca” concentration are too complicated to be described by the Goldman-Hodgkin-Katz constant field equation;174 the analysis of the results suggests the presence of both surface charge effects and competition for a binding site. Additional insight into the properties of the channel has resulted from studies of the permeability of various metal cations. The selectivity among the alkali metal ions, Cs+, Rb”, K+, Nat and Li+ is weak2s95*‘75(Table 6). It has been suggested that the pore is neutral and water filled2 and is possibly lined with high-field strength negative polar groups2’ The selectivity among the alkali earth ions is also weak; however, the sequence Mg2+ > Ca” > Ba2+ > Sr*+ is almost opposite to their mobility sequence, which suggests that these ions interact to some extent with ligands in the channel.’ The transition metal ions Mn’+, Co*+, Ni’+, Zn2+ and Cd*+ are also permeant. The permeability of the ACh-activated channel of chick muscle cells in tissue culture has been measured by examining the extra uptake of radioactive compounds in the presence of carbachol, which is an ACh agonist.13’ This technique has permitted the determination of the permeability of the channel to non-electrolytes. The data are summarized in Table 7. It is interesting that small non-electrolytes such as glycerol and ethylene glycol are permeant. The sequence for the alkali cations is Cs+ > Rb’ > K+ > Na+. The

selectivity is relatively weak, as was also found in the endplate by electrophysiological techniques by Linder and Adams et & Quastel, I75 Gage & Van Helden al.’ This again suggests that the ions may be entering through a channel with a relatively large diameter. Huang et ~1.~~’ concluded that “. . . molecules with positive charge and (or) a hydrogen bonding donating moiety are more permeable than the ones without”. The channel is pictured as a relatively large waterfilled pore containing hydrogen-accepting groups or dipoles and at least one negatively-charged site. The ACh-controlled excitatory postsynaptic potential in sympathetic neurons of the frog seems to be controlled by a large cation selective channe1.278*27g Autoradiographic analysis shows that molecules as large as ethanolamine and agmatine (C[aminobutylJguanidine) are taken up by the cells in the presence of ACh. Electrophysiological measurements of E,,, showed that ethanolamine, glycine amide and agmatine are permeable. The neuromuscular junction of the crayfish is depolarized by stimulation of the excitatory nerve and by glutamate. The reversal potential estimated by extrapolation for the excitatory postsynaptic current produced by ionophoretically-applied glutamate during voltage clamp is about 39 rnV,‘14 and that of the excitatory postsynaptic current is about 24 rnV.“’ The effects of alteration of the concentration of K’, Cl-, Na+ and Ca+ on this potential have been examined. The change in E,,,, following replacement of half of the Na’ by Tris was less than expected from the Nernst equation; it is possible that Tris is somewhat permeant. A three-fold increase in K+ was without effect on E,,,; while the authors suggest that this implies that it is impermeant, the effect of similar changes of K+ concentration on E,,, at the frog neuromuscular junction have been shown to be quite small.2 A ten-fold increase in Ca2 + concentration in a Naf-free Tris solution increased the membrane current produced during voltage clamp by ionophoretitally-applied glutamate. Replacement of Cl- by methansulfonate was without effect on E,,,. Therefore, it is

1352

C. Edwards Table 7. Permeability

ratio of alkali ions and organic

molecules’

3’

R = pe~m~~_iii~y_oftest molecules Transporting Alkali ion Na+ K’ Rb’ Cs’ Ca” One- or two-carbon Ethanolamine Ethylenediamine

molecules

charged

Charged

form

0.72 0.63 0.57* 0.45 0.x2

0.92 0.17 0.12 -0 0.04 -0 0.05 -0

0.1 I

form.

