Chloride channels in epithelia

Biochimica et Biophysica Ac,a, 947 (1988) 521-547

521

Elsevier BBA85339

Chloride

channels

Heinz

in epithelia

GiSgelein

Max-Planck Jnstitut far Biophysik, Frankfurt (F.R.G.) (Received 15 December 1987) (Revised manuscript received 26 April 1988)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Transport mechanisms in epithefia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Definition of ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Nature of the shunt pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

522 522 523 S26

IL Levels of investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chloride flux measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Measurement of membrane potentials with microelectrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cl--selective microelectrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Use of CI- blockers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Single-chann©l recordings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.Reconsfitufion in planar lipid bilayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.The patch-clamp technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

526 526 527 528 528 5"-9 529 529

Ill. Properties of chloride channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Parameters to characterize single channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cl- channels in fluid-reabsorbin 8 epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cl- channels in fluid-secrefin 8 epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Ci- channels involved in volume regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

531 531 534 536 539 539

IV. Resulafion of cpith©lial chloride channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Fluid-reabs :,thin 8 epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Huid-secreting epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

540 540 540

V.

C. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

542

Chloride channel blockers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

543

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

544

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

544

Abbreviations: 9-AC, anthracene-9-carboxylic add; ATP, adenosine 5'-triphosphate; cyclic AMP, adenosine 3': 5'-cycfic monophosphat¢; db.cAMP, N6-2'-O-dibutyryladenosine 3': 5'-¢y¢Ii¢ monophosphate; DCDPC, 3',5-dichlorodiphenylamine-2-carboxylic acid; DG, 1,2-diacylglycerol; DIDS, 4,4'-¢Ffi~ocyanatos61bene-2,2'-disulfonic acid; DPC, diphenylamlne-2-carboxylate; GTPyS, guanoslne 5'-O-[y-ddo]t6phosphate; IP3, inoshol 1,4,5-trisphosphate; NPPB, 5-nhro-2-(3-phenylpropylamino)benzoic acid; PIP2, phosphafidylinositol 4,5-bisphosphate; SITS, 4-acetamido--4'-isothiocyanatostilbene-2,2'-d/sulfon/c acid; VIP, vasoactive intestina~ pepfide; TAL, thick ascending limb. Correspondence: H. G~gelein, Max-Planck-lnstitut flit Biophysik, Kennedyallee 70, D-6000 Frankfurt/Main 70, F.ILG. 0304-4157/88/$03.50 © 1988 Elsevier Sc~'.encePublishers B.V. (Biomedical Division)

522 I. Introduction 1-.4. Transport mechanisms in epit!,elia Epithelia have, amongst other functions, the ability to reabsorb or to secrete fluid. Examples of fluid-reabsorbing epithelia a t ' the amphibian skin, urinary bladder, gallbladder, renal proximal tubule, thick ascending llmb of Henle's loop and collecting duct; whereas typical fluid-secreting epithelia are the exocrine pancreas, salivary gland, lacrimal gland, trachea, shark rectal gland, cornea and choroid plexus. Since no evidence exists for primary active transport of water molecules, transport of fluid must be connected to transport of solutes. In most epithelia, water movement is closely coupled to the transport of Na + and C I ions. In Fig. 1 the arrangement of transport mechanisms in a typical fluid-reabsorbing and -secreting epithelium is shown. One factor that nearly all epithelia have in common is that the energy needed for the net transport of salt is provided by the Na+/K+-ATPase, located in the basolateral membrane. Exceptions from this rule are the choroid plexus, where this active pump is located in the ventricular (apical) membrane [1], and the insect malpighian tubule, which probably possesses an active pump for cations in its luminal membrane [2]. The N a + / K + pump accumulates K + ions inside the cells, and keeps the intracellular Na +

concentration lower than in the egtraccllular fluid. Another common feature of all epithelia studied thus far is an intracellu!ax electrical potential of - 5 0 to - 7 0 mV with respect to the extracelhilar compartment. This electrieal potential difference is mainly generated by diffusion of K + ions across selective pathways in the basolateral cell membrane. As in other tissues, the intracellular concentration of C1- ions in epithelial cells is much lower than in the extraceUular fluid. In some epithelia, the intracellular C I - activity was measured with ion-selective microelectrodes (cf. Table I). It was consistently observed that intracellular C I - ions were above their electrochemical equilibrium, so that uphill transport of C l - into the cells must have taken place. In order to fulfil the different tasks of either reabsorption or secretion of salt and water, ionic transport processes are differently located in reabsorbing and secreting epithelia. In fluid r ~ b s o r b ing epithelia, N a + and C I - have to be taken up by the cell across the apical membrane. A number of different translocation mechanisms have now been elucidated. N a + ions may enter the cell by amlloridc-sensitive ionic channels, as is the case in the amphibian skin, the urinary bladder and the renal collecting duct (reviewed in Refs. 3 and 4). Alternatively, the uptake of N a + may be coupled to the transport of other substances. For example, the apical membrane of kidney proximal tubules

b -" Na* Ha+ 2C[-~E K÷ ~ -

E

Na*=

Na

j

/ LUMEN

Cl" ~

K+

J

~

\ CELL

BLOOD

LUMEN

CELL

2C1" -.-

Na ÷

1 BLOOD

Fig. 1. Ccllul~,rmc,:lelsfor NaCI transport acrossepithelia. (a) Cellular model for Na + and CI- reabsorptionin the thick ascending limb of HenW,e's loop (accordingto Ref. 7). Co) Cellular model for Na + and CI- secretionin the rectal gland of the dogfish Squalus acanthias (accordingto Ref. 12).

523 possesses a N a + / H + countertr~.aasport system, as well as Na ;/substrate cotranspo~ ;ystems, where the substrates may be either glucose, amino acids, sulfate or phosphate [5]. In other tissues, the downhill movement of Na + mediates the uptake of CI- ions into the cells. For example, in the gallbladder a N a + / C I - (or N a + / H + and CI-/HCO~-) cotransporter is present in the luminal membrane [6], whereas.in the thick ascending limb of Henle's loop, the coupled transport of N a + / 2 C I - / K + was observed (Fig. la) [7]. At the basolateral side, C1- ions may leave the cellular compartment passively. The exit step for CI- ions may be mediated by anion-selective channels (cf. Tables I and II and Fig. la) or by electrically silent systems. The latter pathways may be present in renal proximal tubules [8,9] and in the gallbladder [10]. In addition to the transcellular routes, parts of Na + and CI- ions may cross the epithelia via paracellular shunt pathways [11]. In fluid-secreting epithelia, N a + and CI • ions are transported from the blood side to the luminal compartment. In secretory cells, CI- ions have to enter the cell across the basolateral membrane against an electrochemical potential, but may leave the cellular compartment across the luminal membrane by passive diffusion. The individual transport processes were extensively studied in the rectal gland of the dogfish Squalus acanthias (Fig. lb). Greger and Schlatter [12] demonstrated that ions are taken up across the basolateral membrane by a N a + / 2 C I - / K + cotransport mechanism, and that C I - ions leave the luminal cell membrane via anion-selective channels. Na + ions may follow passively across the paracellular shunt. Although similar transport mechanisms were observed in the salivary gland [13] and in the exocrine pancreas [14], it has yet to be shown, whether a general model for all fluid-secreting epithelia exists. Cl--selective channels may also play a role in cellular volume regulation. It is known that exposure of epithelial cells to a hypotonic medium causes cell swelling, followed by subsequent recovery of the cell volume. The latter effect, also called regulatory volume decrease, is associated with a loss of intracellular electrolytes [15]. Recently, it was reported that c l - channels were involved in regulatory volume decrease of cultured kidney cells (OK cells, Ref. 16).

