Ion regulation and membrane potential in tetrahymena and paramecium

C&p. Bi~cbem. Ph~.~il~~. Vol. 76A. No. 1. pp. I to 16. 1983 Printed in Great Britain

~300~9629~83 ~3.00+0.~ IC; 1983 Pergamon Press Ltd.

REVIEW

ION REGULATION AND MEMBRANE POTENTIAL TETRAHYMENA AND PARAMECIUM

IN

JOHN G. CONNOLLY and Cr. A. KERKUT Department of Neurophysiology, School of Biochemical and Physiological

Southampton

University.

Southampton (Receiced

SO9 .5NH, U.K.

I7

December

Telephone:

Sciences, (0703) 559122

1982)

Abstract-l. The electrical potentials across the membranes of the protozoans Tetrahymena and Paramecium are of considerable complexity and although they can be simplified to be regarded as a diffusion resting potential and action potentials, a more accurate understanding may be achieved by considering a more intricate model. 2. The animals are free living and continually interact with the environment. Their electrical potentials; mechanically and electrically elicited hyperpolarisations and depolarisations, resting potential fluctuations and oscillations, plateaus and after potentials, reflect these interactions and often instigate changes in behaviour. 3. The ion fluxes and permeability changes involved in the resting potential are described in terms ion pumps of a diffusion potential dominated by K’ to which Na+, Ca’+ and Cl- and electrogenic contribute. 4. Extending the ideas of many workers. the authors suggest that an electrogenic, ouabain sensitive Na’-K’ Mg?+-ATPase is associated with the contractile vacuole and that it makes an immediate and significant contribution to the resting potential. 5. A model describing the fate of Ca?’ which enters the cilia during ciliary reversal is proposed. In this model the plaques form an isolated, physiologically inert compartment into which Ca2+ is sequestered by a Cal+ -ATPase. Ca’+ is then eliminated from the cell by diffusion or Na+ :Ca?+ exchange. exchange is electrogenic, gives rise to plateau 6. Calculations demonstrate that the Na‘-Ca”+ potentials in paranoiac mutants and contributes to resting potential in these cells. 7. Classically, explanations of nerve activity have been patterned on the squid axon. However the complicated potentials of Tetrahymena and Paramecium may provide a more representative model of events in mammalian brain cells.

flourish in freshwater environments of widely differing solute composition. They are hyperosmotic to the medium (Stoner and Dunham, 1970) and accumulate inorganic cations from the environment (Stein, 1960; Dunham and Child, 1961; Andrus and Giese, 1963; Rosenberg and Munk, 1969; Dunham and Kropp, 1973), although in brackish media internal Na+ concentrations are lower than external Na+ concentrations (Andrus and Giese, 1963). The closely related ciliate Paramecium also exhibits these characteristics (Gelfan, 1928; Akita, 1941; Yamaguchi, 1960; Naitoh and Eckert, 1972). The environment of the cells in Nature is not constant in composition. Also, since unicellular organisms must be self sufficient, there are many processes such as feeding, water expulsion, absorption, excretion and reproduction which must alter the internal environment of the cell. Thus it would be expected that Tetrahymena must have very active and efficient mechanisms for maintaining the osmotic and ionic gradients (Dunham and Stoner, 1967) upon which its electrophysiology (Connolly and Kerkut, 1981) and behaviour (Brown rt al., 1981) depend. Majima (1980) measured the background resting potential fluctuation in Paramecium and found it to Tetrahymena

be l&i00 times greater than that of the squid axon (Fishman et al., 1975). However despite this large flux and the sensitivity of the cilia to changes in Ca*+ and (Naitoh and Kaneko, 1973; Mg2+ concentrations Nakaoka and Toyotama, 1979), the electrophysiological properties of the cell are relatively immune to changes in external ion concentration (Naitoh and Eckert, 1968; Deitmer and Machemer, 1982). Evidence for mechanisms which may influence or effect ion regulation in Tetrahymena, and their possible influence on resting potential, is reviewed briefly below. Several other reviews which discuss the electrophysiology and ion regulation of Tetrahymena and the related ciliate Paramecium have recently been published (Naitoh and Eckert, 1972; Dunham and Kropp, 1973: Machemer and De Peyer, 1977; Eckert and Brehm, 1979; Connolly and Kerkut, 1981; Doughty and Dryl, 1981; Kung and Saimi, 1982; Satow, 1982). ([.I’“& refers to the intracellular concentration of X’, [X’], refers to its extracellular concentration.) STATE OF WATER AND IONS

WITHIN

THE

CELL

The freedom of movement of water and ions within a cell may be restricted to some extent by binding to

2

JOHN G. CONNOLLY

intracellular charged sites or physical and chemical compartmentation. For water, some of which is possibly bound as water of macromolecular hydration (Caille and Hinke, 1972) the more recent, direct, NMR evidence suggests that the bulk of the water is free in biological systems; very little being structured by proteins (Fung et crl., 1975). A concensus exists that in most cells Cl- and K + are free whereas Na+ may be partly bound by intracellular proteins (Lev and Armstrong, 1975). Cal+ and Mg* ’ are mostly inert in Tetrahymena (Patterson, 1976) and Paramecium. The divalent cations are sequestered by chemical compartments, e.g. Rosenberg’s granules (Rosenberg, 1966), physical compartments, e.g. plaques (Plattner, 1975) and mitochondria (Nilsson and Coleman, 1977) and by proteins, e.g. Calmodulin (Suzuki et al., 1979). There is some evidence that Na + and K+ may be present in Rosenberg’s granules (Patterson, 1976). Generally speaking the monovaient ions are free (Stein, 1960; Dunham and Child, 1961; Helm, 1970: Krop, 1971) but most of the divalent cations and phosphate are practically physiologically inert within these protozoans (Yamaguchi, 1960b: Holm, 1970; Naitoh and Kaneko, 1973; Patterson, 1975). This implies that active transport systems are important in ~ontroIling the intracellular concentration of monovaient cations. The extent of their role is difficult to determine as estimates of free ion concentration vary so widely, and are made on systems which have a controlled environment and do not need the ion buffering capacity of freshwater ciliates. ION MOVEMENTS AND

RESTING

POTENTIAL

The differences in free ion concentration across the cell membrane and the differences in the resting permeability of the membrane to those ions is responsible for the resting potential of the organism (Donnan, 1911; Goldman, 1943; Hodgkin and Katz, 1949). If an active ion transporting system (ion “pump”) directly separates the charges across a membrane, the stoichiometry of the process may cause an unbalanced distribution of electrical charges. Such a pump is called electrogenic, and the magnitude of the current it produces may be sufficient to make an immediate contribution to the resting potential. Since ion pumping is an active process it would be expected to show a greater response to temperature changes than would simple diffusion. Thus if an electrogenic process is contributing to a resting POtential, the resting potential will have a greater Q,, value than one would anticipate for a passive process (i.e. 1.035 for a change of IO: C between 15 and 25-C). The Q,* was greater than expected in Paramecium, (1.07, Yamaguchi, 1960a) and in Tetrahymena (Connolly and Kerkut, 1983). In Tetrahymena it was found that without Nai in the medium, Q10 was 1.22, but with 20 mM Na,’ , the Q10 was 1.44, a significant increase (P = 0.1 yO). This is evidence in favour of an electrogenic Na” pump in Tetrahymena, although interpretation of these results does assume that the free ion concentrations and relative permeabilities remain constant during the temperature change. Furthermore, most of the potential change in Tet-

and G. A.