* The published values for Cal’ by A. R. Martin

of I?a +

compound

possible to argue from the data now available that the selectivity of the glutamate-controlled channel at the crayfish neuromuscular junction is similar to that of the ACh-controlled channel at the frog neuromuscular junction. Further, Dekin (personal communication) has found that the channel is permeable to methylammonium (MA), and has estimated PJPNa to be about 1.4. This value is quite close to that found in the ACh endplate channel (1.34, cf Table 6) and very different from that found in the voltage-dependent Na+ channel of the frog myelinated nerve (<0.007, cf Table 3). Dekin also found E,,, to be about +6 mV in muscles treated with tetrabutylammonium ion (which reduces the membrane conductance several fold, and blocks contraction; Fatt & Katz”4). The value of E,,, of t-6 mV is somewhat more positive than that usually found in frog muscle, e.g. -4 mV :’ 74 the difference is probably due to the greater external Na’ concentration in crayfish ringer. The application of ACh to the innervated face of the electroplaque of the eel gives a depolarization similar to that found at the neuromuscular junction.234 This channel is permeable to K+, Na+, Ca” and glucosamine. The relative permeabilities of these ions are 1.0:0.9:0.8:0.14* respectively.‘69 These permeability ratios were calculated from the reversal potential. The permeability of the channel to glucosamine suggests that its diameter is greater than that of the ACh-controlled endplate channel.

been corrected

permeability form

1.0 1.47 1.52 1.91 0.22

Ethylamine Methylamine Ethylene glycol Carbonyl and related compounds Guanidinium Formamide Urea Acetamide Thiourea Other compounds Tris (hydroxy-methyl) amino methane Glycerol Mannitol * Doubly

Uncharged

and glucosamine have (personal communication).

In an identified group of neurons of Apl~~itr, ACh has been shown to produce an excitatory depolarization.’ The inward currents induced by ACh are also present when all of the external Na+ is replaced by Cs’. Mg2+ or Ca2+.15 The only larger cation examined, Tris. seems to block the channel,’ 5.2h8 and so there is not sufficient data to estimate the channel size. Tris6’.‘” and glucosamines5 have also been shown to have a weak blocking action at the AChcontrolled channel at the frog neuromuscular junction. At the locust neuromuscular junction, glutamate also depolarizes the membrane potential similarly to the excitatory transmitter. I’ The reversal potential for both currents is about 3 4mV. The changes in E,,, with changes in external K ’ , Na . Cl- suggest the channel admits cations and not anions. The absence of glutamate current in solutions without Na’ and Ca2 + may be due to the fact that Tris and choline, the replacement cations. have a weak blocking action on the channel (R. Anwyl, personal communication). The channel also passes Ca2+, Li ‘. NH;. methylammonium and guanidinium. The E,,,, in solutions in which Na+ is completely replaced by these cations is about the same as that for the Na+ sohltion.‘2 The data suggest a rather large, cation permeable sclectivity filter. The selectivity of the channel open during the EPSP and the glutamate response at the neuromuscular junction of Drosophil~c larva has been measured. The selectivity ratios, measured from the effects of changes of various ion concentration on E,,,. are P,;,: P,: Pue = 1.0:0.77:3.6.“’ Changing external

The selectivity of ion ciunnels in nerve and muscle Ca’+ from 0.1 to 1 mM has no effect on E,,, and an increase to 1.8 mM produces a negative shift in E,,,; therefore, it was assumed that Pc, is zero. However, given the estimate of Pc,/P,, of 0.22 for the frog neuromuscular junction,z the shift in E,,, expected to be produced by this change in Cazt concentration is quite small. Further, changes in external Ca’ + in this range might alter the internal Ca” level, and therefore could affect the Ca”-activated K+ conductance, as noted above. In any case, it seems unlikely that a single channel admits Na’, K+, and Mg’+ and not Ca’+. The channel involved in neuromuscular transmission in the larvae of the mealworm, Tenehrio molitar, appears also to be a relatively non-selective cation channel. The transmitter is unknown, and so only the excitatory post-synaptic potential evoked by nerve stimulation was studied. This, of course, restricted somewhat the ionic environments that could be examined. E,,, is shifted by changes in Na+, K+, Ca*’ and but not by replacement of ClMg’+ concentrations, by methylsulfate.16’ The cell bodies of the giant reticulospinal neurons (Miller cells) of the lamprey are depolarized by both glutamate and aspartate.‘*’ The reversal potential for the glutamate potential, estimated by extrapolation, varies from - 16 to - 35 mV. Replacement of 90% of the extracellular Na+ by choline shifts the reversal potential in the negative direction, while injection of Cl- into the cell is without effect. The data are consistent with the presence of a large cation channel; however, other possibilities cannot be excluded, because of the incomplete data and the uncertainty resulting from estimating the reversal potential by extrapolation. There appears to be a minimum of two large cation selective channels; the selectivities of the ACh channel at the frog neuromuscular junction and the glutamate channel at the crayfish neuromuscular junction are similar, and the ACh channel of the eel electroplaque is larger.