I-B. Definition of ion channels The expression 'channel" is often used in the literature to describe ion translocation mechanisms whose nature is not precisely known. Therefore, before describing the properties of epithelial anion channels, I shall first discuss what is denoted as a channel mechanism and by which methods these transport pathways can be studied. Passive transport of ions and molecules across cell membranes can be classified as channel and carrier mechanisms. This distinction originates from studies in planar lipid bilayer membranes, where different transport processes of antibiotics were observed. The most prominent ones are the pore former gramicidin and the potassium carrier valinomycin. Both transport processes differ clearly in their mode of action: gramicidin dimers span the lipid bilayer and form a pore, which enables passive movement of partly dehydrated ions across the membrane [17]. Valinomycin, on the other hand, is a smell polypeptide which diffuses in the brayer. K + ions are bound by this antibiotic and carried over the lipid phase of the bilayer membrane [18]. Both transport mechanisms differ markedly in their turn-over rate, which is defined as the number of ions carried across the membrane per second by one transport unit. Gramicidin channels have a turn-over rate of about l 0 T ions per second, whereas the carrier valinomycin transports about 10 4 ions per second across the bilayer [19]. However, from a theoretical point of view, the discrimination between channel and carrier is not strict. IAuger [19] pointed out, that a model, based on the Eyring rate theory, may describe channel and carrier mechanisms as well. On the other hand, both mechanisms differ in their ion-binding ability. Whereas a carrier has a binding site for substrates which is exposed alternatively to the one and to the other face of the membrane, channel proteins possess several binding sites and are accessible to ions from both sides at the same time [19]. The discrimination of passive transport processes in channel and carrier mechanisms is also applied to biological membranes. In contrast to the antibiotics gramicidin and valinomycin, transport molecules in living cells are macromolecules and their polypeptide chains may cross the lipid bilayer

524 TABLE I METHODS USED FOR THE STUDY OF THE EPITHELIAL CHLORIDE CHANNEL Tissue"

Membrane

CI channel function

CI- channel study methods

Refs.

Urinary bladder (rabbit)

basolateral

CI- re.absorption

tracer flux micropuncture CI--selective microelectrodes patch-clamp

22 23

patch-clamp

25

(cell culture) A6 cells (cultured cell line from Xenopus laevis lddney)

-

23

24

apical

CI- reabsorption?

patch-clamp

26

apical

C1- reabsorption?

measurement of dome formation patch-clamp

27 28, 29

apical

CI- reabsorption?

transepithelial potential recording patch-clamp

30, 31 32

basolateral

CI- reabsorption

tracer flux micropuncture use of blockers patch-clamp

33, 34 35 36, 37 Greger (unpublished)

Diluting segment ( Amphiuma kidney)

basulateral

C l - re.absorption

micropuncture C1--selective microclectrode patch-clamp

38 39 40

Epididymal cells (rat, cell culture)

apical

C i - reabsorption?

electrolyte measurement patch-clamp

41 42

apical

CI- secretion

tracer flux mlcropuncmre reconstitufion in planar lipid bilay©r

43-45 45, 46

Pulmonary alveolar cells (rat, cell culture) Renal collecting duct (principal cells, cell culture) Reval cortical thick ascending l~,~b of Henle's loop (rabbit)

Colonic cells (rat)

T84 cells (human colonic carcinoma, cell culture)

47

apical

CI- secretio,I

tracer flux patch-clamp

48, 49 50, 51

HT29 cells (human colonic carcinoma, cell culture)

apical

CI- secretion

micropuncture use of blockers patch-clamp

52 53 53

Cornea (rabbit)

apical

C l - secretion

tracer flux macropuncture Cl--selective microelectrode patch-clamp

54, 55 56, 57 57 58

Malpighian tubule (mosquito)

apical

CI- secretion

electrolyte measurement 59 electron-probe X-ray microanaly~;s 60 patch-clamp 61

TABLE I (continued) Tissue a

Membrane

C1channel function

C1- channel study methods

Refs.

MDCK cells (cultured cell line from dog kidney)

apical

CI- secretion

tracer flux micropuncture patch-clamp

62. 63 64 65

Choroid plexus (bovine)

apical

CI- secretion

tracer flux micropuncmre Cl--selective microelectrode reconstitution in planar fipid bilayer

66 67 67

Rectal gland (shark, Squalus acanthias)

apical

CI- secretion

electrolyte measurement micropuncture CI--selective microelectrode use of blockers patch-clamp

69, 70 71-74 71-74 75 75-77

Trachea (canine)

apical

CI- secretion

tracer flux micropuncmre Cl--selective microelectrode use of blockers patch-clamp patch-clamp

78, 79 80-83 82, 83 84, 85 85-87 50, 88-91

Eccrine sweat gland (secretory cells, cell culture)

apical

Cl- secretion

electrolyte measurement sweat production micropuncture patch-clamp

92 93 94 95-97,162

Lacrimal gland (rat, mouse)

apical

C!- secretion

electrolyte measurement micropuncture Cl--selectivc microelectrodc patch-~lamp (whole cell)

98 99,100 99, lifo 101-105

Pancreatic duct cells (r~t)

apical

CI- secretion

micropancture use of blockers patch-clamp

107 108 109

OK cells (cultured cell line from opossum kidney)

-

volume regulation

measurement of cell size patch-clamp

110 16

(canine, cell culture) (human, ceh culture)

68

" The animal species Oven in brackets refer to the patch-clamp experimen:s.

several times a n d m a y protrude into the a q u e o u s face o n each side c f the lipid bilayer. It turned o u t that also in biological systems the discrimination o f passive transport processes in c h a n n e l s a n d carriers is possible because o f the differences in their turn-over rates [20]. Similar to the antibiotic gramicidin, the turn-over rate o f biological c h a n nels is in the order o f 106 to 10 s ions per second. T h i s high n u m b e r of ions which pass t h r o u g h the c h a n n e l p e r unit of time a n d the fact that ion

channels open a n d close, enable the registration of single c h a n n e l events. O n the other h a n d , the current a m p l i t u d e o f single events o f carrier systems is m u c h too small to be detectable. T h i s is also true for ion p u m p s a n d ion driven co- or c o u n t e r t r a n s p o n systems. In s u m m a r y , in this review a translocation p a t h w a y is denoted as a channel m e c h a n i s m , only w h e n the single c h a n n e l a m p l i t u d e could be resolved by single-channel analysis, or whole-cell

526 recordings or noise analysis revealed corresponding fluctuations in conductance. I-C. The nature of the shunt pathway

As described below, epithelial CI- channels have so far only been observed in cell membranes. It is known, however, that especially in leaky epithelia, large amounts of ions may move across the paracellular shunt pathway [11]. The nature of the translocation systems in the zonulae occludentes is still unknown. Noise analysis in epithelial sheets yielded some information about this transport pathway. With this method, fluctuating channels in the paracellular shunt could not be detected. On the other hand, in the gallbladder epithelium a 1 I f a noise component was/tetected, whenever ionic gradients were present across the epithelium [21]. This noise component was reduced by drugs which reduced the paracellular conductance. Although the nature of this noise component is still unclear, it is evident that this noise is different from fluctuations generated by channels, which yield a Lorentzian component in the power density spectra [3]. In conclusion, it is likely that the translocation mechanism across the tight junctions differs from that of plasma membranes. It is possible that ionic channels exist in the junctional regions, which are either permanently open or which open and close at rates too slow to be d e i f i e d by no~,~e analysis. 11. Levels of investigation The methods employed to study anion transport across cell membranes and to discriminate between the different transport processes are mardfold. In this communication, I shall focus on methods allowing the identification of channel mechanisms, i.e., the incorporation of transport proteins into planar lipid bilayers and the patchclamp technique. The method of noise analysis, which is also able to demonstrate the existence of ionic channel mechanisms, will not be dealt with here, because CI- channels in epithelia have not been studied with this technique in detail. A number of other methods exist which enable the study of anion permeation, without allowing discria~h~ation between channels and other trans-

port mechanisms. Methods such as, for example, tracer flux measurements, recording of electrical potentials with microelectrodes or with ion-selective microelectrodes, were appfied to epithelia long before single-channel analysis was developed. Therefore, the existence of many anion transport pathways, which were later elucidated as channel mechanisms, was known before the application of single-channel measurements was carried out. Because of the great importance of the techniques mentioned above in the investigation of CI- transport, I sh~!l discuss these methods briefly. In Table I, column 1, the epithelial tissues are summarized in which CI- channels were observed. The cell membrane ir which the CI- channel is located (column 2), the function of the channel with respect to the epithelial transport (column 3) and the various methods which were applied to characterize this transport pathway (column 4) are also indicated. 11-,4. Chloride flux measurements

The simplest technique to determine transepithelial electrolyte transport is measurement of the changes in electrolyte concentrations on both sides of an epithelium. This methods yields information on whether certain ion species are secreted or reabsorbed by the tissue. More detailed information on electrolyte transport can be obtained by tracer-flux tecb~iqv.es. In principle, a small amount of a radioactive isotope is added to one side of an epithelium. Then, either the rate of disappearance of this isotope is measured or, alternatively, the appearance of this isotope in another transport compartment is recorded. This procedure yields the unidirectional flux across the membrane. However, in order to obtain insight into the overall movement of one ion species, the unidirectional fluxes in both directions have to he determined. This can be achieved by measuring both unidirectional fluxes simultaneously by adding different isotopes of t h e respective ion species to either side of the membrane. Then, the difference between both unidirectional fluxes yields the net flux across a tissue. With respect to chloride, only the isotope 36C! is suitable for biologic.zl flux measurements. Therefore, the unidirectional fluxes are either measured separately in unpaired experi-