KERKUT

rahymena was inhibited by IO-’ M Oubain, inferring that an Na +--K+-ATPase may be involved. A hyperpolarising electrogenic pump may help stabilize the resting potential and hence behaviour of these cells by short circuiting depolarising spikes which would otherwise be triggered by ~uctuations in the resting potential. CONTRACTILE

VACUOLE

(C-V)

Contractile vacuoles are prominent features of freshwater protozoans (Spallanzani, 1776; Kitching. 1956) and some marine protozoans (Kaneshiro rt al., 1969). This organelle has been proposed to be the site of osmoregulation (Hartog, 1888) and, more specifically, Na+-extrusion, (Prosser, 1950; Dunham and Child. 1961: Chapman-Andersen and Dick, 1962). Dunham and Stoner (1967) stopped the activity of the C-V in Tetrahymena by hypertonic shock with sucrose solution. [Na’ 1, increased, but fell to normal levels after the C-V pump restarted. Kropp (1971) observed that the time course of the changes in [Na +1, correlated well with alterations in pump activity. Kramhoft (I 970) showed that [K ‘1; followed a similar pattern. Schmidt-Nielsen and Schrauger (1963), working with Amneha proteus, discovered that the [Na*] of the C-V was greater than the cytoplasmic [Na ’ 1,whereas the reverse was true for K ’ Josefsson (1966) and Prusch and Dunham (1967) found that the C-V of this cell was positive with respect to the cytoplasm. Thus Na ’ is actively expelled against electrical and concentration gradients, while K + is probably reabsorbed after initially filtering into the ampullae which channel fluid into the C-V (SchmidtNielsen and Schrauger, 1963). Cole (1925) reported that C-V activity in Paramecium was strongly dependent on temperature. Thus cooling may have similar elTects to hypertonic shock. Yamaguchi (1960a), when recording in the region of the C-V complex of Paramecium, noted a 4mV depolarisation just before systole which became a marked 18 mV hyperpolarisation peaking just after systole. Moreton and Amos (1979) recorded a 5-20 mV depolarisation associated with C-V systole in the contractile ciliate Zoothamnium. Although the accessory structures differ in their permanence the function of the C-V in Tetrahymena is essentially the same as that of Paramecium (Patterson and Sleigh, 1976). Potential oscillations with a period similar to that of C-V activity (Patterson. 1976) have recently been observed in Tetrahymena (Connolly and Kerkut, 1981, 1983). The mechanism whereby C-V activity influences resting potential is not clear. In Tetrahymena and Paramecium the cells are covered for the most part in a three-layered pellicle (Fig. l(A)). The C-V is delineated by a single membrane (Schneider, 1960; Elliott and Bak, 1964) which, on discharge, interrupts the pellicle. Moreton and Amos (1979) suggested that Zoothamnium has a similar singular C-V membrane of lower resistance than the pellicle, which short circuits the resting potential upon discharge. Yamaguchi (1960a) speculated that changing ion concentrations as the C-V empties gave rise to a localised alteration in membrane potential. Yamaguchi’s ex-

Ion

regulation

A

and membrane

nI

3

potential

ciliary peripheral central ci I iary

ci

I iary

membrane microtubul microtubules plaques

es

necklace

alveolar

terminal

membranes

plate

ectoplasmic layer------, kinetosome granular

,,,,,,,

,#I,,##

matrix

B

Ca++

1

/

ciliary

membrane

,al microtubules

c

f calmodul i

.L

aversal

100

central

\

microtubules

Na+ Ca++ ciliary

p

/ Ca++

Fig. I. (A) Schematic

diagram

- Na+

exchange

of ciliary structure, (B) diagrammatic representation of Ca’+ during and after ciliary reversal.

planation might reveal why the fluctuations were only observed when recording close to the C-V, and why, in Paramecium, the overall somatic potential is not affected. An implication of Yamaguchi’s work is that Paramecium may not be totally isopotential, but may compartment certain organelles in order to stabilise the somatic resting potential, and thereby the behaviour of the cell. Moreton and Amos (1979) did not specify whether C-V potentials were only found when recording near the C-V, but as they often found the potentials it is possible that the C-V in Zoothamnium is not as well isolated as those in Paramecium. Whatever the mechanism, it would seem that interruption of the pellicle is associated with potential changes. Other sites of discontinuity are the cytopharyngeal membrane, cytoproct, discharged mucocysts of Tetrahymena, discharged trichocysts of Paramecium, parasomal sacs and pinocytic vesicles, (Fig. 2). Activity at these sites may also disturb the background resting potential. The extent of this

of the reaction

path

disturbance will depend upon whether all parts of the cell soma are truly isopotential or whether they are to some extent compartmented. An Na+ extrusion role for the C-V is supported by the finding that Ouabain inhibits its activity (Boggs and Wade, 1972). It also excretes K+ as well as water and probably many other solutes to a lesser extent, (Riddick, 1968; Dunham and Kropp). By comparison with other ciliates it seems probable that the C-V in Tetrahymena will give rise to fluctuations in the membrane potential, especially when recordings are made in the locality of the C-V. Other sites of interruption of the pellicle may also contribute noise to the resting potential, although there may be some compartmentation of these effects. K+ ACCUMULATION

Tetrahymena cells maintain [K+], at a concentration of 20mM in the absence of external potassium (Dunham and Child, 1961). These workers also

JOHN G. CONNOLLY Excitation

Currents

(mechanical, chemical mucocyst

and G. A. KEKKUT

electrical stimuli K+ Ca++

and

+ ANTERIOR

parasomal

channe current

sac

I

carrier Ndf

current

va

A

-

cytostome

( pinocytic vesicle Active

,a3c -47

cytostomal

.!

lip

Transport

“,; Ki-H+

ATPase

Leakage

ta++/tig+fNa+ exchan;e

Currents

Cl-

Mg++ Car

Na +-1(+ ng++

NO

;\TPase

K

cytoproct S;sa:s.‘a.

a. contractile

Fig. 2. Summary

diagram

of probable

found that addition of small amounts of potassium to the medium resulted in a rapid uptake of K + against the concentration gradient. Stein (1960) showed that K+ is lost and Na+ is gained when the cell is cooled. Andrus and Giese (1963) demonstrated that the K+ loss could be up to 50% when the cells were cooled from 25”C, but that normal ion levels were reestablished upon rewarming. Part of the K’ loss (20%) was dependent upon the presence of sodium in the external medium. Metabolic inhibitors could reduce [K+], by 30x, and subsequent cooling caused a further 20% loss. Rewarming the presence of metabolic inhibitors yielded only a 70% recovery of [K + I,. Rewarming in the absence of K+ slows both the reaccumulation of K+ and the extrusion of Na+ against their respective concentration gradients. The rate of K+ accumulation at 20°C was noted to be 3 times that at 15”C, giving a Q,,, value of 3 for this process. There is thus good evidence that there is active K+ accumulation in Tetrahymena. The effect of metabolic inhibitors is suggestive of a primary active transport system although as Patterson (1976) notes, the results could be partly explained by a change in the relative K+ : Na+ affinity of K+ preferring binding sites within the membrane, or by changes in the activity of the contractile vacuole.

and known

vacuole

ion movements

in protozoans.