Stretch of the crayfish stretch receptor causes a depolarization known as the receptor potential, and this normally leads to action potentia1s.s’ Under physiological conditions the principal ion moving through the channel responsible for this potential is Na’.78 However, the presence of a significant depolarization in response to stretch after the replacement of Na+ by hydrazinium, choline, tetramethylammonium, tetraethylammonium and Tris208 or arginine”’ suggests that the channel may be quite large. The currents underlying the generator potential were analysed in vottage clamp experiments by Brown, Ottoson & Rydqvist39 who estimated the ratio of the permeability of arginine to that of Na’ to be about 0.25. In this calculation, it was assumed that Ca*+ was impermeant. However, the receptor potential channel

1353

has been shown to be permeable to the alkaline earth cations, Ca’+, Sr*+, Mg2+ and Ba’+.” The entry of Ca2+ does not explain all of the current formed in the arginine solution and so the diameter of this channel is likely significantly larger than that of the ACh endplate channel. In the muscle spindle of the cat, about 25% of the receptor potential persists in solutions in which Ca2 + and Na+ have been replaced by Tris, glucosamine, tetraethylammonium or spermidine.‘32 Similarly, the receptor potential of the coxal muscle receptors of the crab are reduced to about one third after replacement of Na+ by either Tris or choline.229 This suggests a large channel, probably cation specific, for both receptors. The hair cells in the vertebrate ear give a microphonic potential in response to a mechanical displacement. This transduction process is presumably the first step in audition. The effects of ionic substitution on the current produced by stimulation of the voltage clamped hair cell have been examined5’ in solutions in which the cation was essentially only the test ion. The ratios of the means of the signal amplitudes are NH:: K+ : Rb+: Cs+: Li’ : NaC: tetraethylammonium = 1.3: 1.0:1.0:1.0:0.9:0.9:0.2. The data suggest the presence of a rather large, cation selective channel, The receptor cells in the statocyst of Aplysia may be stimulated by tilt. The depolarizing receptor potential and the accompanying decrease in membrane resistance are abolished foliowing replacement of Na+ by either Tris or Mg2t.97 This suggests the presence of a small, Na+ specific channel; however, the data are rather incomplete. Mechanical stimulation of the membrane of the ciliate Stylonychia produces a depolarization which is usually due to the entry of Ca2c.Za2 Mg2+ can carry the current about as welt as does Ca’*; the permeability of the channel to K” is low (PcJPk 2 19) and the current is blocked by Mn2+.63 Therefore, the behavior of the selectivity filter is neither like that of the large cation channels underlying some receptor potentials (which should be permeable to Mn’+) nor like that of the typical Ca” channel (most of which are impermeant to MgzC). The selectivity filter of the cation selective channel responsible for the receptor potential of the crayfish receptor is larger than the ACh channel at the frog neuromuscular junction, and it may be similar in size to the ACh-controlled channel in the electroplaque. Photoreceptor

potentials

The photoreceptor cells of the rudimentary ventral eye of Limlus generate depolarizing potentials in response to light. rB9 The replacement of extracellular Na+ by Li+, Tris or choline attenuates, but does not abolish, the light-induced current.“’ The effects of these ion substitutions on the reversal potential for the light response were studied with voltage clamp by Brown & Mote.4’ There is no change in the reversal potential when Li+ replaces Na+. Choline is some-