527 ments or the flux in one direction is determined by means of tracer analysis and, in addition, the total flux is evaluated by chemical analysis. Tracer-flux experiments were often performed in conjunction with the transepithelial voltage-clamp technique. With equal solutions on both sides, the transepithelial electrochemical potential difference is zero under short-circuit conditions. If net transport occurs under these conditions, it can be coneluded that active transport takes place. Tracerflux studies can distinguish between reabsorption or secretion of ions. In addition, it can be deduced from flux studies whether passive or active components are involved. The first attempt to investigate anion transport across an epithelium was performed by Uss,.ag in frog skin (reviewed in Refs. 111 and 112). It could be shown that active transport is at least partly involved in chloride reabsorption by tbas epithelium [113]. Since it was shown that intracellular C I - activity is far above its electrochemical equilibrium [114], active uptake mechanisms of CIinto the intracellular compartment must exist. Nagel [114] proposed that active CI- uptake is located in the apical cell membrane. The mechanisms by which chloride ions leave the cell at the basolateral side has not been revealed until now. Although the frog skin was the first epithelium in which ion transport mechanisms were studied, it has not been possible until now to apply singlec b ~ e ! analysis to this tissue. Therefore, the question of whether CI- channels are present in cell membranes of the [rog skin epithelium remains unresolved. As in the amphibian skin, both an active and a passive component for CI- reabsorption in the urinary bladder has been observed by means of tracer-flux measurements [22]. Indeed, later electrophysiological measurements revealed high CIconductance at the basolateral membrane [23] and, recently, single C i - channels were detected in this cell membrane [24]. When colonic cells were stimulated with cyclic AMP, a net secretion of C I - ions was observed [43]. CI- secretion was also observed ill cell cultures derived from human colonic carcinoma (T84) after hormonal stimulation [48,49]. Tracer-flux studies revealed that CI- ions were taken up by these cells at the basolateral side by the K + / 2 C I - / N a + cotransporter. The exit of

CI- across the apical membrane is mediated by passive diffusion [49]. As expected, Cl--selective channels were recently observed using the patchclamp method in the apical memb~za~e of c u l t a r ~ colonic carcinoma cells [50,53]. These examples show that 36C1 tracer-flux gtudies can reveal basic information on CI- transport both in Cl--reabsorbing and Cl--secreting epithelia. Additional examples are summarized in Table I. II-B. Measurement of membrane potentials with microelectrodes

A powerful tool to investigate ionic permeabilities of cell membranes is the t ~ h n i q u e of inserting a microelectrode into a ce:l and recording the electrical potential difference across cell membranes. Membrane potentials arise from the movement of ions through specific pathways in the membrane. These pathways may be ion-specific channels or other electrogenic transport systems, such as for example, the coupled transport of N a + and D-glucose in renal and intestinal epithelia, or the primary active ion transport mediated by the Na+/K+-ATPase. The electrical potential, generated by the permeability of a single-ion species, is dependent on the chemical gradient of these ions across the cell membrane and is given by the Nernst equation RT. [Xl0 where [X]i and [X]o are the activities of the cytosolic and extracellular side, respectively and R, T, z and F have their usual meaning. For membranes where conductive pathways for K +, Na + and CI- ions are present, the membrane potential is described by the Goldman-Hodgldn-Katz equation E = RT PKIK]o+ PNalNa]o+ PcI[CI]I

(23

F Pg[K]i+ Psa[Nah+ Pcl[Cllo where Px is the permeability coefficient for the respective ions. It should be noted that several assumptions were made for deriving the latter equation, for example, independence of ion movement, the existence of a constant electrical field

528 across the membrane bilayer and the absence of net current movement [115]. Eqn. 2 shows that alteration in the ion concentration of a permeant species on one side of the membrane results in a change in the membrane potential. If, however, the membrane potential is dominated by the permeability of other ions, the potential change caused by concentration jumps of the first ion might be too small to be detected adequately. For calculation of the CI- permeability coefficient, the value of the intracellular CI- concentration has to be estimated. In addition, it has to be assumed that cytosolic C1- remains constant during the experimental procedure. Another limitation of this method lies in the generation of liquid junction potentials, which arise at the raeasuring electrode during changes in the C I - activity. Especially in membranes with low CI- conductance, care must be taken to correct for the electrode potential. A further limitation of this method is the possible electrical coupling of the apical and basolateral cell membrane via the paracellular shunt pathway. Especially in leaky epithdia, where the electrical resistance of the shunt is, in general, smaller than that of the cell membranes, voltage changes adsing at one cell membrane also exert effects on the other membrane. In additio11, diffusion potentials arising across the shunt pathway during CI- concentratien changes may influence the membrane potential. Despite all these limitations, CI- concentration changes during recording of membrane potentials re,,ealed Cl--conductive pathways in a number of epithelial cell membranes (cf. Table l). It should be pointed out again that no assumptions about the nature of the conductive pathw.'ly can be made with :his technique. For example, in renal proximal tubules, alterations in contraluminal bicarbonate concentration resulted in a significant potential change across the basolateral membrane [116]. Since this potential change was also dependent on the presence of Na + ions, it could be concluded that an electrogenic coupled cotransport of HCO~- and Na + is present in the basolateral membrane [117,118]. H-C. Cl--selective microelectrodes

Ion-selective microelectrodes are fine glass capillaries, filled with a resin which creates selec-

• -rive permeability to a certain spe~c~ ~f ~cna. Consequently, an electrical potential is generated at this electrode, which is dependent on the intracellular activity of the respective ion. However, the electrical cell potential is also ,ecorded by this electrode. To correct for this potential, it must be recorded in addition with a KCl-filled microelectrode. This is best achieved with double-barrelled microelectrodes, where one barrel is filled with the ion-selective material and the other with a high KCI solution, in order to record the electrical potential. The use of double-barrelled electrodes, however, is restricted to large cells. The application of ion-selective microelectrodes revealed in a number of epithelia (cf. Table I) that intraceUular C I - ions are above their electrochemical equilibrium. This observation was made, for example, in frog skin, renal proximal tubule, the thick ascending limb of Henle's loop and the rectal gland of the dogfish. One can conclude from this observation that C I - ions must be actively taken up by the cell across one cell membrane and may leave the cell by passive transport. For example, it was demonstrated that in the rectal gland of the dogfish, C I - ions are taken up across the basolateral membrane by a secondary active mechanism, which transports one Na +, one K + and two CI- ions simultaneously into the cell. Consequendy, CI- ions are accumulated inside these cells and ~an leave across the luminal membrane via anion-selective channels [72] (Fig. I b ) H-D. Use of C ! - blockers

In contrast to other ion species, where highly selective blockers for channel transport have been known for a long time, the use of CI- channel blockers, in order to identify a channel mechanism, is still in its infancy. For example, epithelial Na + channels are inhibited by the diuretic amiloride in the micromolar range a~d epithelial K + channels are blocked by barium a n d / o r tetraethylammonium ions, among other substances. Consequently, when permeability changes by these substances are observed in cell membranes, it is very likely that channel mechanisms are involved. For epithelial C l - channels, substances which in the micromolar range block these channels have only recently been developed. For example, it was

529 shown that 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) inhibits half-maximally C1- conductance in the thick ascending limb of Henle's loop at a concentration of 8.10 -s m o l / l [37]. The inhibition of CI- channels by this substance was directly demonstrated with the patch-clamp technique in the luminal membrane of the rectal gland [75] and in the apical membrane of colonic cells in culture (HT29) [53]. However, as pointed out recently by Greger [119], the blocking effect of NPPB and related substances on CI- pathways varies considerably from tissue to tissue.' Care must be taken with the substance diphenylamine2-carboxylate (DPC), which was shown to inhibit C I - channels in the thick ascending limb of Henle's loop [36]. It could be demonstrated that this compound also ird',ihits nonspecific channels observed in the basolateral membrane of renal proximal tubules [120].