K+ EXTRUSION

K+ is lost from the cells by leakage down its concentration gradient (Dunham and Child, 1961). K ’ also leaves Paramecium through channels which may be excited electrically, mechanically and by calcium (Kung and Saimi, 1982, review). Similar currents have been seen in Tetrahymena uorax and T. thermophila (Connolly and Kerkut, 1981; Connolly et al., 1983). Moolenaar et al. (1976) measured the background fluctuation in the resting potential of Paramecium caudatum and demonstrated that the power of this fluctuation decreased upon addition of TEA, indicating the importance of K+ channels in the background noise. Adding TEA to the bathing medium of Tetrahymena also reduces background noise, suggesting that K + flux is also important here (Connolly (~2a/., 1983). Dunham and Child (1961) found [K+], followed [K+], between 10 and 60 mM [K+]],; whereas Patterson (1976) found [K ’ 1, to be constant over the same range. If Patterson’s results are correct, then there is some evidence for active K+ extrusion. However, apart from the C-V activity, demonstration of active K’ extrusion awaits further experimental investigation.

5

Ion regulation and membrane potential Na+ EXTRUSION Dunham and Child (1961) monitored the effect of [Na+], on [Na+],. In the absence of external Na+, [Na+], remained constant at 5 mM, 2 mM of which was not exchangeable. Increasing [Na+& to 20 mM did not alter [Na+], Above this concentration [Nat], followed [Na+ I,, Patterson (1975, 1976) obtained comparable data. From their results Dunham and Child inferred that there was active Na+ extrusion when [Naflo exceeded 5mM and that the system saturated at 20 mM. Andrus and Giese (I 963) demonstrated that [Na+ 1, increased 2-fold after cooling the cells from 25 to 6 C. Upon rewarming the cells restored [Na+], to the former lower level against electrical and concentation gradients. However metabolic inhibitors did not affect Na+ extrusion; cooling only was found to be effective. Hoffmann and Kramhoft (1974) found that ethacrynic acid, an inhibitor of the type II electroneutral Na+-Cll pump in kidney (Whittembury and Fishman, 1969) had a slight inhibiting effect on Na+ efflux; although this agent may also affect Nat-K+-ATPase activity (Duggan and Noll, 1965) and membrane permeability (Riordan et al., 1972). These findings imply that Na+ is actively extruded, but that two pumps may be involved, as in the kidney (Whittembury and Fishman, 1969). The operation of a second pump may mask the effect of inhibition of a first pump on ion concentrations. This could partly explain the results of Andrus and Giese (1963) who did not detect any effect of Ouabain on [Nat], at 25 C. These pumps are probably located within the C-V complex.

COUPLING

OF Na+ EXTRUSION ACCUMULATION

TO K+

An enzyme which could actively couple Nat extrusion to K+ accumulation is the Na+-K+ activated (Mg)-ATPase (Skou, 1957). Although it is found in many preparations it is not always simple to determine its presence and activity. Histochemical techniques do not always clearly distinguish between phosphatases and ATPases (Firth, 1978). Ludi et al. (1982) have shown that the activity of the Ca2+-ATPase of the sarcoplasmic reticulum is strongly dependent on the detergent used to solubilize it. The Na+-K+ Mg2+-ATPase is also an integral membrane protein, requiring phospholipids for its function (De Pont et al., 1973) and so its activity may vary with the method of isolation used. The exchange stoichiometry under ideal conditions (3 Nat : 2 K+, Bonting and Caravaggio, 1963) is not fixed but is flexible (Blostein, 1979; Akera et al., 1981). The work of Langer (1974) on quiescent canine myocardium was interpreted by Akera and Brody (1982) as representing a ratio of 2 Na+:5 K’. This variability may be the result of functioning of the pump with some of its sodium binding sites unfilled; interference in the ratio by other ion exchange mechanisms (e.g. Ca2+-Nat exchange, Na+ and Cll or amino acid cotransport) or by partial operation of the enzyme in other transport modes (Schuurmans Stekhoven and Bonting, 198 I ; Akera and Brody, 1982, reviews).

One of the alternative operational modes is the K + activated phosphatase activity (Judah et al., 1962) which is particularly expressed when there is a high concentration of a suitable phosphate and the [ATP] is less than that required for maximum ATPase activity (Wade, 1976; Robinson et al., 1978). Under the conditions so far investigated in squid axon (Mullins and Brinley, 1969) and human red blood cell (Garrahan and Rega, 1972) using maximal or zero [ATP], K+ transport did not occur. Nevertheless, the possibility remains that if phosphatase activity was high but ATP binding to the catalytic site for the ATP

E>.K’

eE,.K+

translocation step was partially protected (perhaps by screening or a lower affinity for non-ATP phosphate), uncoupled K+ uptake could occur, driven by the phosphatase activity of the enzyme. Ouabain is a specific inhibitor of the Na+-K+ (Mg” )-ATPase (Schatzmann, 1953) and its associated phosphatase activity (Inturrisi and Titus, 1970) but its use in assaying activity can be misleading. Akera et al. (1981) found that the pump in cardiac muscle has a reserve capacity which can compensate for partial inhibition by low concentrations of Ouabain (IO-* M). They remarked that lack of inhibition by low concentrations of Ouabain did not necessarily mean the absence of the ATPase. Garrahan and Rega (1972) demonstrated that lO-‘M Ouabain produced a 4-fold increase in passive K+ and Nat flux. Thus the properties of this enzyme and its inhibitor Ouabain make it difficult to determine indubitably the presence and activity of a Na +-K +Mg’+-ATPase. Also, the relationship of this activity to bulk ion movements is even more inscrutable. Dunham and Child (1981) found that bulk movements of Na+ and K+ were relatively independent of each other. They proposed that the ions might even be separated into different compartments. Andrus and Giese (1963) did find some reciprocal ion exchange, but this movement could partly be the result of altered binding site affinity (Patterson, 1976). Andrus and Giese (1963) did not detect any effect of 10ml M Ouabain on the restoration of Na+ and K+ after exposure to cold, despite the effects of Ouabain at this concentration on passive ion permeability (Garrahan and Rega, 1972). Conner (1967) found Ouabain had no effect on K+ efflux from the cell. These bulk movement studies suggest that intracellular binding has a small but significant influence on ion distribution in Tetrahymena. Conner et al. (1963) found an Mg2+ dependent ATPase in homogenates of Tetrahymena pyriformis. Na+ and K+ enhanced its activity by IO-15% and Ouabain did not inhibit it. Whether this behaviour is typical of all the enzyme or is the average of a small portion, very sensitive to monovalent cations, and a larger insensitive portion is not clear. They did find a K+ stimulated Mg2+-phosphatase activity and postulated that this might be responsible for transport activity. Such an enzyme is not known at present, but these results could involve the ATPase operating in an atypical manner as described above. Baugh et al. (1976) found no evidence of the Na+-K+