1354

C. Edwards

what permeant. The shifts in the reversal potential following replacement of Na” by Tris or of NaCl by sucrose are similar. This suggests that Tris may not be permeant. However, it is similar in size to choline, and exceeds it in hydrogen bonding ability, and so it is possible that Tris may block the channel, as was found with some of the large cation channels generating excitatory post-synaptic potentials (see above). The properties of the membrane channel responsible for the light-induced response of the photoreceptor cells of the lateral ocelli of the barnacle have been examined by voltage clamp.38 E,,,, which is about +25 mV, shifts on replacement of Na+ by Tris or K+. The slope of the E,,, vs log Na’ relation is 10-15 mV, and is too small for the channel to be a simple Nat channel. Replacement of Ca2’ by Mg’+ is without effect. It is possible that the channel may be a large cation channel but the data are incomplete and other interpretations are possible. Voltage drpendrnt channels

The membrane of neurons in the ganglia of the snail, L~~~~~ stagnalis, shows a voltage-dependent outward current when the potential is stepped to large positive values with voltage clamp. In internallydialyzed cells the current has been found to be carried by Na+, Csi, Tris and tetraethylammonium ions.42 Therefore, the current appears to be due to a large cation channel. ION MOVEMENTS ACROSS MEMBRANES AT REST In the discussion above, evidence was presented to support the concept that the movements of ions across the cell membrane initiated by chemical transmitters or by changes in membrane potential are uin channels. There is also the question of how ions cross membranes in the absence of these stimuli, i.e. at rest. Physiologically, some cells such as the spinal motor neuron, may never be at rest. Indeed, the endplate membrane of skeletal muscle at rest is apparently constantly exposed to ACh,1”4*‘h2 but since the area of the endplate is a small fraction of the total membrane area, the ion movement induced by ACh is likely to be only a small fraction of the total ion movement found in resting cells. However, there is evidence in the literature which will be reviewed, which suggests that at least some of the so-called resting flux may pass through the voltage-dependent channels. Potassium is usually one of the more permeant ions across the membrane of nerve and muscle cells at rest. As a consequence. an increase in external K+ usually changes the membrane potential in accordance with the Nernst equation. If the channel in the resting membrane through which K’ moves is the voftagedependent K ’ channel, the values of the relative membrane permeabilities estimated from the changes in membrane potential produced by high concen-

trations of various alkali cations should be similar to those in Table 4. Data from several cells in the literature are consistent with this predicted behavior. The perfused giant axon of the squid has been used to investigate the properties of the channels responsible for the resting potential.” The K’ in the usual perfusion solution of isotonic K2S04 solution was replaced by other cations. The selectivity sequence, based on the changes in the resting membrane potential, is K+ > Rb+ > Cs+ z Na+ 2 Lif. The values of the relative permeabilities may be estimated from the published changes in resting membrane potential with the equation:

