Proc. Nati. Acad. Sci. USAVol. 83, pp. 7741-7745, October 1986Biophysics

Single calcium channels in native sarcoplasmic reticulummembranes from skeletal muscle

(Ca2l release/excitation-contraction coupling/patch clamp)

BENJAMIN A. SUAREZISLA*tt, CARLOS OROZCO*, PHILLIP F. HELLER§, AND JEFFREY P. FROEHLICH§*Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892; and §Laboratory of Biological Chemistry,Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224

Communicated by Joseph F. Hoffman, June 13, 1986

ABSTRACT Electrical properties of native sarcoplasmicreticulum membranes from rabbit skeletal muscle were inves-tigated using the patch-clamp technique. Bilayers were assem-bled at the tip of patch pipettes from monolayers formed at theair-water interface of sarcoplasmic reticulum membrane sus-pensions. The membranes were found to contain a spontane-ously active cation channel of small conductance (5 pS in 200mM CaC12, symmetrical solutions) that was selective for Ca2+and Ba2+. Between 50 and 200 mM CaCl2 (symmetrical) theincrease in conductance as a function of [Ca2+J fit a hyperbola(KO.5, 83 mM, and y.., 7.9 pS) that extrapolated to asingle-channel conductance of 0.5 pS at physiological Ca2+levels. The channel opened in bursts followed by long silentperiods ofup to a minute. During a burst the channel fluctuatedvery rapidly with time constants in the millisecond range. Themean burst duration was voltage dependent, increasing from1.8 s at a pipette voltage of +60 mV to 4.1 s at +80 mV. Overthis range, burst frequency decreased with increasing voltagesuch that the fraction of time spent in the open state (fb)remained constant. Application of 1.6 mM caffeine resulted inactivation of the channel that appeared as an increase in meanburst duration. In contrast, 50 jIM dantrolene significantlydecreased burst frequency, whereas 10 ,uM nitrendipine had noeffect. The functional and pharmacological properties of thisCa2+ channel suggest that it may be important in mediatingCa2+ release from the sarcoplasmic reticulum during excita-tion-contraction coupling.

Transient depolarization of the transverse tubular systemduring excitation of skeletal muscle elicits a rapid release ofCa2" from the sarcoplasmic reticulum (SR) into themyoplasm that initiates contraction (1). Although the mech-anism of Ca2+ release during excitation has been postulatedto involve activation of a Ca2+ channel (2-5), experimentalsupport for this hypothesis has been difficult to obtain partlybecause of the inaccessibility of the SR to direct electricalrecording and because of the difficulty in producing Ca2+release in isolated SR preparations at rates compatible within vivo function (6). Investigation of this problem has beengreatly facilitated by the development of the patch-clamptechnique (7) that has allowed direct recording of the single-channel currents in cell membranes and in bilayers assembledat the tip of patch pipettes (8-10). In the present study, weused the latter approach (known as the "tip-dip" method) toinvestigate the behavior of ionic currents in native SRmembranes prepared from rabbit white skeletal muscle. Wepresent here direct evidence for the presence of a channel inthese membranes that appears to be capable of sustaining theCa2+ fluxes necessary for activation of the myofilaments invivo.

METHODS

Membrane Preparation. SR vesicles were isolated fromrabbit white skeletal muscle according to a described method(11) and washed several times in 10 mM Tris/Hepes buffer,pH 7.0, to reduce the level of K+ contamination (<50 ,uM asdetermined by atomic absorption spectrophotometry) andeliminate charge movement through K+-selective channels(12). The washed membrane vesicles were suspended in thesame buffer and used immediately or frozen in liquid nitrogenand stored at -80°C. Separation of the SR vesicles into light,intermediate, and heavy components was achieved by sub-jecting them to isopycnic zonal centrifugation on a continu-ous 20-50% (wt/vol) sucrose gradient as described by Gilbertand Meissner (13). Prior to washing, the unfractionated SRexhibited levels of oxalate-facilitated ATP-dependent 45Ca2+uptake (14) that were 30-50 times higher than those found inthe crude muscle homogenate. The extent of contaminationdue to mitochondrial and sarcolemmal proteins, estimatedfrom the level of succinate dehydrogenase and Na,K-ATPaseactivity present in the unfractionated SR and in preparationshighly enriched in these organelles (13, 15, 16), was 1.9% and1.3% of the total microsomal protein, respectively.