II-E. Single-channel recordings

of patch pipettes by dipping the electrode twice through a lipid monolayer [128-131]. Reconstitution of channel forming proteins into the planar lipid bilayer is possible by two methods: (i) vesicles, c Jnta~ning the channel protein are fused with the planar bilayer; (ii) the channel protein is present in lipid monolayers, which are folded to a bilayer. The first technique was used, for example, to study K + channels from the sarcoplasmic reticulum [132], whereas the latter technique was first applied to the matrix protein from Escherichia coil [1331. Only a few studies have been reported in which epithelial CI- channels were incorporated into a planar lipid bilayer. Reinlaardt et al. [47] fused vesicles derived from rat colonic enterocyte plasma membranes with a planar lipid bilayer and observed anion-selective channels with a single channel conductance of 50 pS. Zeuthen et al. [68] reconstituted a C1--conducting channel from the ventricular membrane of the choroid plexus into a lipid bilayer on the tip of a patch electrode.

II-E.I. Reconstitution of single CI- channels in planar lipid bilayers

H-E.2. The patch-clamp technique

Although single ionic channels were observed first in a black lipid bilayer membrane [121], the investigation of biological channel proteins with this technique remained restricted to some proteins (reviewed in Refs. 122-124). Several methods exist to generate an artifical lipid bilayer membrane. Mueller and Rudin [125] developed the brush technique, where lipids are dissolved in decane and ~ e painted across a septum in a teflon sheet. Under optical control, it can be observed that spontaneous thinning of the dissolved lipid occurs until a bilayer membrane is formed. However, these artifical membranes also contain the solvent decane and, hence, are thicker than biological membranes. This effect is circumvented by the technique of Montal and Mueller [126], where two monolayers are opposed to form a bilayer membrane. Only small amounts of apolar solvent (mostly hexadecane), which is needed to pretreat the edge of the septum, is present in these membranes. The moaolayers may be spread from artificial lipids, dissolved in hexane or, alternatively, they form spontaneously on the air/water interface of a vesicle suspension [127]. Most recently, artifical lipid bilayers are formed on the tip

There are several excellent review articles describing the details of the patch-elamp technique [134-138]. Therefore, I shall present only a briet outline of this method. A fire-polished micropipette with a tip opening of about 1 /tin is connected to a current-to-voltage converter of high resolution and low noise. The preamplifier (probe) of this instrument is mounted on a micromanipulator. Several types of micromanipulator are in common use, for example: hydraulically driven (Narishige MO 103, Tokyo, Japan), or electrically driven (Zeiss, F.R.G., Merzhliuser, Wetzlar, F.R.G.) or, more recently, piezostepper (Physik Instrumente, Waldbronn, F.R.G., List, Darmstadt, F.R.G.). The patch pipette, which is filled with physiological saline, is gently pressed onto a cell membrane of a tissue preparation. The tissue is kept in a temperature-controlled measuring chamber, mounted on the stage of an inverted microscope. The patch pipette is then guided to the cell membrane at 400 × to 600 x magnification and, after applying slight suction to the interior of the micropipette, part of the cell membrane is pulled into the pipette forming close contact with the glass wall. This establishes a high electrical

seal between the membrane and the glass, so that the input resistance rises from about 5-10 Mohrns (pipette resistar..ce) to about 50-100 Gohms. This high input resistance has two implications: (i) the Nyquist noise is low enough that currents as low as about 0.2 pA can be resolved. (ii) A command voltage, now applied externally to the patch pipette, leads to a voltage drop across the membrane patch on the tip of the electrode, whereas the voltage drop across the electrical resistance of the pipette and of the bath are negligible. Although the input resistance of a sealed membrane is high, it is much lower than that expected for a pure lipid membrane. For example, in bovine ehromaffin cells, it was observed that the total input resistance is composed of 50% by the seal and of 50% by the membrane patch [139]. The fact that the total input :esistance is composed of the seal resistance, Rs, and the resistance of the membrane patch, Rp, was studied by Fischmeister et al. [140] in more detail. These authors investigated coupled chick embryo cardiac cells with two patch electrodes, where one was in the cell-attached and the other in the whole-ceU configuration of a neighboring cell. By this means, it was possible to measure R s alid Rp separately. It was demonstrated that R s could be 16-times higher than the apparent input resistance and that the patch resistance, Rp, was influenced by the electrode-filling solution [140]. The unexpectedly low resistance of the membrane patch may be due to (i) currents through channels that are too small a n d / o r too fast to appear as single-channel events, (ii) electrogenic carrier systems, Off) currents through permanently open channels and (iv) leakage currents due to mechanical damage of the membrane patch. A resistance of the membrane patch lower than that of the seal was also observed in renal proximal tubules. As a consequence, the cell potential could be recorded with good precision in cell-attached patches [141]. A prerequisite for obtaining high resistance seals is a clean membrane. In epithelial tissues, several experimental approaches were used. For example, in order to study membranes of the lacrimal gland with the patch-clamp technique, the tissue has to be disrupted by collagenase treatment in order to obtain acinar cells or isolated cells [102-104]. In order to study the luminal membrane in renal

tubules, some investigators cut open isolated tubules longitudinally with fine needles [142,143]. An alternative method was developed to investigate renal proximal tubules and ducts of the rectal gland of the shark. Tubules were perfused on one side with a perfusion system [144], while the other end was lying on the bottom of the measuring chamber. At the open end, the lateral membrane was free and could be reached by the patch pipette. Alternatively, the pipette could be inserted through the open end into the tubule lumen and could be attached to the luminal membrane [145]. A powerful method for studying epithelial cells is the use of cell cultures. This technique has been applied mainly to established cell lines. For example, C I - channels were studied in M D C K cells (derived from dog kidney [65]), in A6 cells (derived from Xenopus laevis kidney [26]), in OK cells (derived from opposum kidney [16]) and in the human colonic carcinoma cells HT29 and T84

[50,53]. In order to study the selectivity ot C l - channels with respect to cations, a gradient for NaCl is generally applied across the membrane patch. The reversal potential of the singie-channel current is recorded and the selectivity ratio is calculated by means of the Goldman-Hodgkin-Katz equation (Eqn. 2). However, care must be taken bceause of the possible generation of diffusion potentials across the membrane patch. For example, in the basolateral membrane of renal proximal straight tubules, it was observed that a diffusion potential was generated across the membrane patch, when the bath NaCI concentration was changed on one side of the membrane patch. This potential could be recorded in two independent ways: (i) the patch-clamp amplifier was set to the current-clamp mode and the input potential was recorded. (ii) A current-voltage relation was plotted for the leakage current (Io), as shown in Fig. 2. With 150 mM of NaCI on both sides, the straight line crossed the voltage axis at about 0 mV. Loitering the bath NaCI concentration to 70 raM, caused a shift of the lo/r curve to the right and yielded a reversal potential of 15.2 + 2.6 mV (n--5) (corrected for liquid junction potentials). This indicates that the membrane patch has a conductance which is about 30-times higher for Na + than for CI-. The nature of this conductance is as yet unknown. If single

-so - ~ Vc

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i

i

i

~,~

.~-

~s

~o

Fig 2. Cunent-voltage curves of the leakage current in a cell.-cxcisedpatch of the basolateral membraneof the straight proximal tubuleof rabbit kidney. <>. NaCI-Ringersolutionon both sides; , , NaCI-Ringersolution in the pipette and I/2 NaC1.Ringersolution(505 of NaCI was replacedisoosmolarly by mannitol)in the bath. The shift of the reversalpotential to the rightindicatescationselectivityof the leakagecurrent. ionic channels are present in the patch, this diffusion potential will influence the single channel current. Consequently, the reversal potential of the single channels has to be corrected by the diffusion potential, in order to obtain the correct permselectivity of the ion channel. It should be pointed out that this diffusion potential is not dependent on the type of measuring electrode, but originates from intrinsic membrane properties. m . Properties of chloride channels

and Fig. 3), so that these channels are denoted as outward rectifiers*. For rectifying channels, the single-channel conductance is not constant in the entire potential range. Therefore, how the single-channel conductance was calculated should be stated. Tv, o possibilities for defining the single-channel conduct=rice are commonly used: (i) data points for positive and negative voltages in the i / V curve are fitted separately by linear regression and bo~h values are presented, (ii) the sin#e-channel slope conductance is given at a fixed voltage which is mostly the resting membrane potential. An example of such outward rectifying channels recorded from the human colon carcinoma cells H'I'29 is shown in Fig. 3. Linear regression of data points at positive potentials yielded ~ = 91 pS, and at negative voltage g was 40 pS. Another important parameter is the dependence of the single-channel open-state probability, P0, on the potential, which reflects a possible voltage-dependence of the macroscopic CI- current (Table II, column 3). P0 is defined as the time dunng which the channel is found in its open state, divided by the total observation time. In most cases, the open-state probability is obtained from the single-channel amplitude histogram (cf. Fig. 3). With this method, the mean probability of one channel being open can easily be calculated, a!so from multi-channel recordings, with the equation N

III-A. Parameters to characterize single channels Single ionic channels can be characterized by a number of parameters, for example, the singlechannel conductance, g, the open-state probability P0 and kinetic parameters, such as the time constants obtained from open- and closed-time histograms. The single-channel conductance, g, is generally calculated from the current-voltage ( i / V ) relationship. However, for some epithelial CI- channels the i / V curves are not linear over the voltage range studied, but display rectification in one current direction. For a number of CI- channels, the single-channel amplitude for outward currents was larger than for inward-directed ones (cf. Table II

where N is the total number of channels in the patch and P(n) denotes the probability that level n is occupied. However, the assumption that all individual channels present in the patch have the same mean open probability Po has to be made. The kinetics of channel gating can be described by time constants calculated from open- and closed-time histograms. Since this analysis was * The direction of electrical current flow is referred to the movement of positively charged ions. Consequently,with respect to CI- movementoutward currents means inward flow of CI- ions,i.e., from the extracellularto the cytosolic side.

