Channels activated by stretch in neurons of a helix snail.

Channels activated by stretch in neurons of a helix snail ELAINEB ~ E I P ~AND R H )CATHERINE E. MORWHS~

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 07/27/12 For personal use only.

B i s b g y Bepamnenb, University of Ottuwa, Otfu~la,Ont., Canada K I N 6N5 Received June 14, 1991 BBIIARD, E., and MORRIS, C. E. 1992. Channels activated by stretch in neurons of a helix snail. Can. 1. Physiol. Phrrnacol. 70: 207-213. Single-channel recordings from central neurons of the helix snail. Cepuea rremsra8ks, revealed two types s f channels that could be activated by stretch (i.e., by the membrane deformation produced when suction is applied to the patch pipette). One, a K" channel (58 pS in physiological solution), was evident in excised and cell-attached patches. Its conductance in symmetrical [K'] solutions indicated a channel of high K + permeability (P, = 3.4 x 10- l 3 cm/s). Though osmoregulation has been suggested as a function for such channels, comparisons among mslluscs indicate osmotic milieu does not govern their expression; Cqaeca is terrestrial, and stretch-activated K+ channels similar to those described here occur in aquatic and marine molluscs. The second type of channel, observed only in excised patches, was C1- permeant: it had a large conductance (130 pS) and was inactive prior to patch excision. Membrane tension may not be the physiological activator of either the R' or Cl-- channel; the channels are designated as stretch-activated channels on the basis of their experimental behaviour during single-channel recording. Key tvords : # channel, CE- channel, snail neuron, stretch activation. +

B%DARD, E., et MORRIS, C. E. 1992. Channels activated by stretch in neurons of a helix snail. Can. J . Physiol. Pharmacol. 70 : 207-213. Des enregistrernents de canaux individuels de neurones centraux de l'escargot Cepaea nernnrnll,~ont rCv6lC deux types de canaux qui pourraient &re activks par l'dtirement (c.-h-d. par la dkformation de la membrane produite lorsqu'une succion est appliqude a la pipette en contact avec la portion de membrane). Ee premier canal, un canal K + (58 pS dans une solution physislogique) a CtC observC dans les portions likes aux cellules et dans les portions exeisCes. Sa conductance dans des solutions [K'] symCtriques a indiquC qu'il s'agissait d'un canal trks permkable au K' (P, = 3,4 x 10-'%rn/s). Il a dCjh 6tC suggCrC que de tels canaux pourraient jouer un r6le dans l'osmoregulation; toutefois des comparaisons entre mollusques indiquent que le milieu osmotique ne dicte pas leur expression; C c p ~ e aest un animal terrestre, et I'on retrouve des canaux K + activCs par l'etirement, similaires & ceuw dkcrits ici, chez les mollusques marins et aquatiques. Ee second type de canal, sbservC seulement dans les portions excisCes, Ctait permCable au GE-,avait une forte conductance (130 pS) et etait inactif avant l'excision de la portion. La tension membranaire pourrait ne pas Ctre I'activateur physiologique des canaux CI- ou K9;les canaux ssnt qualifies de canaux actives par 1'Ctirernent d'aprks leur eomportement expdrimental durant un enregistrement de canaux individuels. M ~ t sc!&s : canal K + , canal CB-, neusone d'escargot, activation par B9Ctirement. [Traduit par la rddaction]

lntr~ductisn Stretch-activated (SA) channels akin to those first discovered in vertebrate skeletal ( G and Sachs ~ 1984) ~ are ~ nonselective catioa (SACat) channels. SA channels in molluscan cells, i.e., heart cells and neurons of the pond snail (LymMea stagnajis) (Brezden et al. 1986; Sigurdson et 198Tb; sigurdson and ~~~~i~ 1989a), are broadly similar to s*cat channels (see Morris (1990) and Sachs (1988, 1990) regarding membrane density, voltage sensitivity, and kinetics) but are K f selective and hence are termed SAK channels. Having initially observed SAK channels in an aquatic organism, we wondered if they represented specialized machinery related to the ssmoregulatory demands sf a dilute milieu. In addressing this question, we used a comparative approach, looking for SAK channels in marine and terrestrial as well as aquatic gastropod molluscs. The Helicideae are land snails often used as neurophysiological models; a locally available helix, C q a e a nernomlis, was used in this study. We show that Cepwea neurons, like those of pond snails (Lymnaea) and of the marine gastropod Apkysia (Vandorpe and Morris 1991) possess SAK channels. Recent findings from the latter two species (Morris and Horn 1991; Vandorpe and Morris 1992) suggest that questions about 'Comespndence may be sent to the author at the following address: Neurosciences, Ewb Institute, Ottawa Civic Hospital, 1053 Garling Are., Ottawa, Ont., Canada KIY 4E9. Printed in Canada 1 Imprime au Canada