Electrophysiological Recording. Gigaseals (10-20 Gohm)were obtained with the tip-dip method (8-10) from themonolayer spontaneously formed at the air-water interfaceof a suspension of the SR membranes. Unless otherwiseindicated, SR vesicles were preincubated for 5 min at 0°C ina medium containing 200 mM CaCl2 and 10 mM Tris/Hepes,pH 7.0, and were added to the bath maintained at 22-24°C ata protein concentration of 2-8 mg/ml. High resistance sealswere formed by using pipettes with a minimum open tipresistance of 10 MQl to ensure the formation of reproducibleseals and stable patches. Drugs were applied either byinjection from a separate pipette with a 30- to 40-,m tipopening held near the patch or by perfusing the chamber withthe appropriate buffers and modifiers.

Single-channel currents were recorded with a patch-clampamplifier equipped with a 50-Gfl feedback resistor in its headstage (EPC-7, List Electronic, Darmstadt, F.R.G.) at alow-pass filter setting of 10 kHz. The output from theamplifier (500 mV/pA) was recorded on FM tape with a DCto 10 kHz bandwidth and later post-filtered at 500 Hz with an8-pole Bessel filter and digitized at 5 kHz with a Nicolet 4094oscilloscope. Analysis of the current amplitudes and dwelltimes in the open or closed states was carried out with a PDP11/23 computer using a double-threshold capture program(17). After filtering, the minimum current step and shortestopen-close transition that could be unequivocally resolvedwere 0.1 pA and 2.5 ms, respectively. Analysis ofthe channel

Abbreviation: SR, sarcoplasmic reticulum.tTo whom requests for reprints should be addressed.tPermanent address: Centro de Estudios Cientificos de Santiago,P. 0. Box 16443, Santiago 9, Chile.

7741

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7742 Biophysics: Suarez-Isla et al.

slow bursting behavior was done by hand from recordsdisplayed on chart paper with a minimum time resolution of100 ms. Measurements were limited to patches that containedapparently only one active channel during recording periodsthat lasted 6-10 min and included between 11 and 50 bursts.Operationally, a burst was defined as any period of seeminglycontinuous channel activity separated from others by a gap atleast five times longer than the longest closure within any ofthem (18).

RESULTSSingle-Channel Current Measurements. Representative ex-

amples of patch-clamp recordings of single-channel currentfluctuations in unfractionated native SR membranes obtainedin 200 mM symmetric Ca2l and 10 mM Tris/Hepes, pH 7.0,are shown in Fig. LA. The channels, which were observed in98% of the high-resistance seals that lasted more than 2 min,opened spontaneously and remained active for periods up to20-30 min. Current fluctuations were only seen with positivevoltages applied inside the pipette indicating the presence ofstrong rectification or a marked dependence of the channelgating mechanism on voltage. Fig. 1B shows the single-channel current-voltage relationship determined with equal(symmetrical) or unequal (nonsymmetrical) concentrations

A

+80 mV

+100

+120IL" ~̂~ ~ ~~~Jd"wiMJ~~~~~~~~~ -ar TS

1 pA250 ms

B

pA

0.51

mV

FIG. 1. (A) Records of currents from SR membrane patches atdifferent pipette potentials. Currents were recorded at constantpotentials between +80 and +120 mV (with the bath solution atground) in symmetrical solutions containing 200 mM CaCl2 and 10mM Tris/Hepes, pH 7.0, at 220C. Current signals were filtered at 500Hz and digitized at 5 kHz. Downward deflections correspond tochannel opening events. (B) Single-channel current-voltage relation-ship in the presence of (e) or absence (o) of a Ca2' gradient. Thepipette and bath solutions each contained 10 mM Tris/Hepes bufferat pH 7.0. All other experimental conditions are given in the text.

of CaCl2 in the pipette and bath solutions. Because of thefrequent appearance of two or more current levels at highpolarizations of the patch pipette, it was necessary to limitmeasurements to test voltages below +100 mV. In 200 mMsymmetrical Ca2+ (Fig. 1B), the unitary current amplitudeincreased linearly from 0.1 pA at a transmembrane potentialof +20mV to 0.4 pA at +80 mV, yielding a slope conductanceof 5.6 ± 0.5 pS (mean ± SEM; n = 9).Under our experimental conditions (200 mM symmetrical