The author has estimated the ratios to be P,:PRb:PCq:Phla:Pf.i = 1:0.89:0.l5:0.12:0.~. The cation selectivity of intact squid axons has also been estimated from changes in the membrane potential produced by substitution of cations in external solution.io4 The average permeability ratios, calculated from the Goldman-Hodgkin-Katz equation are P,,:PK:PRb:Pcs:PNa = 1.8:1.0:0.72:0.16: ~0.08. The sequences are the same as that found for the K+ channel of the frog myelinated axon,*2o although the numbers are somewhat different. The cation selectivity of the K’ channel in large identified neurons in the buccal ganglion of the marine mollusk ~uzlunu~ in~~~~s has been measured.L73 Salicylate was added to block the permeability to Cl-. The changes in membrane potential in solutions containing only the test ion were measured, and the values were used to estimate the relative values of P,. are equation 3. The mean values by PK:PRb:PCs:PId:P& = 1.0:0.71:0.15: <0.01:<0.01. At pH 7.7, the resting membrane of the muscle of the barnacle Balanus ~14bi~~{s Darwin is mostly permeable to cations.‘ih The permeability sequence has been measured from the voltage changes after ionic substitution. The conductance sequence was determined by measurements of the membrane conductance in the same solutions. The permeability and conductance sequences are identical, K+ > Rbf > Cs+ > Na’ > Li+. The K’ channel in the cells used in the above ex~riments, the squid axon, the neurons of ~al~~n~~ and the barnacle muscle, is voltage-dependent. In at least some of the measurements quoted, the membranes were depolarized by high concentrations of the test cations. Therefore, the voltage-dependent K’ channel was probably open, and so the measured permeabilities represent a summation of unknown contributions from the voltage-dependent K+ channels opened by depolarization and the K+ channels which contribute to the resting potential. Similar measurements have been made on two cells whose K+ channels are not voltage-dependent. In the neuroglia in the optic nerve of Nectnrus, the average

The selectivity of ion channels in nerve and muscle relative permeabilities are P,,: P,: P,,: Pc,: PNH, = 2.3:1.0:0.55:0.34:0.16.35 The permeabilities of Na+, Li’, guanidine and tris are too small to measure. The permeabilities of the membrane of the dark-adapted photoreceptor of the barnacle, Bakwus, have been ratios measured in two ways. 4o The permeability measured from the relationships between the membrane potentials and the ion concentration are P,:P,,:P,,:P,,,:P,, = 1:0.87:0.31 :O.l:O.l. The ratios measured from the membrane conductances in the various test solutions are P,: P,,: P,,: P,,: PLi = 1:0.64:0.38:0.08:0.06. In these two cells, the sequence of ions is the same as that found by Hille12o for the voltage-dependent K+ channel of frog nerve, and the numbers are similar, although not the same. Therefore, the data are consistent with the proposal that the channel whose permeability is responsible for the resting potential is the voltage-dependent K+ channel. The effects of depolarization on the K+ channels can be minimized by conducting the experiments at membrane potentials at, or close to, the resting level. This is done by determining the concentration of the ion replacing K+ required to give a membrane potential equal to that found in the usual bath solution. In that case, the relative permeability is simply Px _=_ PK

CKI, cn ’

assuming the ion conductances not to be altered by the presence of the test ion. In preliminary experiments on frog muscle, the permeabilities for Tl+, Rb+, and NH: are about the same as those given in Table 4 (Muniak and Edwards, unpublished). In the perfused squid axon, measurements of the permeabilities to a number of ions have given the ratios P~~~~m;~midin~~P~u;~nidinc~PLi~PNs~Pme~hylguanidine~ 2.81:1.21:1.05:1.0:0.96:0.92:0.72.1z3 pCx: Pcholinc = These ratios were calculated from the changes in the membrane potential produced by changes in ion concentration in the bath. The sequence and the relative selectivities are quite different from those of the voltage dependent Na+ channel which is responsible for the rising phase of the action potential. The authors suggest that the “. . . resting Na+ may operationally be different from the voltage-dependent Na+ channel”. However, in the squid axon membrane at rest P&P, is about 0.035, and therefore, the chance that the test ions cross the membrane uiu the K+ channel should be 30 or SO times the chance of their crossing via a Na+ channel. Indeed, formamidine, guanidine, methylguanidine and cesium are all slightly permeant through the K+ channel (Table 4) and this could explain the data. The results of measurements of the flux of various alkali cations across the resting membrane of frog muscle are also consistent with the proposal that these ions enter via the voltage-dependent K+ channel. Electrical measurements show this channel is