ssmoxnsing by molluscan SA channels may be moot; normally the channel; may be controlled not through tension but through receptor-mediated processes. Even if molluscan SA channels ~ ~ ~ "O' 'espond primarily to cellular mechanics*they are abundant in the membrane and a better understanding of their special traits is warranted. Stretch represents a convenient way to reved a class of channels that might otherwise be silent until their regulators were While the SAK channel. we also noted and briefly characterized a type of "-

Methods Ce66 preparation Cepaea stemomlis were obtained from Boreal (St. Catharines. Ont .), housed in a terrarium at 20-24"C, and fed romaine lettuce. Active animals were used for preparing cells for recording. Circumoesspkageal ganglia were agitated in 0.25% Sigma type XIV protease in normal Cepaen saline (NCS; Trams et al. 19665; see Table 1) for 1 h and then transferred to NCS plus antibiotic (Table 1). Sheath remnants were disrupted and cells were dispersed with fine needles on a poly-L-Iysine coated glass coverslip. Neurons (diameter, 30-80 pm) were maintained at room temperature (20-24°C) for 1-3 days before use.

Experirnentab procedures Recording conditions were similar to those used previously (Sigurdson et al. l987b); positive or negative pressure was applied to the patched membrane with a tmnsducer (model DPM-1; Bics-Tek,

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CAN. J . PHYSIOL. PHARMACOL. VOL. 70, 1992

Burlington, Vt.) connected to a pipette holder via a three-way valve (for pressure application or equilibration to atmospheric pressure). SyBgard-coated thick-walled (Corning 7052 glass was u s d for pipettes; they were 10- 13 MQ after fire polishing. A List EPC-7 amplifier's output was Bessel filtered (usually 2 kHz) and recorded on FM tape. For display, records were replayed to a digital storage sscilloscope connected to an XY plotter. For cell-attached patches, the membrane potential, Vm,was calculated as -50 nlV (an approxirnation of V,,,, the resting potential) minus the applied pipette potential (V,), and for inside-out patches, Vm = -5. Experinaents were done at room temperature (28 -24°C).


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A~la!y~is of data For current -voltage (I-- r/) rehtions, records were replayed to a storage oscilloscope and amplitudes of r 5 fully resolved singlechannel events per potential were measured. Stretch sensitivity was confirmed at each potential. Single-channel conductance (g) was obtained from linear regression of I-V data aver near-ohmic ranges; data from separate patches were not lumped. Membrane tension was varied using monotonically increasing steps of suction ( - I n~iraeach). As described previously (Sigurdson et al. 198'78), an index of the open probability (pope,,)was determined from the time-averaged current over the single-channel current. An index, not a true probability, is reported because the number of ehannels in the patch was not necessarily known; the largest index go,, value is a minimum estimate of channel number. A one-channel recording was obtained, from which time constants were extracted as before (Sigurdson et al. 1987b); records (filtered at 4 kHz) at two pressures were digitized at 40 kHz. Throughout, means are given with standard deviation and sample size (which, unless noted, refers to the number of different patches). Significance (5% level) of differences between means was determined with a a-test.