6

JOHN G. CONNOLLY

Mg2+-ATPase in Tetrahymena ciliary preparations, nor Doughty (1978) in Paramecium. Dentler (1977) identified an Na+-K+ Mg2+-phosphatase in the ciliary basal bodies of Tetrahymena using a histochemical method, but not an ATPase. Unfortunately none of these latter works encompassed the membranes of the C-V, which is the most likely site for high ATPase concentrations. The Na+ -K+ Mg” -ATPase has been detected in Entamoeba inuadens but the surface membrane of this parasitic protozoan has many unique properties, (McLaughlin and Meerovitch, 1975). Furthermore, Ouabain was not found to alter the membrane potential of Amoeba proteus (Josefsson, 1966) or Pelomyxu carolinensis (Riddle, 1962) however in the latter case the concentration was not specified. The lack of cilia and their related ionic movements may make ion regulation in Amoeba incomparable to that in Tetrahymena. Hence the bulk of the foregoing evidence does not support the presence of an Na+-K+ Mg’+-ATPase. However, as discussed above, these negative arguments are not conclusive. Positive evidence for the transporting ATPase was provided by Boggs and Wade (1972) when they studied the effect of Ouabain on the anterior contractile vacuole of Paramecium multimicronucleatum. While lo-* M Ouabain did not affect the C-V, 2-5 x lo-‘M Ouabain slowed pump activity and doubled the diameter of the C-V. They compared this effect to Ouabain’s diuretic action on the kidney, i.e. inhibition of the Na+ pump prevents Na+ resorption and leads to water retention. Hoffmann and Kramhsft (1974) found that ethacrynic acid (EA) has a biphasic effect in Tetrahymena altering Na+ influx more than efflux, implying the presence of a Type II Na+ pump (Hoffmann and Kregenow, 1966). However EA also affects Na+-K+-ATPase and membrane permeability in frog muscle (Riordan et al., 1972). More recently, Connolly and Kerkut (1983) have shown that Ouabain (10m5 M) reduces both the magnitude and Q,, value of the membrane potential of Tetrahymena. The anion sensitive HCO;-Cl ~-ATPase involved in acid secretion in frog gastric mucosa (Kasbekar and Durbin, 1965) is not sensitive to Ouabain and is not thought to be electrogenic. A Ouabain insensitive K+-H+ ATPase has been identified in gastric mucosa (Forte et ul., 1967; Limlomwongse and Forte, 1970; Ganser and Forte, 1973 ; Lee e/ ul., 1974). The enzyme has considerable phosphatase activity but is apparently electroneutral (Sachs et al.. 1976). The presence of these enzymes in Tetrahymena is uncertain, but possible, given the digestive processes which these cells undertake. Their electroneutrality means that it is unlikely that they make an immediate contribution to the membrane potential. These studies suggest that intracellular binding and compartmentation may contribute to the total cell content of Na+ and K+. However the major, free proportion of monovalent cations are probably governed by active transport processes, one of which may be an Na+-K+ Mg’+-ATPase located on the membranes of the contractile vacuole and its ancillary structures. There is evidence that these processes make an immediate electrogenic contribution to the resting potential.

and G. A.

KERKUT

It has been suggested that hyperpolarising electrogenie Na+ extrusion may have an antiarrhythmic effect upon the heart (Gadsby and Cranefield, 1979). At resting potential an electrogenic Na+-K+-ATPase will reduce the frequency of spontaneous depolarising Ca’+ spikes possibly triggered off by such activities as C-V pumping. Thus in addition to the functions of maintaining ionic and osmotic gradients across the cell membrane, the Na+-K+-ATPase may also have a stabilizing influence on protozoan behaviour by short circuiting spontaneous discharges which would otherwise give rise to ciliary reversal. Na+ ACCUMULATION

Dunham and Child (1961) noted that when [Na+], < 5 mM, [Na+], remained constant at 5 mM, even though 3 mM of this was exchangeable. Dunham and Kropp (1973) showed that if C-V activity was inhibited by sucrose addition, thus preventing Na+ efflux, [Na+], was greater than [Na+lO, even when [Na+], exceeded 5 mM. Kropp and Dunham (1971) monitored 24Na+ uptake with increasing concentrations of Na,’ The initial part of the curve showed a steep rise, saturating at 0.8 mM and showed inhibition with external K+. They interpreted this as evidence for a carrier mediated uptake mechanism, although intracellular binding and compartmentation could also account for this. Hoffman and Kramhaft (1974) found an inward Na+ flux which was sensitive to ethacrynic acid. Andrus and Giese (1963) interestingly noted that when dinitrophenol (DNP), a phosphatase uncoupler, was added to Tetrahymena cells [Na+], increased. This suggests that an ATP or phosphate independent accumulation mechanism was unmasked when Na+ extrusion was halted. Rasmussen (1973) and Hoffmann et al. (1974) demonstrated that Tetrahymena can accumulate nutrients without forming food vacuoles. Earlier Hoffman and Kramhoft (1969) had discovered that Nat and amino acid transport was coupled. Slater and Tremor (1962) showed coupling of Na+ and PO:- exhibited coupled uptake. Na+ and sugar uptake are also coupled (Cirillo, 1962; Aomine, 1974). The Q,, value for phenylalanine uptake between 12 and 20°C was 2.14 (Stephens and Kerr, 1962), and similar to the value for arabinose. (Cirillo, 1962). DNP will reduce amino acid uptake (Schaeffer, 1970). Stoner and Dunham (1970) also reported direct and indirect coupling of amino acid to cation transport. These observations have led several authors to suggest that there are widespread and diverse mechanisms of Na+ accumulation, at least some of which are in some way energy dependent and carrier mediated. These systems are thought to be essentially similar to those found in higher organisms (Geck and Heinz, 198 1, review). Cd+

EXTRUSION

IN THE RESTING STATE

Browning and Nelson (1976) induced a net uptake of Ca2+ in Paramecium by reducing temperature from 22 to 0°C. They inferred that a Ca’+ extrusion process had been inhibited by cold. This uptake had the properties of a channel rather than a carrier, and was enhanced by the addition of K+ extracellularly.

7

Ion regulation and membrane potential They also poisoned the cells at room temperature with cyanide and azide. Although this drastically reduced ATP levels, the efflux was unaffected. They proposed that an Na+-Ca2+ exchange carrier, similar to that found in heart (Reuter and Seitz, 1968) and squid axon (Baker ef al., l969), might be responsible for Ca*+ extrusion, as the mechanism is independent of ATP. They also thought that Ca2+ uptake was a passive process. Cd+ AND Mg*+ ACCUMULATION AND PYROPHOSPHATE DEPOSITION IN THE RESTING CELL