1355

quite permeable to Rb+ (P,,/P, = 0.92)“’ but not ratios estimated from to cs+.izo The permeability measurements of the rate of passive uptake are: RRb/PK about 0.543*242 and PcJPk about 0.005 (using a value of P,,/P, of about 0.01 derived from electrical measurementslz6 and P,JP,;, of 0.57 from flux measurementsz5 The Na+ influx in cells at rest is down electrical and chemical potential gradients and there is evidence that at least some of this influx may be via voltagedependent Na+ channels. About half of the Na+ influx in axons of the squid, Loligo, is blocked by tetrodotoxin (TTX). 17*231 Indeed Baker et ai.” note that: “This suggests that a significant portion of the resting Na+ influx into squid nerve takes place through the Na-selective channels which are involved in the action potential”. This suggestion is supported by the finding that TTX also hyperpolarizes the resting membrane potential by about 5 mv, and that the effect of TTX is said to resemble that of complete Na+ remova1.s8~223 In frog nerve studied with voltage clamp, TTX reduces slightly the current and the amplitude of the Na+ current fluctuation spectra at the resting potential. An estimate of the minimum number of Na+ channels that are open in the resting membrane of the squid axon may be calculated by comparing the resting ionic flux with that during an action potential. The TTX-sensitive Na+ influx, which is a measure of the passive Na’ influx, is about 16 pmol/cm’ s. The net Na+ influx during the rising phase of the action potential is about 1.63.1 pmol/cm2.36 The duration of the rising phase of the action potential is less than 1 rnslz8 and the influx during this period is 5-10 times the passive influx in 1 s. Therefore, if it is assumed that all of the Na+ channels are open during the rising phase of the action potential, the random opening of somewhere around lo-’ of the voltage dependent Na+ channels could account for the passive, TTXsensitive Na+ influx. In the node of Ranvier of frog nerve, it has been estimated that somewhat more than 10e4 of the Na* channels are open in the absence of an applied voltage.5 l The efflux of K+ from cells is usually considered to be passive (see review by Mullinszol). In the giant axon of the squid, the resting efflux is about 45 pmol/cm’ and the efflux during an action potential is about 2.9 pmol/cm’. Most of the efflux takes place in about 2ms but not all of the K+ channels are opened at the peak efflux. lz8 Thus, again, the resting flux can be accommodated by the opening of about 1% of the voltage-dependent K+ channels. The data on frog muscle are also consistent with the idea that much of the passive Na+ and K+ flux could use the voltage-dependent channels if about 0.1% of the channels were open. The Na+ influx at rest is about 3.5 pmol/cm’ s and the net influx during an action potential is 16 pmol/cm2.125 Thus the Na+ influx during an action potential, which lasts several ms, is about equal to the passive influx during 4.5 s.

1356

C. Edwards

The K+ outflux at rest is about 5.4pmoljcm’s. while during a spike is about the net K+ outflux 9.6 pmol/cm’. ’ ” Therefore, the K’ outflux during an action potential is about the same as the passive outflux in 2s.

AFTERWORD The data summarized above suggest that the selectivity properties of the channels studied to date are consistent with the proposal that the number of channels in nature is not too large. At this point. the number could be estimated to be a dozen or so. It is likely. of course, that other channels will be discovered. and that, indeed, some of the already described channels may turn out to be more complicated than they appear today. Further, the fact that channels show similar selectivities does not require that they be identical in structure. The channels may be opened by a number of transmitters for postsynaptic channels, possibly by hormones or other compounds or by changes in membrane voltage for voltage-dependent channels. The opening of some channels is controlled by changes in intracellular ion concentrations. such as the Ca” dependent K ’ channel. The mechanism controlling the gating of channels in sensory receptors is unknown, although Ca”+ may also play a role here. Nevertheless, a very large number of channel-selectivity control combinations is possible. This was first suggested for transmitter-controlled channels by Swann & Carpenter.“’ It remains to be determined whether all of the possible combinations are present in nature. There may be rules which preclude some combinations, but too little is known at present even to guess about possible rules. As noted above. the data for anions requires the existence of at least three selectivity filters. However. no measurements have been made where the internal anion levels have been manipulated in a known, quantitative manner. In the experiments on nerve cells, the anions were injected ionophoretically. and so the internal anion concentrations after the injections were unknown. Therefore the anion permeabilities cannot be calculated from E,,,. The recent advances in cell perfusion techniques make it likely that more quantitative data on anion selectivity may soon be available. The data for both the Na+ channel and the K+ channel can be ht fairly well by assuming a single selectivity filter for each channel. Indeed, the selectivity of the voltage-dependent Na’ channel in tunicate eggs is very much like that of the frog nerve membrane. and the selectivity of the voltage-dependent K+ channel in starfish eggs is also similar to that of frog nerve. There are complicated effects with both channels when two or more ions are present; however. these are usually explained by assuming the presence of binding sites and/or barriers in the channel.