their mechanosensitivity , soluble cytoplasmic molecules and deep cytoskeletal elements were not implicated. The nonlinear 1- V curves (e. g ., Fig. 2, circles) for excised patches (70KCS in the pipette, NCS in the bath) had extrapolated zero-current potentials at $6 k 5 rnv (n = 41, close to the Nernst prediction (72 mV) for K+, confirming the channel's selectivity for K9 over Na+ and C1- (EN,and Eel were -44 and O mV, respectively) . Under only limited conditions did SAK current behave as if K+ transit were governed simply by the K+ electrochemical driving force (i.e., according to the Goldman, HoQgkin, Katz (GHK) current equation with K9 as the sole permeant species, e.g., Benham et al. 1986) (Fig. 2). Using the "limiting conductance" (BK/V) for symmetrical [K'], we solved for the single-channel permeability coefficient, PK (cmis). Ideally, IK(V)/V = limiting g a constant. In practice, even with symmetrical solutions (7OK, Table I), some rectification occurred (Fig. 2, squares), so limiting g (92 3 pS (n = 3)) was taken from the steepest linear region (inward current), yielding PK = 3.4 x 18-l%cm/s. Inward current with O mM Na+ at the inner face (Na9 substituted by choline) fell on the curve predicted by the GHK equation (Fig. 2, triangles), but with internal Na9 present (Fig. 2, circles), inward current was diminished except near the extremes of driving force (i.e., near Ke,, the reversal potential, 42 mV, md, at the other extreme, as the limiting g for [KS],,, was approached). If the "fast?' (i.e., nonresolvable) nongermeant block of the SAK channel lumen occurred at an inward-facing Naf-binding site (Yellen 1987), the inward M currents at intermediate potentials (e.g ., around -38 mV) would be reduced and $/,,,,,, should ,B be unaffected, consistent with tthe I-V relations with and without Hntracellarlar Na+ in Fig. 2 (circles vs. triangles). Relief of Na+ block with hyperpolarization would explain the observed redress of the single-channel conductance at hyperpolarized potentials. SAK channel conductance did not increase linearly with [K+loUt.Cell-attached recordings with [K+lPi,, at 4, 40, 70, and 158 mM (see Table 1) gave g = Q pS (i.e., undetectable), 71 -k 10pS (n = 6), 76 f 13 pS (n = 8), and 81 $ 5 pS (n 5): respectively; g was evidently saturated by 70 rnM M9 and reached half saturation between 0 and 48 mM. At pipette concentrations of 1 mM, the K + channel blockers, tetraethylammonium (TEA) (see Hille 1984) and 4-aminopyridine (Meves and Pichon 1977) did not detectably alter SAK channel currents. Qarinidine at 1 mM, however, increased the flickeriness and reduced the amplitude of SAK channel currents approximately fourfold. These aspects of the Cepaea SAK channel (lscal anaesthetic-like block by quinidine and lack s f efkct of f rnM TEA and 4-arninopyridine) are consistent with observations on Lymnaea heart SAK channels (Brezden et al. 1986: Sigurdson 1990).

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Results SAK channels7 near-phy.riodogicad conditiot?s In cell-attached recordings with NCS in the pipette, stretch always elicited outward currents in patches depolarized from k/,. SA currents were never seen at Vr',,,, or at hyperpolarized potentials. The I-V relation (Fig, 1) for the SA events was approximately linear from -40 to 40 mV, with g = 58 k 3.7 pS (n = 5). Linear extrapolation of the foot yields a zerocurrent potential ( -55 f 3.6 mV, pa = 5 ) close to the average intracellularly measured V&,,,,of Cepcaea neurons (-50 mV; Trams et al. 1965). EKand EcI both fall near Kc,,; to determine which ion carried the SA current, [K+lpipettewas increased from 4 to 70 mM while maintaining [Cl-Ipipetae constant (70KCS, Table I). This substHtution caused I-V curves to shift to the right and reverse near O mV as expected for a largely K+-selective channel (Fig. 1). Substitution of 70 mM KC1 (in 7OKCS) by M+ salts of acetate or gluconate (anions that showed no permeability through Cepaea Clchannels, see below) did not affect SA currents. It is concluded that these SA events, like those in Lyn~naeacells (Sigurdssn et al. B987b; Sigurdson and Morris 1989a), reflect the activity of a SAK chagZI%e1. SAK channel permeation properties Additional properties sf the SAK channel were evident from studies on inside-out patches. With NCS in the pipette (and bath), stretch elicited outward currents in cell-attached patches, but $A currents were not evident following excision (symmetrical NCS). This was not a consequence of excision per se but o f the low concentration of permeant ions. With elevated [K+] (see solutions in Table 1) in the pipette and NCS in the bath, patches exhibited %Acurrents whether cell attacked or excised. Since, in excised patches, channels retained