Patterson (1976) noticed that Ca’+ was accumulated by the cell when present in only trace amounts in the medium. Earlier, Rosenberg (1966) had noticed the appearance of numerous granules in Tetrahymena under starvation conditions. These contained a salt consisting of a complex Mg*” and Ca2+ pyrophosphate. Associated with the membrane surrounding the granules was a Mg2+-activated, Ca2+-inhibited, pyrophosphatase. Rosenberg and Munk (1969) demonstrated that rapid uptake of Ca2+, Mg2+ and PO:-, and simultaneous deposition of granules, depended on having all three ions (Ca*+, Mg2+ and PO:-) present in the external medium. Accumulation was much slower if one of the ions was absent. Munk and Rosenberg (1969) found that the extracted granules had a Ca2+ : Mg *+ : PO:- ratio of 1: 1:2. Coleman et al. (1972) determined the ratio to be 1.5: 1 for CaZi :Mg*+ in situ. They deduced that the phosphatase preferred Ca2+, and that most of the Ca’+ was labile. With phosphate deprivation the granules were broken down (Munk and Rosenberg, 1969). Ca’+ was lost before Mg2+, supporting the contention of these authors that the phosphatase is Ca’+ preferring. No evidence was found in these studies for active Ca’+ and Mg’+ extrusion; although Rosenberg and Munk (1969) suggested Ca*+ efIlux was an active process. A possibility suggested by Nilsson and Coleman (1977) is that concentrating Ca*+ in the lipid matrix of the granules reduces the gradient for extrusion of Ca*+ when granules become associated with cell membranes. They also found Ca2+ deposits in Initochondria and in lipid vesicles. The proximity of mitochondria to parasomal sacs in Tetrahymena (Allen, 1967) and a Paramecium (Patterson, 1978) led Patterson to suggest that this was a possible route for Ca’+ extrusion, perhaps by simple diffusion out of a very concentrated Ca’+ compartment. Patterson (1974) showed that the granules could account for most of the Ca’+ and Mg’+ stored in the cell, and that monovalent cations (Na’, K+) reduce Ca*+ uptake. Na’ and K+ may become incorporated into the granules, and may thus be competing with Cal+, but they may also be stimulating a carrier mediated extrusion process. All of these authors have recognised the importance of the deposits in divalent cation regulation. Satir and Gilula (1970) discovered a ring of membrane particles at the base of metazoan gill cilia. Satir et al. (1972) later described a similar ciliary necklace in Tetrahymena (Fig. l(A)). Dute and Kung (1978) found that the ciliary necklace region in Paramecium

bound ferritin, so that the granules were probably associated with Ca2+ deposits. Plattner et al. (1973) reported intramembranous particle aggregates (plaques) in ciliary replicas. In 1975 he correlated these to Ca2+ dependent electron dense deposits and suggested a role for them as Cal+ depots. Ogura (1976) confirmed their presence in isolated Paramecium ciliary membrane and Satir et al. (1976) identified them in intact Tetrahymena cilia. They also showed the plaques to be membrane bound, so that their contents are compartmented between the intraciliary space and the ciliary membrane (Fig. l(A)). Dute and Kung (1978) showed that the plaques in Paramecium bound fertitin. Ca’+ dependent electron dense deposits were found in the same region by Przelecka et ul. (1977). Electron microprobe analysis showed Ca2+ is present in the plaques if it is included in the media (Plattner, 1976; Fisher et al., 1976; Tsuchiya, 1976). Plattner (1976) also detected phosphorus and sufphur in the deposits. However Fisher et al. (1976) did not find phosphorus but chloride instead. It is not certain therefore, whether Ca*+ in the plaques is free or whether it is sequestered in a phosphate or pyrophosphate salt. The mechanism of incorporation of ions into the plaques is also uncertain. A Ca*+ ATPase has been associated with the plaques (Baugh et al., l976), but not a phosphatase as yet. Calmodulin (CaM), a Ca2+ binding protein and a secondary messenger for Ca2+ in many systems (Cheung, 1982, review), has been detected in whole Tetrahymena (Suzuki et al., 1979) and in their cilia (Jamieson et Ql., 1979, 1980). CaM from Volvox, a protozoan-like cell, can activate erythrocyte Ca2+-ATPase (Kurn and Sela, 1981). Fhtphenazine, a CaM inhibitor, was shown by Kurn (1982) to inhibit phosphate uptake in Volvox. This result was thought to indicate that CaM may regulate phosphate uptake--either directly or by its effect on cation pumps. If Ca2+ in the plaques is not incorporated into an inert pyrophosphate it may be bound by a specialized Ca *+ binding protein, comparable to caisequesterin of the sarcoplasmic reticulum of skeletal muscles (MacLennan and Wong, 1971). Tsuchiya and Takahashi (1976) located Ca”+ dependent deposits in the cilia of Paramecium. Przelecka et al. (1977) identified similar deposits on the radial arms of the axoneme of Tetrahymena between the central and peripheral microtubules. The alveolar sacs, whose inner and otiter membranes form two layers of the pellicle, have been proposed as a site of Ca*+ sequestration by Satir and Wissig (1982). Cd+

REGULATION

DURING

EXCITATION

When Paramecium is depolarised, Ca’+ enters the cell through voltage sensitive channels (Naitoh er (II., 1972). A similar system occurs in Tetrahymena (Connolly and Kerkut, 198 1). Eckert (1972) proposed that Ca2+ flows down its concentration gradient into the cell, and raises free intraciliary Ca’+ transiently, leading to ciliary reversal. Normal beating was thought to be restored when [Ca*+], was reduced by active membrane pumps. Sites of Ca”+ entry 45Ca2+ does enter Paramecium

during excitation

M

JOHN G. CONNOLLY

(Browning and Nelson, 1976) but there is also considerable Ca*’ influx in the resting cell (Yamaguchi, 1960b; Browning and Nelson, 1976). There is also considerable steady state Ca*+ influx in Tetrahymena, only 35-407; of this being via the cilia (Kusamran et ul., 1980). Ogura and Takahashi (1976) and Dunlap (1977) have shown in Paramecium that voltage sensitive Ca’+ channels are located exclusively in the cilia. Nandini-Kishore and Thompson (1979) showed that glucose stimulation increased total cell Ca* + by 25:& which they considered a small increment. This figure may be misleading, for Na+-glucose uptake may not depolarise the cell enough to open voltage sensitive Ca’+ channels, and Ca” could be entering by other routes such as the somatic mechanosensory receptors (Ogura and Machemer. 1980). There may well be other Ca’ ’ channels on the soma which are sensitive to ionic and chemical stimuli, perhaps glucose or osmotic challenges. Many Ca’+ channels. including some voltage sensitive ones. will be open in the unstimulated cell. Thus Ca” can enter all over the cell surface through a variety of channels. giving rise to the background influx, of ‘$Ca’ ’ observed by Yamaguchi (1960b) and Browning and Nelson (1976). A depolarisation would then lead to an additional CaZ+ influx through the cilia. The electron dense particle arrays (Fusion Rosettes) surrounding the trichocysts of Paramecium have been proposed as sites of Ca’+ entry (Satir and Oberg, 1978). Trifluoperazine, a CaM antagonist, will block discharge of the trichocysts (Satir et uf., 1980). CaM is also a major component of discharged trichocysts (Rauh and Nelson, 1981). These workers also found CaM-like proteins in discharged mucocysts (secretory organelles in Tetrahymena). These results add evidence for the view of Satir et N/. (I 980, review) and other workers that following Ca” entry at various sites, the Ca’+-CaM complex is an important secondary messenger for many cell processes in the protozoans. The presence of these alternative entry sites for Ca’+ may explain why Kusamran et ul. (1980) found that “Ca” was rapidly equilibrated all over the cell whereas Berger (1976) and Hiwatashi et cd. (1980) did not see coordination of ciliary activity in conjugating Paramecium after several hours. If in the first result it is not assumed that Ca’+ entry only occurs through the cilia then the latter experiments may be taken as evidence that ciliary Ca’* does not mix freely with cytoplasmic Ca”, as their authors suggest. This once raises the question of ciliary compartagain mentation. at least chemical if not to some extent electrical compartmentation.