Many cell membranes contain a Na+ + K+activated enzyme which uses the energy of ATP to move Na+ out of, and K + into the cell, against electrochemical gradients (see review by SkouZ43). Several ions can replace K + in activating the enzyme; the sequence of effectiveness is Tl+ > K+ > Rb+ > NH: > Cs+ > Li’. It is interesting that this sequence is identical to that for the permeability of the K + channel in frog nerve and elsewhere (Table 4). It is possible, therefore, that the protein responsible for the K+ selectivity filtermay be quite similar to that responsible for activation of the Na+ + Ki activated the K’ enzyme. Voltage-dependent Ca” channels are quite numerous in nature; however. the analysis of the properties of these channels has not proceeded as rapidly as has that of the Nat and K ’ channel. Some of the problems arc discussed in detail by Hagiwara & Byerly.‘“’ and only a few brief remarks will be made here. To date, no preparation has been found that is as useful to studies of the Ca2+ channel as the squid axon is for studies of the Na+ and K+ channels. However. the perfused molluscan neuron1h1.172 looks promising. and some selectivity data from this preparation were given above. The problem of separation of the inward Ca” current from the outward K’ currents has not been solved satisfactorily. The K’ currents arc complicated and may consist of several components. and the sensitivities of the components to various pharmacological blocking agents are not necessarily identical. The very large transmembrane gradient of Ca’+ (the ratio of external to internal concentrations is of the order of 105) makes E,,, quite positive. The Ca2+ current is small compared to the outward currents at positive potentials, so E,,, cannot be measured. In addition there are only a small number of ions that can be used to probe the Ca2’ channels. The largest divalent cation that can be used, Ba2’. is usually permeable. The smallest organic divalent cation is ethylene diamine. and is so different in structure from Ca2+ that it is likely not an adequate probe. The results of studies with cations smaller than Ca’+ were discussed above. The possible relationship between the transmembrane fluxes in quiescent cells and the voltage-dependent selective channels was also discussed above. The question is whether cell membranes have different channels for the so-called resting fluxes and for the voltage-dependent fluxes, or whether one set of channels is responsible for both lluxes. With this in mind, there is also the question of the existence of noise. or random openings, not only in the voltage-dependent channels. but also in some or all of the channels controlled chemically, either by transmitters or by internal substances. All of the channels are primed to open quickly following the appropriate trigger, and it may be possible that they occasionally open spontaneously in the absence of the trigger.