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1szsensitiplit-y ~f SAK channels 80 tons and membrane potential The Cepaea SAK channel was essentially insensitive to Ca2+. With permeant ions ( i s . , K +) available on at least one side, SA currents were elicited equally well for any permutation of intra- or extra-cellular exposure to 10 rnM Ca2+ (XCS) or Bow pea2+] saline (LaCaCS, see Table 1; note also that the 7QM solution has nominally O mM @a2+).SA events could be elicited with zero internal Na+ (CCS, 70K), indicating that tthe channel is unlike those K + channels whose activity is governed by internal Na+ (e.g,, Luk and Carmeliet 1990).


BEDARD AND MORRIS

FIG. I . H-V relations for SAK channels in examples of cell-attached patches, NCS in the bath. @,I NCS in the pipette; o,70KCS in the pipette. Recordings of stretch-activated events are shown adjacent, with the membrane voltage indicated. The left and right columns are from patches with NCS and 70KCS in the pipette, respectively; scale: 2 pA, 20 ms. TABLEI . Solutions used (mM) Component NaCl KC1 CaC12 w 2 3 2

Hepes Glucose Choline CI pH' 2

70KCSh

40KCS

70K

CCS

80

14

4

70 10 5 5

44 40 10 5 5

70 -

4 10 5

-

-

7.8 (Na)

7.8 (K)

NCS"

10 5 5 10 7.8 (Na)

7.8 (K)

5

5

-

80 7.8 (K)

'Glucose vias inchded for isolating and culturing cells but not for recording. LoCaCS had 0.2 mM Ca2' and slpM EGTA. '~SUKCSwas similar, but had 0 m M Na61 and 150 m M KCI. 'pH of solutions was adjusted using 1 M NaOH or KOH as indicated in parentheses.

On each of the 10 patches, depolarizatiebn beyond 4- 120 rnV increased SAK channel activity. Strong depolarization also promoted Lymnaea neuron SAK channel activity (Sigurdson and Morris 1989b). In both species, the effect is of ghysiologic d interest only in a negative sense: SAK channel activity should be insensitive to voltage excursions within the physioIsgicd range. Sensitivity offhe %AK chrane&to stretch Over the physiologicd voltage range, Cepaea neuron SAK channels were typically quiescent without suction. Suction that increased pop, altered neither g nor F.;,,,,,,l (I-V relations of spontaneous and suction-evoked events in a given patch were indistinguishable); the permeation pathway is apparently unaffected by membrane stress. Negative (Fig. 3) or positive pressure (not shown) increased the channel p,,, for the duration of the applied stimulus. p,,,, subsided to near-zero immediately on release of pressure. Sometimes, a small degree of activation persisted for several more seconds. At very Isw and saturating pressures, the number of simultaneously open channels varied less than at intermediate pressures

(i .e . , near half-maximal stimuli). Maximal variance in the region of hdf-maximal activation reflects the stochastic nature of the mechanosensitive channel gating; variance (current fluctuations) of an ideal (two state) channel peaks at popen = 8.5, whereas at maximal pOF,, variance is zero. The traces retained a high frequency noise component at saturating stretch, indicating that as with Ljmnaea and other SA channels (GtaEaaray and Sachs 1984; Sigurdssn et al. 19818b; see also Rarnirez et al. 1989), the Cepaea neuron SAK channel makes stretch-insensitive as well as stretch-sensitive transitions. Saturating pressures typically yielded current from seven to eight channels. The rneswaPrane area associated with these channels can be estimated using S a h a n n and Neher's (1983) correlation sf patch area (visual measurements) and pipette resistance. Given our pipettes and the reduced conductivity of our s01utions, we estimate a patch area on the order of 5 pn2, corresponding to a channel density of 1-2 pn-*, in keeping with other SA channels (Morris 1990). pop,, versus pressure relations for the Cepaea SAK charnel were steeply sigmoidal (Fig. 3, graph). In practice, the steep activation region could be located during a cursory check