Naitoh and Kaneko (1973) found that ciliary reversal occurred when [Ca’+], was raised from IO-’ to IO-‘M in triton extracted Paramecium. Nakaoka and Toyotama (1979) obtained reversal in only 10-xM Ca’+ when they reduced [Mg’+& from 10 to 5 mM, or raised [K+],, from IO to 20mM. They suggested the reversal depended upon an increase in free ATP concentration, but their result could also be explained as a reduction of competitive inhibition of Ca’+. Andrivon (1970a), using Ni’+ as a probe for Ca?+. showed that approximately IO-‘M Ni’+ was

and G. A. KEKKUT required for complete ciliary immobilisation in Paramecium. The discrepancies in these results may be partly due to differences in the pH and ionic composition of the preparations. pH especially would affect the dissociation constant of the Ca*+-EGTA complex, and hence the free Ca*+ concentration. Both of the latter parameters are extremely difhcult to estimate with any degree of certainty at such low concentrations. Noguchi et al. (1979) noted that the properties of the Ca*+-ATPase of Paramecium changed upon solubilisation and therefore will probably be affected by the Triton detergent used to extract these cells. This again means that the in L&J Ca’+ requirement for ciliary reversal may differ from the values obtained by these experiments. Triton extraction may also reduce the concentration of endogeneous modulators of Ca2+ activity, such as Calmodulin (CaM) within the cilia. CaM has been shown to confer Ca’+ sensitivity on the ciliary dynein ATPase (Blum e? ~11.. 1980) which otherwise prefers Mg’+ as an activator (Gibbons, 1966). CaM has been shown to inhibit the assembly of rat brain microtubule at a concentration of Ca’+ (IOpM) which on its own will only slightly restrict assembly (Marcum et a/.. 1978). Thus CaM could reduce the Ca’ ’ requirement of the cilia for the reversal process, which probably involves microtubule disassembly. Further evidence for the involvement of CaM in ciliary reversal has accumulated from the use of neuroleptic drugs. A detailed study by Shimizu et ul. (1982) shows that chlorpromazine and trifluoperazine displace Ca’” from CaM and render it physiologically inert. Rauh et ul. (1980) showed that these CaM antagonists blocked Ca’ ’ dependent ciliary reversal in Paramecium. Conversely, earlier workers found that low concentrations of chlorpromazine reduced motility and induced ciliary reversal in Tetrahymena (Nathan and Friedman, 1962) and Paramecium (Dry1 and Masnyk, 1971; Dryl, 1974). However the reversal in Tetrahymena was accompanied by a permeability increase. probably due to the local anaesthetic action of the drug. This change in permeability may have led to a depolarisation, thus reducing motility and inducing reversals. At the low (IO ‘M) concentrations used, this may well have overshadowed the anti-CaM action of the drug. [Ca” 1, is usually under tight control in living systems, and is quickly inactivated near its site of entry (Rose and Lowenstein, 1971; Baker, 1972: Brehm and Eckert, 1978). If [Cal+], is excessive, it is lethal to cells (Lee and Klosek, 1975). Hence it is unlikely that free Ca’+ brings about ciliary reversal, and the view has arisen among the above authors (Satir et d., 1980. review) that the Ca“-CaM complex is the mediator of ciliary reversal, although its exact mechanism is uncertain (Doughty and Dryl, 198 I, review). Reduction

of’ intraciliury

Ca’ ’

Reduction of intraciliary Cal+, whether free or complexed to CaM, is a prerequisite of restoration of normal ciliary beating (Eckert, 1972; Naitoh and Kaneko, 1973). This could be achieved by binding to substrates such as Ca’+ binding protein and phospholipids, by incorporation into inert compartments such as plaques, or by direct extrusion from the cell.

9

Ion regulation and membrane potential Brehm and Eckert (1978) noticed that Paramecium cytoplasm had a “buffering” effect upon intracellular injections of Cal+. Kilburn et al. (1977) observed that cilia became occluded with electron dense material when subjected to osmotic shock, suggesting that there is some simple Ca*+ binding. Andrivon (1970a) supplied evidence of active processes in Paramecium involving Ca*+. Ciliary immobilisation by Ni*+ could be slowed by metabolic inhibitors. However Chua et al. (1977) working with whole Tetrahymena, found that known ATPase inhibitors did not affect the activity of the Ca*‘-ATPase they had extracted. Ni’+ uptake was also shown to be temperature sensitive (Andrivon, 1970b) and could be inhibited by Nat and K+ (Andrivon, 1972). There have been several reports of Ca* ’ -ATPases in Tetrahymena and Paramecium which appear to fall into two classes (reviewed in Riddle et al., 1982). The first is a membrane bound Ca*+-ATPase found throughout the cell, and the These may be second is a dynein Ca *+-ATPase. involved in the process of renormalisation of ciliary beat. Evidence from work on mutants supports this view. Baugh et al. (1976) isolated a mutant strain of Tetrahymena which had no plaques and showed a ciliary membrane I OO-fold reduction of that intraciliary Ca’+-ATPase, suggesting Ca’ ’ -ATPase was associated with the plaques. Later Byrne and Byrne (1978) correlated a greatly reduced incidence of plaque formation with the paranoiac mutation of Paramecium. This mutant is behaviourally characterised by prolonged backward swimming during its avoidance response (Kung et al., 1975). especially in Na’ solutions. If free Ca’+ is responsible for prolonging this backward swimming, then the plaques, and hence the Ca’+ -ATPase, play an important role in reducing its level after excitation. A Ca’+-ATPase may pump Ca’+ into the compartment of the plaques, which is adjacent to the ciliary membrane. The form of Cal’ in the plaques is not clear, but it is extremely concentrated, and a downward gradient may exist which can cause Ca’+ extrusion from the cilia by diffusion, a carrier mediated process or direct ion pumping. Tada and Katz (1982) have reviewed control mechanisms for the Ca’+-ATPase in cardiac membranes. When the endogenous protein phospholamban is phosphorylated by a protein kinase it will enhance the activity of ion pumping Ca’+-ATPase. There appear to be two protein kinases which can phosphorylate phospholamban. One may be activated by the CaaCaM complex and the other by cyclic AMP, (c-AMP). c-AMP is a secondary messenger for the catecholamines. Nadini-Kishore and Thompson (1979) showed that Cal’ entry leads to adrenalin release in Tetrahymena, and thereby increases [cAMP]. The Cal ‘UZaM complex may also act directly on the Ca’+-ATPase. The membranes of Tetrahymena and the cardiac sarcoplasmic reticulum may be comparable. Hence at least three possible positive feedback mechanisms are known which would enable organelles such as the plaques to alter their Ca’+ storage rate in accordance with fluctuations in [Ca’+ 1, during excitation. Browning and Nelson (1976) showed that at 22-C