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534 performed with only a few epithelial C1- channels [24,47,53,75], it will not be discussed here. The selectivity of CI- channels can be expressed as the permeability ratio of C1- versus cations or as a permeability sequence of various other anions. With the exception of the big anion channels, the ratio Pcl/P~a is usually larger than 10, which means that the channels are highly selective for anions over cations. Therefore, in Table II the latter value is only given for the high conductance channels.

III-B. CI-channelsinfluid-reabsorbingepithelia Although transcellular CI- transport has been observed in most reabsorbing epithelia, single CIchannels have been recorded in only a few tissues. This is mainly due to technical difficulties. For example, the basolateral membrane of the frog skin has been inaccessible until now because of the underlying connective tissue. In renal tubules, the basement membrane has in most cases prevented the application of the patch-clamp technique. In proximal tubules of the rabbit kidney, the patch-clamp method was applied to the lateral membrane at the tom-off end [120]. In cell-attached patches, only cation xchannels could be observed [146,147]. It is very likely that this cell membrane does not possess anion-selective channels, but that CI- exit is mediated by other translocation nwcilatfi~ as. In the basolateral membrane of rabbit urinary bladder, as well ~ in cultured cells derived from rabbit urinary bladder, a high conductance anion channel was observed [24,25]. Both channels differed somewhat in their voltage-dependence. In the in vitro preparation, the channel was active only within 5:20 mV, whereas in the cell culture preparation channel activity between - 8 0 and +20 mV was reported. In the rabbit urinary bladder, the channel had to be evoked usually by large voltage steps. Since the channel was inactive at the physiological membrane potential the authors assumed that this channel was normally inactive in situ. Consequently, its physiological role remains unclear [24]. A large conductan:t anion channel (g = 360 pS), active in the small voltage range of 5:20 mV, was observed in apical membranes of A6 cells, a

cell line derived from Xenopus laevis kidney [26]. These cultured cells have some properties in common with tight epithelia and are able to reabsorb Na + [748] and probably also CI-. Nelson et al. [26] speculated that a downhill driving force for CI- uptake from the lumen into the cells could occur at high transepithelial potentials. Under these conditions, the channel described could be involved in CI- reabsorption. This assumption needs further experimental conformation. In cultured pulmonary alveolar cells, a similar maxi CIchannel (g = 382 pS) was recorded. This channel became inactive at potentials above + 40 mV and below - 50 InV. It consisted of multiples of smaller conductance levels with conductances of 60-70 pS [29]. The authors suggested that the channel consists of six conducting pathways in parallel (so called co-channels), which share a gating mechanism so that all can synchronously be conductive or nonconductive. A similar gating mechanism was recently proposed for K + channels in early distal tubules of Amphiuma kidney [149]. The role of tiffs maxi C I - channel in transepithelial CItransport has still to be clarified. In primary cultures of collecting duct principal cells, a CI- channel with a conductance of 120 pS was recorded [32]. The conductance of this channel had exceptional characteristics: it increased gradually between 0 and +40 mV and then remained approximately constant up to at least + 140 mV at 120 pS. The physiological role of this channel was not discussed. In the diluting segment of Amphiuma kidney, a CI- channel with a conductance of 150-200 pS was recently observed [40]. Its open-state probability decreased at positive and negative potentials (values are not reported). Therefore, this channel exhibits similar characteristics than the large conductance channels, although its singlechannel conductance is somewhat smaller. Whether this channel accounts for the CI- conductance observed [39] has still to be proven. A brief report exists about CI- channels in cultured rat epididymal cells [42]. In vivo, this tissue reabsorbs CI- ions. However, the channel was observed in the apical membrane of the cultured cells. The author speculates that the channel serves for passive uptake of CI- ions from the lumen into the cells. To my knowledge, no data

535

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exist o n intracellular C I - activity in this tissue which would allow calculation o f the driving force for (31- ions across the luminal m e m b r a n e . Therefore, the physiological role o f this c h a n n e l is still unclear. T h e c h a n n e l h a d a low c o n d u c t a n c e a n d exhibited strong o u t w a r d rectification. I n this respect, it is very similar to m a n y luminal C I channels in secreting epithelia, as discussed below. Small c o n d u c t a n c e C I - c h a n n e l s of basolateral m e m b r a n e s were only observed in the rabbit urinary bladder [24] a n d in the cortical thick ascending limb of Henle's loop (Greger, u n p u b lished data). In b o t h preparations, the C I - con-

d u c t a n c e was clearly demonsta'ated with other m e t h o d s (Table I). Therefore, it is very likely that these c h a n n e l s a c c o u n t for basolateral C I - permeability a n d mediate t h e C I - exit step. In summary. I n fluld-reabsorbing epithelia, C I c h a n n e l s with very different properties were observed. I n a n u m b e r o f cultured cells the channels are located in the upward-facing (apical) m e m brane. H o w these l u m i n a l C1- c h a r n e l s are incorporated into t h e task o~ C I - reabsorption h a s still to be determined. In a few epithefia (urinary bladder, thick a s c e n d i n g limb of Henle's loop, Amphiuma diluting segment), basolateral C I -

536 TABLE III HORMONAL REGULATION OF CHLORIDE CHANNELS IN CI- SECRETING EPITHELIA Tissue

Hormonal regulation of fluid secretion

Intracellular messenger

CIconductance dependence on the intracellular messenger

Colonic cells T84 (human colonic carcinoma cell culture) HT20 (human colonic carcinoma, cell culture) Com~ Malpighian tubules

VIP

cAMP

cAMP, Ca 2+

VIP prostaglandin E2

cAMP

cAMP, Ca 2+

yes

50,51,168

VIP epinephrine insect diuretic hormone

cAMP cAMP

cAMP cAMP

no

52, 54 55-57

cAMP

-

MDCK cells (cultured cell fine from dog kidney) Rectal gland (shark. Squalus acanthias ) Trachea (canine) (human, normal) (human, CF) (human, normal) (human, CF) Eccrine sweat gland, Secretory cells (human, normal) (human, CF) Lacrimal gland Pancreatic duct cells

Ca2 +-dependence of C1channels

Refs.

44,47,167

59, 61

epinephrine

cAMP

cAMP

VlP

cAMP

cAMP

no

74,169, 7~

epinephrine epinephrine no isoproterenol no

cAMP cAMP no cAMP cAMP no cAMP

cAMP cAMP, Ca 2+ no cAMP no

no yes yes no no

78, 79, 86, 87 50, 89, 91 50, 8~, 91 88 88

epinephrine isoproterenol no aeatylcholine adrenaline cholceystokinin

cAMP no cAMP Ca 2+ IP3 cAMP (?)

cAMP no Ca 2+ GTPyS, 1P3

yes yes yes

95, 98,170 95.170 99-102,104 103,105 107.109,171

62, 64, 65

Abbreviations: VIP, vasoactive intestinal peptide; cAMP, adenosine 3':5'-cyclic monophosphate; IP3, inositol 1,4,5*trisphosphate; GTPyS; guanosine 5'-O-[y-thio]triphosph.tte; CF, .~3'sticfibrosis. channels were observed, which can explain the macroscopic C1- c o n d u c t a n c e a n d the passive C I exit step postulated from other eleetrophysiological data (Table I, Fig. la). O n the other h a n d , reabsorbing epithelia seem to exist where t h e contr',duminal C I - exit step is solely carrier mediated. F o r example, in rabbit renal proximal tubules, the patch-clamp technique could n o t detect Cl--selective channels u n d e r physiological conditions (own observations). I I I - C . CI - channels in fluid-secreting epithelia