The selectivity of ion channels in nerve and muscle The Na’ and K’ channels are quite selective. The K+ channel passes ions only in a certain size range, i.e. it has a cut-off for both large and small ions, The Na+ channel probably functions similarly, but since Li”, the only ion smaller than Na*, that can be tested is permeant, a cut-off for small ions has not been demonstrated. For both channels the ions that permeate give up some of the bound waters. In contrast, the Cl- and large cation channels admit all ions up to a certain size, with no cut-off on the small side. Anions are relatively unhydrated; further the ions that pass the large cation channel under physiological conditions, Na+ and K+, likely retain most of their bound water molecules because of the large size of the channel. Therefore, these channels function somewhat differently from the way the voltage-dependent Nat and Kt channels function The Ca* ’ channel may be intermediate between these two classes. Small ions, such as Mgz* are relatively impermeant. The largest cation in terms of crystal radius, Baz* , is quite permeant, and so no cut-off for large ions has been demonstrated, The channels are usually permeable to Ca’* Sr”, and Ba2’, and so appear to be less selective t6an are the Na’ and K’ channels. There are several large cation selective channels. The selectivity filter for the ACh-controlled channel in the electroplaque and that for the receptor potential in the crayfish stretch receptor are both somewhat larger than that for the ACh-controlled channel at the frog neuromuscular junction. The first two channels

1357

may be similar in size, but the data are not adequate to say whether or not they are identical. Probing the selectivity of these channels will require the finding of satisfactory cations larger than those (molecular weight < 200 daltons) used so far.2+7i*183~‘q6 Possible obvious lines of future research of great interest are the intensive study of the properties of the channels and the models required to explain the pro~rt~es.10i.‘22 Perhaps more exciting developments will come when the channel molecules are isolated and their chemical properties and structures can be studied. Some progress along these lines has already been made on the ACh-controlled channel from the electroplaque. Acknowledgments-1

am indebted to the following for discussing some of the ideas contained herein and for reading various versions of the manuscript: M. Anderson, D. Carpenter, M. Rekin, H. R. Guy, S. Hagiwara, 8. Hi&, J. Jacklet, B. Lindley, R. Llinas, L. Mullins, P. O’Day, T. Slater, H. Tedeschi and K. Zierler. L. Welch graciously typed the several versions and the numerous alterations. Parts of this were written while I was visiting the Institute of Physiology of Czechoslovak Academy of Sciences under sponsorship of the National Academy of Sciences-Czechoslovak Academy of Sciences Exchange Program; and while visiting Kyushu University and Shimane University under the sponsorship of the Japan Society for the Promotion of Science. While this was written, my research was supported by grants from the Muscular Dystrophy Associations of America and the National Institutes of Health (NS-07681 j.

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The selectivity

of ion channels

in nerve and muscle

1365

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1981)

Note added in proof--Some recently published data on the selectivity of the voltage dependent Na+ channel in the squid giant axon determined from E,,, and voltage clamp are: Phydrazln.:-PLi: P,,: Phydrorylumine:Pgusnidine: P,: P. ,m,nnguanidine:PCs = 1.4: 1.1: 1.0:0.5:0.32:0.04:0.04:0.01 [data of Rojas E. & Taylor R. E., published in Rojas E. (1981) Ion permeability. In Membrane Transport (eds Bonting S. I & dePont) pp. 61-106, Elsevier/North Holland Biomedical Press, Amsterdam]. The data are somewhat different from those in Table 3 for frog nerve. This is surprising, because the other data for the squid axon, reviewed above, agree with Table 3. The selectivity of the Na+ channel of neuroblastoma cells in culture is decreased in the presence of veratridine, batrachotoxin, dihydrograyanotoxin and scorpion and sea-anemone toxins [Frelin C., Vigne P. & Lazdunski M. (1981) The soecificitv of the sodium channel for monovalent cations. Eur. J. Biochem. 191, 437442j.l WC.7,6-c

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In the GH cell of Onchidium, the rising phase of the action potential has both Na’ and Ca’ components. The Nat guanidium and ammonium ions. The channel is quite permeable to Li+, and is somewhat permeable to formamidinium, Cal+ channel is permeable to Sr’+ and Ba’+, but not to Mn’+ and Mg’+. The Cl- controlled by acetylcholine is permeable to chlorate, but not to formate and propionate ions [Edwards C., Sawada M., Kato M., Akaike N., Shimizu M. & Oomura Y. (1982) Camp. hiochem. Physiol., in press]. These somewhat incomplete data suggest that the selectivity of the three channels are similar to channels described above.