there is a powerful extrusion mechanism for Ca’+ which is not sensitive to metabolic inhibitors such as cyanide and azide. They suggested that a Ca’+-Na+ exchange pump, as has been detected in squid axons (Baker et al., 1969), barnacle muscle (DiPolo, 1973) and heart sarcolemmal membranes (Reuter and Seitz, 1968; Langer, 1982, review) may also operate in Paramecium. Hansma (1979) demonstrated that Na+ influx was associated with prolonged backward swimming in paranoiac mutants. Saimi and Kung (1980) investigated this phenomenon further and showed that prolonged inward Na+ current was both Ca’+ dependent and voltage dependent and was diminished by internal application of Na+ and EGTA. These are features of an Na+-Ca’ + exchange as well as a Ca’+ dependent Na+ channel. Deitmer and Machemer (1982) found that replacing artificial seawater with Na+-free seawater made the membrane potential of the marine ciliate Purumecium culkinski 15 mV more negative. Input resistance rose 3-fold to 70 MR and rectification increased too. However, they concluded that spike properties were not affected by changes in [Nat lo. In this cell Nat addition is associated with depolarisation and a conductance increase; results which are typical of an electrogenic (>2: 1) Na +:Ca*+ exchange. Saimi and Kung (1980) examined the plateau potentials which followed the depolarising spikes of paramecia placed in Na’ solutions. In 8mM Na+, with a resting potential of -26 mV, wild type cells achieved a plateau of - 10 mV, while a paranoiac mutants maintained a plateau of -2.6mV. The inward Nat current associated with the plateaux was maximal when the cells were voltage clamped at - 17 mV which may be the reversal potential of the current. However the plateaux only depolarise by 0.8 mV for a 2 x change of Na, from 2 to 4 mM, whereas a simple, Ca’ ’ activated Na+ channel would be expected to change plateau magnitude by 17 mV according to Nernst. The reversal potential (E,) of an Na + : Ca’ + exchange mechanism may be estimated using an equation provided by Mullins (1979, review), E, =

n E,, - 2Ec, n-2 -

where n is the coupling ratio of Na+ :Ca’+ The following assumptions are made: (i) [Na ’ I0 = 8 mM, [Ca” I0 = I mM (Saimi and Kung, 1980). (ii) [Ca’+], = IO ’ M at sites of entry during reversal. This is slightly higher than the estimate of Eckert (1972) of at least 10mh M as the Ca” is not assumed to be distributed throughout the intraciliary space. (iii) [Na’ 1, = 3 mM when [Na +1” is between 0 and 20mM (Dunham and Child, 1961; Patterson, 1976; Hansma, 1979). The real activity may well be lower than this. (iv) The coupling ratio, n = 4. A value of 334 is acceptable theoretically (Mullins, 1979) and experimentally (Langer, 1982). However some experimental estimates may be lower due to interference from other processes involving Ca’+ and Na+ ions.

JOHN G. CONNOLLY and G. A. KERKUT

10

E, for 4 Na+:Ca2+ is then found to be -1OmV during reversal, the same as the wild type plateau value of Saimi and Kung (1980). Alternatively if - 17 mV (Saimi and Kung, 1980) is used as E, in the equation above then n = 3.7, an acceptable value. Whatever the exact internal concentrations of ions and coupling ratio involved, the above calculations indicate that the plateau and inward Na+ current could be substantially accounted for by an Na +: Ca’+ exchange. This would take up Na+ and expel Ca” when the cell was hyperpolarised below E, and [Ca?+], > 10-h M, i.e. n (ENit - E,,,) > 2(E,, - I$,,), (Mullins, 1979) where En, is the resting membrane potential (approx -25 mV, Saimi and Kung, 1980). The fact that the inward Na+ current peaks at - 17 mV may be explained by assuming that if the cell is more depolarised than - 17 mV, the potential approaches E, for the carrier, and the current will reverse, whereas if the cell is more hyperpolarised, insufficient Ca2+ will have entered the cell to allow the inward Na+ current develop. At resting potential, [Ca’+], = lo-* M (Eckert, 1972), E, = - 100 mV, and so the carrier will expel Na+ and take up Ca2+. However, if [Ca’+], was compartmented from the free intraciliary space, perhaps in the plaques (Nilsson and Coleman, 1977), and concentrated in that compartment, perhaps by the Ca2+-ATPase (Baugh et al., 1976), then the Na+ :Ca’+ exchange could still export Ca2” from the cell at resting potential. The prolonged reversal of ciliary beat which is characteristic of paranoiac behaviour may be associated with several different mutations. However a decreased number of plaques (Byrne and Byrne, 1978) and a reduced concentration of a Ca2+-ATPase to compartment Ca2+ into them (Baugh et al., 1976) could lead to a prolonged raised concentration of free intraciliary Ca’+ and hence sustained ciliary reversal. This would present a greater workload to electrogenic Na+ :Ca’+ exchangers outside the plaques, resulting in the prolonged enhanced Na+-currents and plateaux potentials observed by Saimi and Kung (1980). Incorporating the work and ideas of the above authors it is therefore suggested that the raised intraciliary [Ca” ] following a depolarising spike may be reduced by compartmentation into the plaques and the high concentration of Ca” then allows Na ’ : Ca’ + exchange to eliminate Ca’ ’ from the cell without affecting resting potential. There may also be Na’ :Ca” carriers outside the plaques and in the paranoiac mutant, which has a reduced ability to compartment Ca’+, these may assist the plaque Na+ : Ca’ + carriers to slowly eliminate Ca’+ from the cell, giving rise to plateau potentials and a slow inward Na+ current. The essential features of this model are illustrated in Fig. l(B). OTHER

IONS

[Cl~ 1, in Tetrahymena is generally less than [Na+], and less than in the medium (Dunham and Child, 1961; Dunham, 1964). They also showed that [Cl 1, remains constant over a wide range of [Cl 1”. They concluded that the bulk of Cl was passively distributed although some active extrusion may occur via the contractile vacuole (Dunham and Stoner, 1967) or the cytoproct.

An enzyme which could possibly transport Cl ions is the anion sensitive HCO;&--ATPase (Ganser and Forte, 1973). Food vacuoles are known to become acidic shortly after leaving the cjtopharynx (Kitching, 1956). Another enzyme mentioned earlier which is could affect acid secretion the K +-H+-ATPase. These H+ pumps may also contribute to acidification during digestion. The accompanying anion is usually Cl , which may then be eliminated via the cytoproct. An Nat-H+ exchange carrier similar to that in snail neurones (Thomas. 1976) may also be present. These processes could result in an unequal distribution of H’ across the cell membranes, and so H+ may also contribute to the resting potential. Orthophosphate is a very important anion in Tetrahymena metabolism and may be accumulated actively without food vacuole formation (Pruett, 1965). The Q,, for its uptake was 1.7 between 18 and 28-C. (Pruett et al., 1967). This is too high for a passive process, but not a clear indication of primary active transport because orthophosphate is metabolically sequestered and this may drive uptake. There is some evidence of coupled Na+-PO:uptake (Slater and Tremor, 1962) suggesting a possible carrier mechanism. Orthophosphate is also related to Mg2+ and Ca2+ uptake because of their common incorporation into Rosenberg’s granules (Rosenberg, 1966) and has been proposed to be the major anion extruded by the C-V (Dunham and Kropp, 1973). As there is some evidence for an Na+ :Ca2+ exchange mechanism it seems probable that there will also be an Na+-Mg2+ exchange mechanism, similar to that of squid axon, (Baker and Crawford, 1972; De Weer, 1976). Addition of 1OOmM sucrose brought an increase from 56.5 to 114.9 mM amino acid/kg cells (Stoner and Dunham, 1970). The changes in intracellular ion concentration were less significant, but this osmoregulatory device could affect membrane potential by altering the Donnan equilibrium. The many behavioural and digestive functions carried out in Tetrahymena will involve some ion movement. The net result of this may not be electroneutral, although small in magnitude, and so there may be a great many processes participating in the resting potential of protozoans. AREAS FOR FURTHER