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0 F ~ G2.. GHK analysis of SAK channel currents. 1-Y relations for inside-out patches with 40K in the pipette. L, 7OK in the bath (straight line corresponds to a conductance of 92 pS); o , NCS in the bath (zero-current potential is near 70 mV). A, CCS (a zero Na+, chsline-substituted NCS, see Table 1) in the bath (the overlying curve is predicted by GHK equation 2, using 3.4 >( cm/s for P,; the predicted zero-current potential is 72 mV). ( r and o represent means of four patches). Inset: %/, = -30 mV, with NCS (top trace) and CCS (bottom trace) in the bath. Scale: 4 pA, 40 ms.

immediately after obtaining a seal (e.g ., Fig. 3, trace), even if input-output was not characterized in detail. Most patches activated below -60 m H g (half saturation was -55 f 10 mmHg for five patches in whish input-output was quantified) (1 mmHg = 233.322 Pa), but some were more recalcitrant. These high-threshold patches were obtained when seals formed spontaneously (i.e., with no suction) and showed half saturation at - 100 f 5 mmHg (three quantified patches). Perhaps suction engenders greater disruption at the membrane cortical cytoplasm interface, thereby increasing channel stretch sensitivity (Morris and Horn 1991). A recording was obtained with and without stretch from a patch with at most one channel open. Single open time and triple classed time distributions were observed (Fig. 4); stretch altered pop,,, not the number of kinetic states. As with Lyrnraaea SA and stretch-inactivated channels (Sigurdson et aB. 1987b; h4orris and Sigurdson 1989)p stretch seems to alter pawn largely via the rate s f leaving the Bong closed state between bursts.

SACl channel in excised patches of Cepaea neurons While studying SAK channels in excised patches, we noted that suction frequently ( 25 % of excised patches) triggered activity 0%a large conductance channel distinguishable from the SAK channels by the foIlowing criteria: large currents flowed under conditions of near-zero electrochemical driving forces for K + (e.g., symmetrical NCS), flickery (i.e., in the millisecond range) transitions were rare, md the modd (i.e., most common) conductance was interrupted by a multiplicity s f other levels (Figs. 5 and 6). With 114 mM C11- in the bath (NCS) and 70 rnM C1- (TOK) in the pipette, inward and outward currents (Fig. 5) that reversed at Ecl (+ 12 mV) were seen. When glucoraate and acetate were the pipette anions, outward currents were absent, though, in the same patches, bath Cl- carried inward current through SAC1 channels. Unlike the SAK channel, whose pop,, changed incrementally with increasing stretch, SAC1 channel activation was all or none. SAC1 currents were never observed in the

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t

I

-80

-4 8

I

-8 20

Pressure (rnrnHg) FIG. 3. The effect of stretch on the open probability of SAK channels. Upper left: an example of a quick test of stretch sensitivity (cell-attached, 70KCS in the pipette, k.', = -40 mV, about -80 mmHg applied at the bar, scale 2 pA, 200 ms). The graph, in which different syrnboIs denote different patches, illustrates three cases in which the effect of suction saturated and two in which the patch ruptured prior to saturation. As described in the Methods, values s f the index go,, > 1 indicates > 1 SAK channel in the pipette.