Colonic cells are k n o w n to secrete C I - ions w h e n they are stimulated with vasoactive intesti-

nal peptide (VIP), a h o r m o n e which increases the intracellular cyclic A M P level [44]. Single C l c h a n n e l s were recorded in p l a n a r lipid bilayer m e m b r a n e s after fusion with vesicles, derived from epithelial cells of rat colonic mucm,~ [47]. A s the vesicle p r e p a r a t i o n ' c o n s i s t e d o f apical a n d basolateral m e m b r a n e s , the origin of the C I - channels was n o t determined. However, the vesicle possessed a N a + permeability which was highly sensitive to amiloride, w h i c h is k n o w n to occur only in the apical m e m b r a n e . C o n s e q u e n t l y , the a u t h o r s suggested that the CI- channels observed originated f r o m the l u m i n a l cell m e m b r a n e . M o r e direct s u p p o r t for the existence of apical C I c h a n n e l s c a m e f r o m p a t c h - c l a m p studies, per-

537 formed in the cultured cell lines T84 and HT29, derived from human c,~lon carcinoma ceils [50,53]. In both cell types, single CI- channels were recorded in the upward-facing membrane of confluent monolayers, correspondin~ to apical call membranes. Most experiments were performed when cells were stimulated with forskolin or cyclic AMP, in order to "_:nduce C1- secretion. In both cell lines, outward-rectifying C1- channels were recorded with comparable conductances (Table II). It was also found consistently that the openstate probability increased with depolarizing potentials in both tissues. However, differences exist in the permeability sequence for anions (Table II), and, more markedly, the depeadeaee oii cytosolic Ca 2÷ seems to be different (Table III). In T84 cells, it was shown that opening of the channels could be evoked in cell-attached patches, when the Ca 2+ ionopbore A23187 was added to the bath and, in addition, direct activation of Cichannels by 180 n m o l / l of Ca 2+ in cell-free patches was demonstrated [50]. On the other hand, in cell-free patches of HT29 cells, complete removal of bath Ca 2+ did not impair CI- channel activity (own unpublished results). The CI- channel from rat colonic mucosa also exhibited rectifying properties [47]. However, since the orientation

of the protein in the bilayer was not known, the polarity of the rectification remained undetermined. Only brief reports in abstract form are available on two other C l - channels in Cl--secreting epithelia, namely the ral'~bit cornea [58], and the mosquito malpighian tubule [61]: in both epithelia, the cham~zl was present in the apical membrane and its conductance was 66 pg and 25 pS, respectively. Madin-Darby canine kidney (MDCK) cells are an established cell fine derived from dog kidney which exkfibit CI- secretion after stimulation with adrenaline |62]. Patch-clamp studies revealed a maxl mnon channel ( g = 456 pS) in the apical membrane which was about 5-times more permeable for C i - than for N a + [65]. Since opening of this channel could be evoked by adrenaline in cell-attached patches, the authors suggested that it was involved in (2!- secretion. The channel became inactive at high positive and negative potentials, similar to some maxi CI- channels in reabsorbing epithelia. I shall next discuss C l - channels in secretory glands, such as the rectal gland of the shark, the trachea, the sweat gland and the lacrimal gland. In the exocrine pancreas and the salivary gland, the

tO-

5-

0o 0 20o ~=

g|pS)

Fig. 4. Two types of singleCI- channel in a cell-excised(inside-out)patch of the luminal membraneof the dogfish rectal gland. (a) The single-channelrecordingsreveala largerconductancechannel(g ~ 45 pS) with fast closingevents,as well as a smallconductance channel (g ~11 pS) with slow open and clo~! ki~.etics.NaCI-Ringersolution was present on both sides. (b) Frequencydistribution of the single channel slope conductancesclearly reveal two distributionscorrespondingto the two channel types (reproduced from ReL 75).

538

O-

o-

2p^ lLe o _

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Voltage-dependentCI- channels in the luminal membrane of a cell-excised(inside-out) patch of the shark rectal gland. The pipette and bath were filled with NaCI Ringer solution. (a) Single-channelcurrent traces at different clamp potentials (indicated at the right). 0 ~ denotes the closed state of the channel. (b) The current-voltage relation was fitted by linear regression, yielding a single channel conductance of 44 pS. (c) Voltage-dependenceof the open-state probabifity, P0, of the channel. The solid line was drawn by eye (reproduced from Ref. 75). Fig. 5.

presence of C1- channels was postulated [150], but has not been detected with single-channel analysis until now. The electrical events which occurred after hormonal stimulation were intensively studied in isolated in vitro perfused segments of the rectal gland o f the shark. Greger and Schlatter [74] demonstrated that the primary event of hormonal stimulation was the increase in luminal C I - conductance, evoked by cyclic A M P . Later on, patch-clamp studies in cell-attached luminal membranes directly demonstrated the induction of

C I - channels after hormonal stimulation (Fig. 4) [76]• In excised m e m b r a n e patches, two types of C I - channel with very different characteristics were studied (Fig. 4). The larger channel had a conductance of 45 pS and was activated by depolarizing potentials (Fig. 5). In contrast, the small channel (Re 11 pS) was slightly more active at hyperpolarizing potentials. The kinetic properties were also quite different: the larger channel exhibited bursts of activity with fast closing events, whereas the small channel had long-lasting open times

539

without any flickering (Fig. 4). Finally the larger channel was blocked by NPPB, whereas this drug had no effect on the smaller channel. Two types of CI- channel of high and low conductance were also observed both in canine and human tracheal cells, in the colonic carcinoma cells HT29, as well as in secreting cells of the sweat gland. In primary cultures of dog trachea, Shoemaker et al. [86] observed four different types of anion channel, which differed in their singlechannel conductance and the potential dependence of the open-state probability (cf. Table II). In contrast, Welsh [87] provided evidence that only one channel type exists in a similar preparation. However, this author observed significant variations in the absolute size of individual channels, especially at depolarizing potentials. Both groups also investigated primary cultures of human airway epithelial cells from subj~ts with cystic fibrosis and from controls. Although the conclusions concerning the regulation of the CIchannels are similar (see below), the properties of the channels differ somewhat. Frizzell and coworkers [50,89] observed two types of channel with conductances of 20 pS and 50 pS. The larger channel exhibited outward rectification, whereas the smaller one had a linear i / V relationship. Dependence of the open probability on voltage was not reported. In contrast, Welsh and coworkers [88,90] observed but one channel type with g = 26 pS at 0 mV potential. This channel had outward-rectifying properties and its activity decreased with depolarization. A salient difference exists between the Ca 2+ dependence of the channels: whereas Frizzell et al. [50,89,91] observed a pronounced dependence of channel activity on cytosollc Ca z+ in cell-excised experiments, Welsh and Liedtke [88] observed no such effects. In the secretory segment of the sweat gland, CI- channels were recently investigated. Until now only reports in abstract form exist. In primary cultures of human sweat glands, Schoumacher et al. [95] recorded two types of channel (g = 25 pS and 50 pS), where the larger one was outward-rectifying. Welsh et al. [96] performed whole-cell studies and observed CI- conductance with outward-rectifying properties and inactivation with depolarization. In lacrimal glands, only whole-cell recordings

of the C1- conductance were performed [101-103]. From noise analysis, Marty et al. [101] estimated a single-channel conductance as low as 1-2 pS, a~d observed activation of the channels with depolarizing potentials. These authors also observed current fluctuations in cell-attached basolateral membrane patches of lacrimal glands, which they interpreted as C1- channels, having a single-channel conductance of about 5 pS. However, until now no clear evidence has been provided that the observed fluctuations are really due to CI- conductance. In conclusion in all Cl--secreting epithelia studied with the patch-clamp technique, C1- channels were observed in the luminal cell membrane only. Consequently, the model demonstrated in Fig. lb seems to be of general validity, at least with respect to the CI- exit step. In most secreting epithelia, CI- channels with small as well as with higher conductance values were observed. Maxi CI- channels were only recorded in MDCK cells and in the canine trachea. III-D. C I - channels involved in volume regulation

Only one brief communication about single CIchannels involved in volume regulation in epithelial cells exists. Kolb et al. [16] observed activation of CI- channels in cell-attached patches of OK cells (cell culture from opposum kidney) when they were exposed to hypotonic media. Interestingly, this anion channel could also be activated by inducing mechanical stretch to the membrane patch. III-E. Summary