CaCl2 and positive voltages inside the pipette), patch record-ings gave downward deflections that could correspond eitherto the "inward" movement of Ca2+ (pipette to bath) or to the"outward" movement of Cl- (bath to pipette), the two mainconductive species. Nonsymmetrical conditions were used toidentify the charge-carrying species and to test the selectivityof this channel for mono- and divalent cations. With 100mMCaCl2/1 mM NaCl in the pipette and 1 mM CaCl2/150 mMNaCl in the bath, the unitary current increased less steeplywith voltage (slope conductance, 2.2 pS) and gave an I-V(current to voltage) curve that extrapolated linearly to a zerocurrent value of -70 mV (Fig. 1B). This is similar to thecalculated equilibrium potential for Ca2+ of -59 mV but is inthe opposite direction to ENa (+11 mV) and Ec1 (+129 mV).A similar experiment carried out by lowering the CaCl2 in thebath from 200 to 25 mM while maintaining 200 mM CaCl2 inthe pipette gave a slope conductance of 8 pS and anextrapolated reversal potential of -15 mV, close to theNernst potential for Ca2+ of -25 mV (data not shown).Efforts to eliminate the contribution from anionic channels byadding 2 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) (19) to the pipette and bath solutions orby replacing Cl- with the impermeant anion gluconate did notalter the activity of the 5-pS channel, although larger fluctu-ations of 10-15 pS were less frequent. These results indicatethat the 5-pS fluctuations result from Ca2+ movementthrough a channel that selects for Ca2+ over Na+ as well asCl-.

Further evidence that the channel is selective for Ca2+ wasobtained by examining its behavior in the presence of Ba2+that is known to substitute for Ca2+ as the current-carryingspecies in a variety of different Ca2+ channels in excitablecells and tissues (20). Substitution of 100 or 200 mM (sym-metric) Ba2+ for Ca2` as charge carrier produced no apparentchange in the bursting behavior but increased the single-channel conductance by about 30%, similar to the increasefound in other tissues (20). In contrast, addition of 1 mM ofthe chloride salts of either La3` or Cd2+-ions known toblock Ca2+ currents in cell membrane patches (20) and inbrain synaptosomes incorporated into lipid bilayers (21)-was sufficient to block irreversibly the 5-pS channel afteronly a few seconds of bath microperfusion (data not shown).The dependence of the single-channel conductance on

Ca2+ concentration was investigated under conditions wherethe [Ca2+] was varied symmetrically between 50 and 200mM.Over this range the single-channel conductance displayed anonlinear dependence on [Ca2+] that appeared to be levelingoff(Fig. 2). Below 50mM CaCl2 the signals became too weakto accurately resolve. A reasonably close approximation tothe conductance vs. concentration relationship was obtainedby fitting the data with the aid of a computer (22) to ahyperbola with a maximum conductance, y of 7.9 pS and ahalf-saturation constant, Ko.5 of 83 mM Ca2+. Extrapolationof these data to a Ca2+ concentration of 5 mM, close to themaximal estimated intraluminal concentration under physi-ological conditions (23), indicated a single-channel conduc-tance of approximately 0.5 pS.Unitary current fluctuations similar to those found in the

unfractionated preparation were observed in the light, inter-mediate, and heavy fractions of SR. Although a detailedcomparison of the Ca2+ channels in these preparations was

Proc. Natl. Acad Sci. USA 83 (1986)

Proc. Natl. Acad. Sci. USA 83 (1986) 7743

8

v-C

C.i,)¢j

50 100 150 200Ca> mM

FIG. 2. Single-channel conductance as a function of Ca2l con-centration. Single-channel currents were recorded in symmetricalsolutions containing 50- to 200-mM CaCl2 and 10 mM Tris/Hepes,pH 7.0, at pipette voltages ranging from +50 to + 150 mV. Each datapoint represents an individual experiment. Data were fitted to ahyperbola by computer using a nonlinear least-squares curve-fittingroutine (22).