Q

1

2

3

4

Open time (ms)

FIG.4. Open time histogram. Pipette suction, -90 mrnHg, I/,, = 0 mV, SQKCSin the pipette, cell-attached. The solid line is the best fit to a single exponential (g,,, = 0.39 vs. 0.04 at 0 rnrnHg). Inset: examples of events at 0 mmHg (top two traces) and -90 mmHg (bottom trace). Scale 2 pA, 10 ms. Kinetic constants in milliseconds (f errors from 95% confidence limits on the fits): (i) 0 mmHg: Topen, 0-81(f 0.013); TC,, 0.14(f 0.857); rcz, 0.496k0.093): 7 ~ 3 , 84.2(&5.2); (ii) -90 mmHg: T,,,,, 8.57(+0.05$); T ~ , 84.06(+0.@07); T,,, 0.28(+0.029); TC,, 1.42(f 0.038).

cell-attached configuration, though suction was always applied before and aker excision. This suggests that disturbances which accompany excision (e.g., chaotrogic disruption of cytoskeletsn by CI- (Baurngald et al. H981), changes in ligand and enzyme availability) may promote the SAC1 channel's stretch sensitivity. Spontaneous activity (i.e., events not stimulated by stretch) of the SAC1 channels did not occur during times (often several minutes) before and between stimuli, nor was activity induced by voltage changes.

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BEDARD AND MORRIS

FIG. 5. SACl channel currents from an inside-out patch. Pipette solution, 7OK, bath solution, NCS; a constant suction s f -35 mmHg used to activate channels; membrane potential as indicated. The dotted line indicates the zero-current Bevel and arrows denote the modal (130 pS) level. Scale: 4 pA, 10 s.

Suction generally activated the Cl- channel to the modal conductance level (130 pS), in which it spent 93 5 % (n = 3) of its open time. Whether the modal state is the highest conductance state was not clear. Suction sometimes induced brief 268-pS events. These may represent a second independent channel, an additional "barrel" in a mltibarrel channel (Miller and Richard 1990) or the highest conductance state of a unitary permeation pathway. When the channel was in a given state, further brief ( < % s) suction sometimes activated one or several other states (Fig. 6 ) ; these usually disappeared immediately at the cessation of suction, and the channel returned to its initial level where, as described below, it tended to remain active for about 10 s. Whatever the hndamental unit of this anion conductance mechanism, conductances >260 pS were not induced by suction. The threshold for activating the SAC1 conductance, using stimuli of II s duration, was -3% to -40 rnanhfg. Applied 22 times to a quiescent patch, stimuli of -40 mmHg activated a channel 83% of the time. Upon suction release, the time course of inactivation (measured as p,,,,,, the conditional probability of the channel being open, given that it was open at e = 0 (time of suction release)) had an exponential decay constant 12.5 s (log-linear regression coefficient, 0.98; the zero-time p i n t , with its predetermined value of 1, was excluded). Once activated by transient stretch, the channel showed occasional transitions among various open substates and short-lived closed states. The decay constant refers, therefore, to the channel's sojourn in a set of active states, in the absence of applied stretch. Upon inactivating, SAC1 channels were not refractory to mechanical stimuli (as might be expected if inactivation were due to disassembly of multimers); stretch reapplied within seconds of inactivation reactivated the channel (e.g ., Fig. 6 ) . SACl channel behaviour is consistent

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FIG.6. SAC1 channel behaviour during transiently applied suction and following suction release. Five inside-out patches with pipette solution, 38K,bath solution, NCS; pressures in the range of -35 to -50 mmHg were applied during times indicated by bars; membrane potentials were (a) 70 mV, (b) 30 mV, (c) 58 mV, (4 30 mV, and (e) 48 mV; the zero-current is indicated by the dotted line. Scale: 2 pA (d) or 4 pA (u,b,c,e);1 s (a,c,d,e) or 2 s (b).

with a large free energy difference between the inactive state(s) and a transition state(s) (hence the lack of spontaneous activation) and a smaller, but not inconsequential, free energy difference between the open states and the transition statebs) (hence the large decay constant). Though our observations indicate that the transition inactive -- active is mechanosensitive, we do not h o w if the reverse transition is also mechanosensitive. Comparison o j the SAK chkenrsel with other invertebrate SA channels The SAK channels of the helix snail Cepaea (terrestrial pulmonate; suborder Stylomrnatophora) closely resembled those of the aquatic pulmonate Lj~mnaea(suborder Bassrnrnatophsra) (Sigurdson et al. 198'7b; Sigurdson and Morris 1989w), suggesting that the channels are homologo~rs. As well as having stretch-sensitive open probabilities, both are highly selective for K + over Na+, both have moderately high conductances under physiological conditions (58 pS for Cepaea; 30 -45 pS for Lj~rnnerea,whose fluids are more dilute (Sigurdson 1990)), both are stretch sensitive via a stretch-induced decrease in a long closed state, both are blocked by I mM quinidine but are insensitive to I rnM extracellular TEA and 4-AP, both occur at a density on the order of 1 pM-2, both are insensitive to voltage in the physiological range, and both are present in virtually all central. neurons examined. Neurons of Cepaea like those of Lj~rnnae~ and Aplysia (Vandorpe and Morris 1991) did not exhibit SACat channels. All three species, however, possess SAK channels. In idewtified Apkysia neurons, we have shown that the SAK channels and the serotonin-modulated channel or '%-channel9' are the same entity (Vandorpe and Morris 1992).