According to their single-channel conductances, CI- channels can be classified in at least three categories: (i) magi channels with g = 200-450 pS, (ii) channels with intermediate conductance of 40-60 pS and (iii) small channels with g = 5-30 pS. The maxi channels exhibited p,-onounced voltage-dependence: they became inactive at large positive and negative potentials. The voltage range, in which the channels remained active, was highly variable. Another common feature of these channels was the existence of substates. The voltage-dependent maxi C1- channel

540 was observed both in apical and basolateral membranes, in fluid-absorbing as well as in fluidsecreting epithelia. Its physiological significance was not always clear. With respect to the singlechannel conductance and the voltage-dependence, these high conductance CI- channels resemble the voltage-dependent anion channels (VDAC) from outer mitochondrial membranes [151,152], and the matrix protein (porin) from Escherichia coli [153]. e l - channels with high conductance were also observed in a number of nonepithelial cell membranes, for example in cultured muscle cells [154], cultured Schwann cells [155], molluscan neurones [156], peritoneal macrophages [157], basophilic leukemia cells [158], cultured astrocytes [159] and Xenopus oocytes [160]. In epithelial tissues, a relatively large number of e l - channels with intermediate conductances of about 50 pS exist. Most of them exhibit outwardrectification and appear in apical cell membranes of fluid (and CI-) -secreting epithelia. On the other hand, inward-rectifying channels and those with no rectifying properties with g = 50 pS were reported (Table II). The third category, namely C1- channels with a single-channel conductance of between 5 and 30 pS, were observed in apical membranes of C I secreting epithelia. Apparently, these channels have no rectifying properties.

latter stimulates adenylyl cyclase, leading to an increased cyclic AMP level, aldosterone exerts its effect via new synthesis of transporting proteins (reviewed in Ref. 163). The effects of both ho.."m.ones on apical Ha + chamlels have bccn intensively studied (reviewed in Ref. 163), but effects on the CI- conductance of these tissues are not reported. In the medullary thick ascending limb of Henle's loop (TAL) of mouse and rat kidney, Sasaki and Imai [164] demonstrated that the antidiuretic hormone stimulated active NaCl reabsorption. With isolated perfused segments of mouse medulla TAL, Schlatter and Greger [165] showed that the primary action of cyclic AMP was to increase basolateral CI- conductance. In rat alveolar epithelium, CI- absorption was increased by cyclic AMP and by the fi-adrenerglc challenge of isoprotenerol [166]. However, in a number of other epithelia, like the cortical thick ascending limb of Henle's loop of rabbit kidney, the Amphiuma diluting segment and epididymal cells, fluid secretion is either not or only minimally regulated by hormones. The dependence of C I - channel activity on intracellular Ca 2+ has only been investigated in the urinary bladder. Hanrahan et al. [24,25] reported that cytosolic Ca 2+ had no effects on the two types of CI- channel they observed in this tissue. These observations were made in excised membrane patches.

IV. Regulation of epithelial chlorioe channels

IV-B. Fluid-secreting epithelia The gating of ionic channels can be regulated by the electrical membrane potential a n d / o r by chemical compounds. The influence of the electrical potential on epithelial CI- channels was discussed in the preceding section. In this section some effects of exogenous hormones and their intracellular messengers will be presented.

IV..A_. Fluid.reabsorbing epithelia In fluid-absorbing epithelia, little is known about the regulation of the CI- channel. In some tight epithelia, such as the urinary bladder, the cultured cells of toad kidney, A6 cells and the renal collecting duct, fluid reabsorption is regulated by the mineralocorticoid aldosterone a n d / o r the antidiuretic hormone. Whereas the

In all fluid-secreting epithelia discussed in this paper, the transport of fluid and electrolytes is under hormonal control (Table III). The intracellular signal transduction can be mediated by the following pathways: (i) the adenylyl cyclase-cyclic AMP system (Fig. 6a), where the intracellular messenger cyclic AMP activates the catalytic subunit of the protein kinase A and this enzyme activates the channel protein by phosphorylation (reviewed in Ref. 172); (ii) the phospholipase C system (Fig. 6b), where the intracellular messengers inosito! 1,4,5-trisphosphate (IP3), and 1,2diacylglycerol (DG) can be formed. IP3 releases Ca 2+ from intracellular stores [173], and these ions can directly activate ionic channels. In addition, DG activates the protein kinase C which

541 Hormone

a

Interstitium

1

o

b

Hormone

Interstitium

o /

/ II

Cel{ J

t.ego,o,o,, co,o,.,,c "-.. /

ATP

Rough Endoplasmic Reticuium

~ ~

~........... I / { 2 / - IPhosphoryteti0n~

.~o'~/'c,-/

CeU

CtLumen Lumen Fig. 6. Modelsof intracellularsignal transductionand CI- channelactivation.(a) The adenylyicyclasc-cyclicAMP system(aozording to Ref. 172). (b) The phospholipaseC system.Two possibilitiesare shown: (i) channel proteins,which are already present in the cell membrane, can be activated. (ii) Channels, present in the membranes of zymogen granules, are activated before fusion of the zymogengranuleswith the cell membrane(accordingto Refs. 174,179 and 180).

can phosphorylate CI- channel proteins (reviewed in Ref. 174). It is possible that all the mechanisms described are present in the same tissue and that interactions between these signal-transducing pathways t ~ e place. In most tissues, as shown m Table III, secretagogues lead to at~ inere~ed cyclic A M P level so that phosphorylation of CI- channels by protein kinase A can be expected. In most tissues, it could be dir~tly demonstrated that an increase in intracellular cyclic A M P increased the luminal CIconductance. Fig. 7 demonstrates an experiment performed in a cell-attached luminal membrane of the shark rectal gland. After administration of the stimulants (cf. legend to Fig. 7) (21- channels were evoked in the membrane patch, as shown in the upper part of the figure. The lower panel of Fig. 7 demonstrates how the open probability of the single channel, as well as the transepithelial potential difference increased with thne. As shown in Fig. 6a, it is assumed that CIchannels are activated by phosphorylation by the catalytic subunit of protein kinase A. Indeed, activation of CI- channels by this enzyme in the presence of ATP was demonstrated directly in cell-excised patches of the shark rectal gland [175] and in human tracheal cells [91]. Frizzell and coworkers [91] observed that this activation of CIchannels was not possible in tracheal cells from patients with the diseas¢ cystic fibrosis. Moreover,

in the latter preparation, it was not possible to evoke CI- conductance by the fl-adrenergic stimulant epinephrine [89] or by isoprotencrol [88], whereas these hormones activated apical C1channels in noncystic fibrosis cells. On the other hand, C l - channels could be observed in cell-excised patches of cystic fibrosis cells as well as in controls [88,89], and the properties of the C l channels in both preparations were identical. The discrepancy, that C l - channels were not present in intact cystic fibrosis cells after hormonal stimulation, but appeared in cell-excised membrane patches, was explained by Frizzell and coworkers [89]. These authors observed that the CI- channels in tracheal cells were activated by cytosolic Ca 2+ ions and they assumed that the activation of the channels in excised patches of cystic fibrosis cells was due to the high calcium concentration in the bath. On the other hand, Welsh and Liedtke [88] reported that the C I - channels in the sarne preparations were independent of cytosolic Ca 2+. This discrepancy in the Ca 2+ dependence of tracheal C l - channels between both groups awaits further explanation. One important conclusion of these experiments is that in cystic fibrosis cells, the CIchannels are still present, but there is a defect in the activation of the channel protein. F:iizzell et al. [91] presented evidence that this defect lies beyond the level of cyclic AMP-dependent protz;a ldnase; that is, at the channel itself or at some channel-as-

542

time(s)

2pA~___ 200ms [

Stimutotion

,.Io

O.S c~0.6 0.4 0.2 0

~.---?d 0 50

Popenr PDt.... y.~d

.o.o

, , f 100 150 200 Time i s )

-6 -.4Z :~ 0

Fig. 7. Activationof a CI- channel in a cell-attachedluminal membrane patch of tl~e shark rectal gland. The time (s) after stimulation (in mol/h 10-4 dibutyryladenosine 3',5'-cyclic monophosphate, 10-4 adenosine, 10-e forskolin) is given at the right side of each recording. The lower panel shows the time-dependence of the open-state probability and of the transepithelialpotential(PDte)(reproducedfrom Ref. 76).

has been shown previously that this compound released Ca 2+ from the endoplasmic reticulum [173]. This indirect action of IP3 was confirmed in the experiments of Llano et al. [103], in which the effect of IP3 was absent when intracellular Ca 2+ was strongly buffered to 10 - s mol/l. Fig. 6b demonstrates that several possibilities exist to increase luminal CI- conductance in exocfine glands by the phospholipase C system. One possibility is that inactivated CI- channel proteins, already present in the cell membrane, are activated either by Ca 2+ ions or by phosphorylation. Alternatively, C1- channels can be present in zymogen granules which fuse with the plasma membrane after stimulation of the cell [176]. Although it has been impossible to discriminate between these processes until now, the latter is supported by the fact that in isolated zymogen granules o f rat pancreatic cells appreciable CI- conductance was observed when the animals were treated with secretagogues before removal of the pancreas [177]. In contrast, in untreated animals, no C I - conductance was present in the zymogen granules. These observations are supported by experiments in permeabiliT.ed isolated acini of the rat exocrine pancreas. Fuller et al. [178] observed that protein release by the zymogen granules after stimulation with secretagogues was reduced in absence of C1- ions.