INVESTIGATION

From this present review it is evident that further work must be done to establish the presence and contribution of electrogenic Na+-K ’ (Mg’+ )ATPase and Na+ :Ca’+ ion exchange systems. The activities of the digestive system and contractile vacuole are among several processes which may influence resting potential. H +. Mg’+. Cl-, PO: as well as Na’ , K ’ and Ca2 ’ may participate in the membrane potential. Coupled transport of Na+ and metabolites such as amino acids may also be significant. Ionic fluxes may give rise to electrogenic or diffusion potentials or both. The electrogenic component will be sensitive to modulators and potential and may change direction during depolarisation. Until more is known about the nature and extent of the contribution of each of these components to the resting potential it seems premature to attempt a full math-

Ion regulation and membrane potential ematical description of it. Such a model will be far more complex and dynamic than the description of membrane potential and events in the squid axon, (Hodgkin and Huxley, 1952a,b; Hodgkin et al., 1952). Thus the impression which emerges is that protozoan electrophysiology is not the product of fixed interactions between relatively few, well defined and unchanging participants. Rather it is a dynamic entity with a great many components whose properties and interrelationships are in a constant state of flux, changing as the activities (e.g. reversal, mating, C-V pumping) of the whole cell change and susceptible to the influence of internal and external modulators (e.g. temperature, pH, Noradrenalin, ATP. Calmodulin). The authors do not believe that these characteristics isolate protozoan electrophysiology from mainstream neurophysiology, but note the similarities between protozoans and leech neurones (Jansen and Nicholls, 1973) snail neurones (Kerkut and Thomas, 1965; Marmor, 1975; Thomas, 1976) Barnacle smooth muscle (DiPolo, 1973) and heart sarcolemmal membranes (Reuter and Seitz, 1968. Langer, 1982, review). Ca*’ spikes and prolonged action potentials have also been described in neurones of the mammalian CNS (Krnjevic and Lisiewicz, 1972; Wong et al., 1979; Fulton and Walton, 1981; Hotson and Prince, 1981). Hence protozoans may be a more useful model for understanding basic events in brain cells than the squid axon. Protozoans have already proved their usefulness in the study of membrane biochemistry (Thompson and Nozawa, 1977). the genetic dissection of behaviour (Kung er ul.. 1975; Kung and Saimi, 1982) and electrophysiology (Satow, 1982). The ease with which they can be easily grown and harvested in large quantities renders them very useful for parallel experiments in genetics, biochemistry and electrophyisology. In this direction studies on the effects of drugs and membrane composition and fluidity in Paramecium (Forte et al., 1981) and Tetrahymenu thermophilu (Connolly et al., 1983) have already yielded promising and interesting results. Protozoans may also be useful system for investigation of Ca’+-dependent events and the agents involved in their control. Thus although protozoans are difficult to define electrophysiologically, this very complexity may make them important and instructive models for a deeper understanding of events in mammalian nerve and muscle cells. SUMMARY

1. Water, K ’ and Cl- are essentially free within the cell. Na+ may be partially bound while Ca’+, Mg2+ and PO:- are extensively compartmented by physical or chemical means. 2. K+, Na+, PO:- and amino acids are actively accumulated by the cell, while Na+, Ca’+ and Cl- are actively extruded. There is positive evidence for primary active transport utilising the Na+-K+-ATPase and the Ca2 ’ -ATPase. Among the secondary active transport systems there appears to be a Na+ :Ca2+ exchange and Na+ coupling to amino acid and PO:uptake. The authors suggest that these processes are electrogenic and make an immediate contribution to the resting potential and that the dominant system in

II

the wild type cell is the Na+-K+-ATPase. Its hyperpolarising action may have a stabihsing effect on the resting potential and hence behaviour of the organism. H+, Mg2+, Cl- and PO:- are probably not passively distributed and may significantly participate in the resting potential. Negative evidence from bulk ion studies is often inconclusive because of the difficulty of isolating single transport mechanisms from others involving that ion, and because of the flexibility in activity and stoichiometry of the transporting systems. 3. The C-V is a major site of Na’ extrusion from the cell and this function may be associated with an Na+-K +-ATPase. Events which disrupt the pellicle, such as discharge of the C-V, can disrupt the resting potential of the cell. This effect seems to be screened to some extent, suggesting that not all parts of the cell are isopotential. 4. Ca” is deposited at many sites within the cell: Rosenberg’s granules, lysosomes, mitochondria, alveolar sacs, ciliary plaques, ciliary necklace and ciliary axoneme. This deposition is often coupled to Mg’+ and PO:deposition and is associated with Ca’+-ATPase and Ca’+-phosphatase activity. 5. Calmodulin is an important modulator of Ca’+-ATPase activity and other Ca’+ dependent events within the cell. There are probably additional regulators of Ca2+ processes such as noradrenalin, cyclic AMP and small peptides comparable to phospholamban. 6. Cal+ enters the cell via the electrically excitable ciliary membrane, mechanically excitable somatic membrane, trichocysts, mucocysts, active transport systems and possibly chemically excitable routes as well. The Ca’+ requirement of the cilia for reversal is not easy to define, as detergent extracted preparations may have very different properties when compared with in Ct’o enzymes. The requirement is thought to be between 10m6 and lo-‘M Ca’+. This may be accounted for by Ca2+ influx during the depolarising spike although the possibility of further Ca2+ release from intraciliary stores such as plaques cannot be ruled out. The Ca2+-CaM complex may be the actual form of Ca2+ which instigates cihary reversal. 7. Based upon the resuits and ideas of many workers a model is proposed whereby reduction of raised intraciliary Cal+ due to a depolarising spike is achieved initially by compartmentation of Ca’+ into the plaques by a Ca’+-ATPase. A raised Ca’+ concentration in the plaques allows the Na+-Ca2+ exchange to expel Ca’+ from this compartment and hence from the cell. Nat-Ca’+ exchange may also occur along the rest of the ciliary membrane, and this may more directly influence resting potential than exchange within the plaques. The reversal potential of a Na+-Ca’+ exchange is calculated to be approximately the same as the reversal potential of the inward Na+ current observed by Saimi and Kung (1980) in wild type and paranoiac paramecia. This implies that the Nat-Ca’+ exchange may be responsible for the plateau potentials observed in these cells, especially in paranoiac mutants where exchange is expected to be enhanced due to the reduced Ca’+ compartmenting ability of this strain. 8. The membrane potential of Tetrahymena and to

JOHN G. CONNOLLY and

12 a lesser diffusion it also affected

extent Paramecium is not purely a simple potential. It is suggested by the authors that contains electrogenic components and is by physiological processes such as digestion,

nutrition, C-V activity and mucocyst discharge. The most important electrogenic components are thought to be an Na ’ -K’ -ATPase and an Na ’ Xa’ ’ exchange which may help stabilize swimming behaviour. The Ca*’ -ATPase associated with the plaques may exert an electrogenic effect by transporting Ca’+ into an inert compartment. These processes are summarised in Fig. 2. 9. Protozoans have already proved their worth as subjects for the integrated study of the electrophysiology, biochemistry and genetics of excitable membranes. They may be a more complete model for detailed membrane studies than the squid axon: and have many features analogous to mechanoreceptor cells, cardiac muscle cells and some neuronal cell bodies of the mammalian C.N.S. Acknowledgemenr-The authors would like to thank Professor M. A. Sleigh for his help and comments during the preparation of this paper.

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