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CAN. S. PHYSIBL. PHARMACOL. VBL. 70, 1992

In embryonic and larval Brosopha'la muscle, channels very like the molluscan SAK channels have been described; the insect SAK channels represent the largest (in terms of conductance and abundance) sf four classes of K+ channel, with a sonductance sf 90 pS in 140 rnM K + (Zagotta et al. 1988). NO function has yet been ascribed to these channels, Leech central neurons exhibit SACat channels of unknown function (Pellegrino et al. B 990). Crustacean stretch receptor neurons have two types of SACat channels (Erxleben 1989). The one that gates at lower pressures (half maximal, < 15 mmHg suction; lower than the Cepaea SAK channel, but in the same range as the Lymnaea stretch-inactivated channel (Morris and Sigurdson 1989)) is postulated to underly the cell's mechanosensitivity (Erxleben 1989), though this remains to be proven (see Morris 1990).

Peraneation characteristics of thr SAK channel Current and s,1,6'. data under various conditions indicated that, of the normally available ions, only K+ permeated the Cepaea SAK channel to an appreciable degree. The driving force for K + was not, however, the sole determinant sf permeation. Conductance was not linear with [K+],, and sublinearites not explained by GWK electrodiffbasion occurred in the 1-V relations, suggesting that ion binding occurs in the permeation pathway. The SAK chanxael conductance did not change when [K+] was increased from 70 mM (zipproximately the cytoplasmic concentration) to 150 mM, suggesting that the channel operates at maximal conductance when carrying outward current. Its PK was comparable to that of a s a vertebrate maxi-K channel (e.g., 4.5 >( 1 0 - 8 ~ c m / for Ca2+-activated K+ channel; Benham et al. 1986). The Lymnaea SAK channel PI( is also in this range (4.9 x 10- l 3 cm/s (Sigurdson 1998). Function of SAK channels Neither their phyletic nor cell-type distribution provide clues linking SWK ckannels to discrete functions; versions of SAK channels have been found in fish embryos (Medina and Bregestovski 1988), cochlear hair cells (Li et aH. 1991), amphibian kidney (Sackin 1989), and smooth muscle (Ordway et al. 1990). The channels probably serve diverse roles. While it is often assumed that the functions of %Achannels relate to rnechanotransgiucth, there is little evidence at the cellular level to support this view (Morris and Horn 1991). We have not demonstrated to our satisfaction that nxsrnbsane tension activates SA channels in a physiological context (Morris et al. 1989; Morris and Horn 1991; Morris and Moore 1992; Steffensen et al. 1991). Moreover, we have found that a known receptor-mediated M+ channel in Aplysia neurons (the S-channel) whose neurornodulatory role is perfecly explicable without recourse to stretch activation can be activated by stretch under single-channel recording conditions (Vandorpe and Morris 1992). Though preliminary findings (e.g ., Li et al. 1991 ;Tseng 1991 ; Ghazi et al. 1991; Yang et d.1991) suggest that for some SA channels physiological roles may exist, they have yet to be substantiated (Morris 1992). Nonetheless, stretch is a useful diagnostic, enabling one to reveal otherwise quiescent channels. We are presently asking whether molluscan SAK channels other than that in Aplysia sensory neurons are receptor-n~ediatedchannels. It is noteworthy that second messenger or receptor-mediated channels susceptible to stretch have recently been found in teleost embryos (Medina and Bregestovski 1991) and mammalian hepatoma sells (Bear and Li 1991).