IV-C. Summary sociated regulatory protein which is also present in the excised membrane patches. In the secreting epithelia discussed thus far, the intraceliular signal was transduced by the adenylyl cyclase-cyclic AMP system. It is well established that in exocriue glands, like the pancreas and the lacrimal gland, intracellular signal transduction is mediated by the phospholipase C system (Fig. 6b). In whole-cell patches of the lacrimal gland, Evans and Marty [105] observed a potentiation of muscarinic responses on K + and CI- channel activity when the cells were dialyzed with guanosine 5'-O-[3-thio]tdphosphate (GTPyS), a nonhydrolyzable GTP analogue. Stimulation of the CIcurrent was also recorded after addition of inositol 1,4,5-trisphosphate (IP3) to the pipette. It

In most fluid-secreting epithelia, activation of luminal CI- channels by the adenylyl cyclase-cyclic A M P system was observed and in a few cases direct activation of the channels by the catalytic subunit of protein kinase A was studied [91,175]. No general conclusion about the role of intracellular Ca 2÷ can be drawn for these cells. In the literature, Ca2+-dependent as well as Ca2*-inde pendent CI- channels have been reported. S~imulation of luminal t A - channels via the phospholipase C system have thus far been observed only in rat lacrimal cells [103,105]. As expected, these CIchannels were strongly dependent on intracellular Ca 2+ activity. In fluid-reabsorbing epithelia, regulation of CI- conductance by exogenous substance was reported only for the cortical TAL of the mouse [165].

V. Chloride channel blockers Blockers of ionic channels are classified in three categories according to their binding kinetics [115]: ' slow' blockers dissociate from the channel protein at a lower rate than that of channel closing. These blockers increase the closed time of the cha,'mel but do not induce additional closing events (flickering). 'Intermediate' blockers have faster binding and dissociating rates than the normal closing rates of the channel and therefore induce flickering. 'Fast' blockers bind and dissociate s6 fast that their kinetics cannot be resolved in singlechannel experiments and, therefore, they apparently decrease the single-channel amplitude. It is generally assumed that blocking agents penetrate partly into the channel opening, bind to a site and inhibit the flow of the permeant ions. However, interaction of the blocking agent with a site of the transport protein located outside the channel mouth and indirect action on the gating mechanism may also be possible [181]. The effect of CI- channel blockers was recently reviewed by Greger [119] and will, therefore, be treated briefly in this review. The use of blockers in cell-excised membrane patches is summarized in Table II, column 6 and the structural formula of CI- chamM blockers are presented in Fig. 8. The amino-reactive agent 4-acetamido-4'-isothiocyanatostilbane-2,2'-disulfonic acid (SITS) inhibited the high conductance CI- channel (g = 360 pS) in A6 cells [26], and the related compound 4,4'-diisothiocyanatostilbene-2,2 '-disul fonic acid (DIDS) blocked the small (g = 64 pS) as well as the large ( g = 3 6 2 pS) CI- channels in rabbit urinary bladder [24]. Whereas in the latter preparation the kinetics of the block were not reported, in A6 cells Nelson et al. [26] observed an increase of the channel closed time, then induction of flickering and finally irreversible disappearance of the channels after application of 1 mmol/l SITS. However, great care must be taken in the application of these drugs, since they may also affect other transport systems. For example, it is well established that the stilbene disulfonates inhibit anion-exchange systems in red blood cells (reviewed in Ref. 182), in the apical membrane of the gallbladder epithelium [183] and in renal basolaterai membrane vesicles [184]. Moreover, it

SITS so;

DIDS sO~ H

COO-

[~-. -~

Diphenyt daci omine-2(DPC) carboxytci H CO0NO?

5*Nitre-2-(3-phenylpropylamino)-benzoic acid (NPPB)

COOH Anthrocene-9cerboxytic acid

Fig. 8. Chloridechannel blocker.

has been demonstrated that SITS inhibits file HCO~- exit step in renal proximal tubules [ii6,ii7]. Stilben~ disulfonates may also affect other systems than anion transporters. Recently, it was observed that these substances activate nonselective cation channels in the basolateral membrane of the rat exocrine pancreas [185]. However, these unexpected effects were only observed when the drugs were apphed to the cytosohc side of cell-excised membrane patches. Another class of CI- channel blockers applied to epithelia are diphenylamine-2-earboxylate (DPC) and related compounds. These novel types of CI- channel blockers have been studied extensively in isolated perfused segments of the rabbit thick ascending limb of Henle's loop [36,37]. The effects of these substances on single CI- channels were investigated in human and canine tracheal cells. Welsh [87] reported that 1 mmol/l DPC decreased the single-channel amplitude of tracheal C l - channels without affecting its kinetic properties. This implies that DPC probably acts as a fast blocker on this channel. The DPC analogue 5n i t r o - 2 - ( 3 - p h e n y l p r o p y l a m i n o ) b e n z o i c acid (NPPB) was tested on single CI- channels of the rectal gland [75], of the human colon carcinoma cells HT29 [44], and of the trachea [85]. Pro-

544

0-

.50 n Fig. 9. Effect of 5-nltro-2-(3-phenylpropylamino)benzoieacid (NPPB) on CI- channels in HT29 cells. 0 -* denotes the closed state of the channel. Note the induction of brief closing events (flickering)by the compound (according to Ref. 187).

nounced effects were observed at concentrations o f about 10 /tmol/l. As demonstrated in Fig. 9, this compound induced fast flickering of C I channels in HT29 cells and, therefore, it can be classified as an intermediate blocker in this preparation. It is interesting to note that N P P B inhibited only the larger c o n d u c t a n ~ channel ( g - 45 pS) in the rectal gland but had nc effect on the smaller channel ( g = 11 pS) [75,77]. The specificity of these blocking agents, with respect to other channel types, has still to be determined. Recently, we could demonstrate that DPC, NPPB and, even more so, 3'5-dichlorodiphenylamine-2-carboxylic acid inhibited nonselective cation channels of the rat exocrine pancreas [1851. Anthracene-9-earboxylic acid (9-AC) (Fig. 8) inhibits the chloride conductance in the apical membrane of the canine tracheal epithelium [84]. With the patch-clamp techni:lue, Welsh [87] observed that 4 m.m.ol/l of 9-AC decreased the ..,i,~l~-.,ha,nel toiidiict~tK~ of C I - channels in this tissue to 68%. A similar decrease of the singlechannel conductance was obse,wed with I m m o l / l DPC. These doses are high compared to the effects of NPPB, which inhibits single C I - channel conductance completely at a concentration o f 0.1 m m o l / l in the same tissue [85]. In whole cell recordings of the rat lacrimal gland, it was shown that 1 m m o l / l furosemide and 0.4 m m o l / l bumetanide inhibited C I - channels [1061. These drugs are known to inhibit the N a + / 2 C I - / K + cotransport system, observed in a

number of tissues [186]. However, much lower concentrations of furosemide and bumetanide are needed to block the cotransporter than those used for the C I - channel in the lacrimal gland. Therefore, there is doubt as to whether the observed effects of these drugs o n the C I - conductance in lacrimal glands are specific. Recently, we observed that 1 m m o l / 1 furosemide inhibits nonselective cation channels in the rat exocrine pancreas (own unpublished results). In summary, SITS, DIDS, 9-AC, D P C and N P P B have been tested thus far as C I - channel blockers in single-channel experiments. Detailed studies of the kinetics o f blocker action are not available. Substances, comparable to the action of amiloride on epithelial N a + channels or of Ba 2+ or tetraethylammonium on K + channels are, unfortunately, lacking for epithelial (21- channels. Acknowledgements I am deeply indebted to Professor Dr. K.J. UUrich and Professor Dr. R. Greger for their readin s the mare,script and for the many stimulating discussions. I would like to thank Dr. C.M. Fuller for revising the English. References

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