SAC[ channels are qualitativ~6yHdflerent fr~nzSAK channels Cepaea SAC1 channels responded differently from SAK channels to pipette suction. Activity induced by a brief ( - B s) suprathreshold stimulus persisted for about 10 s. In contrast with the all-or-none onset -offset behaviour of %A@]channels, SAK channel activity was graded with the stimulus intensity an$ Po,, abated immediately upon removal of the stimulus. If the mechanosensitivity s f the SAC1 channel has some physiological relevance, it is not likely to be for a process demanding fine or rapid control. Since the Cpiaea SACl channel was activated by suction only after patch excision, it may be functioning abnormally. An extreme possibility is that it is not a channel at all, but a "crippled" carrier with malfunctioning (i.e., chronically open) cytoplasmic-side gates. A comparable proposition has been made for a channel from a yeast H + ATBase mutant (Rarnirez et al. 1989). The Cepaea Cl- channels may be related to those described in Eyntnaea neurons by Geletyuk and Kazachenko (1985) (200 pS maximum, multiple ccsndmctance, seen only in excised patches, erratic activation pattern). This channel was also seen in our hands in Lymsaaea (BCdard et al. 1988); unlike that in C ~ p a e a it, was not reproducibly stretch sensitive. In a preliminary report on large (multi)conductance lymphocyte Cl - channels, Pahapill and Schlichter (1990) report that the bizarre behaviour characterizing the channels under non physiological conditions (excised patch, room temperature) disappears when channeHs are studied cellattached at physiological temperature. In the absence of evidence about their physislogica% roles, multiconductance Cl- channels in excised patches (see also Krouse et al. 1986) are perhaps best regarded as 'disruption-induced. ' ' It has not been established, and ought not to be assumed, that mechanical stimuli normally gate SACl and SAM cham nels. Stretch does, however, serve to reveal their presence. Given their potential for carrying substantial currents when activated, the channels deserve further attention.

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Stretch-activated cation channels with large unitary conductance in leech central neurons.

Characterization of stretch-activated ion channels in Xenopus oocytes.

Stretch- and volume-activated channels in isolated proximal tubule cells.

Stretch-activated channels in human retinal Muller cells.

Stretch-activated composite ion channels in Bacillus subtilis.

Hypoglycemia-activated K+ channels in hippocampal neurons.

Lead ions close steady-state sodium channels in Helix neurons.

Piezo1-dependent stretch-activated channels are inhibited by Polycystin-2 in renal tubular epithelial cells.

Eucalyptol induces hyperexcitability and epileptiform activity in snail neurons by inhibiting potassium channels.

Distribution of alpha CDCP-immunoreactive neurons in the central nervous system of the snail Helix aspersa.

Actions of APGW-amide and GW-amide on identified central neurons of the snail, Helix aspersa.

Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons.

Stretch-activated channels in single early distal tubule cells of the frog.

Stretch-activated non-selective cation channels in the antiluminal membrane of porcine cerebral capillaries.

Parathyroid hormone activation of stretch-activated cation channels in osteosarcoma cells (UMR-106.01).

GABA-gated anion channels in intact crayfish opener muscle fibres and stretch-receptor neurons are neither activated nor desensitized by glutamate.

Gadolinium ion inhibits loss of metabolites induced by osmotic shock and large stretch-activated channels in bacteria.

Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells.

Calcium-activated SK channels control firing regularity by modulating sodium channel availability in midbrain dopamine neurons.

NSAIDs modulate GABA-activated currents via Ca2+-activated Cl- channels in rat dorsal root ganglion neurons.

Sensitivity of stretch-activated K+ channels changes during cell-cleavage cycle and may be regulated by cAMP-dependent protein kinase.

Transmembrane proteoglycans control stretch-activated channels to set cytosolic calcium levels.

Divalent cation effects on lens conductance and stretch-activated cation channels.

Degradation of barley glucan by a purified (1yields4)-beta-d-glucanase from the snail, Helix pomatia.