not attempted, the available evidence indicates uniformity intheir single-channel properties. However, significant differ-ences were observed in the frequency with which theyformed stable high-resistance seals. The percentage of suc-cessful trials was consistently lower in heavy SR (14.6%, 82trials) as compared to the intermediate (41.7%, 60 trials) andlight fractions (32.9%, 76 trials). The unfractionated prepa-ration that was used in the majority of these experimentsformed seals with a frequency between that of the heavy andlight SR (21.1%, 209 trials). The source of this variation isunknown but may be related to an intrinsic property of themembrane that affects its ability to form a stable monolayerat the air-water interface (24).A salient feature of this channel is its peculiar activation

pattern exemplified by the current trace shown in Fig. 3A. Ata steady polarization of +80 mV with 200 mM CaCl2 presenton both sides of the membrane the channel typically exhib-ited bursts lasting several seconds followed by quiescentperiods of up to one minute. While active, the channelfluctuated rapidly with open-close transitions in the millisec-ond range. The histograms of the open and closed lifetimedistributions corresponding to the slow and fast burstingpatterns are shown in Fig. 3 B-D. For the slow burstingbehavior, the distribution of open-state lifetimes is adequate-ly described by a single exponential (Fig. 3B), although analmost equally good fit was obtained with a sum of twoexponentials in which about 6% of the bursting events weredistributed in a component with a mean lifetime of 12 s. Incontrast, the long silent periods between bursts have abiexponential distribution (taking into consideration closedtimes <20 s) with mean lifetimes of 0.74 and 11.32 s (Fig. 3C).The possibility that the distribution of closed times mightinclude a third component of even longer duration is sug-gested by the presence of an appreciable fraction (10%) ofclosures with lifetimes greater than 30 s.Comparison of current traces obtained at different pipette

potentials indicated that the channel slow bursting activity isvoltage-dependent. This was apparent in the mean burstduration that increased from 1.8 ± 0.1 to 4.4 ± 0.7 s (P <0.001) as the holding potential was raised from +60 to +80mV (Fig. 4A). Over this range, the burst frequency declinedfrom 7.7 ± 0.7 to 3.4 ± 0.5 min- (P < 0.001) so that the ratioofthe time spent in the active phase to the total recording time(defined as the fractional bursting time, fb) remained essen-tially unchanged. Above +80 mV multiple current levels

A

VantoC

C.0-zc0eo

itI

It., -- - -

_- 7--

1 pA

2 s

V)C

0

0

10 20 30 >30 100 200Time, s Time, ms

FIG. 3. (A) Characteristic bursting behavior of the SR Ca2"channel on a slow time base. The trace was recorded in symmetrical200 mM CaCl2 and 10 mM Tris/Hepes, pH 7.0, at a pipette potentialof +80 mV. Horizontal lines indicate long duration channel openingsassociated with the slow bursting behavior. (B and C) Histograms ofdistributions of open (B) and closed (C) times corresponding to theslow bursting activity of the Ca2+ channel. The histograms wereconstructed from 147 bursts pooled from 12 experiments. The solidcurve represents computer-generated mono- and biexponential fits tothe open and closed duration histograms (22). In B the singleexponential decay function has a mean open lifetime of 3.1 s. (D andE) Histograms of open (D) and closed (E) time distributions for thefast bursting activity.

began to appear precluding analysis of the voltage depen-dence at more positive test potentials.The brief interruptions within long open periods that

characterize the channel fast bursting behavior were ana-lyzed from a single current trace that contained a seeminglycontinuous active phase with 915 fluctuations. The openduration histogram constructed from this recording (Fig. 3D)had two components with associated time constants of 3.2

8. 40..0 :T

UE z -

C5.0~~~~~~~~~~~1.

m 1.0 X

+60 +80 Control DantrolenemV

FIG. 4. (A) Voltage dependence of the Ca2+ channel slowbursting behavior. Points represent mean + SEM for data pooledfrom seven different patches that included between 11 and 50 burstsover recording times that varied between 283 and 678 s at eachvoltage. Asterisks denote differences that are statistically significantto P < 0.001. Paired t-test comparisons for individual patchesbetween +60 and +80 mV also gave significant differences (P <0.001). Burst frequency (0); mean burst duration (o); fractionalbursting time (A). (B) Effect of 50 /.M dantrolene on the slow burstingpattern. Experimental conditions were identical to those in Fig. lAexcept that 50 AuM dantrolene was either present (dantrolene) or not(control) included in the pipette and bath solutions. Data wereobtained from paired control (n = 5) and test patches (n = 6) and areexpressed as mean ± SEM. Symbols are as in A.

Biophysics: Suarez-Isla et al.

7744 Biophysics: Suarez-Isla et al.

and 22.7 ms. Unlike the slow bursting pattern that containsevidence for the existence of two closed states, the distribu-tion of closed times for the fast bursting activity was bestdescribed by a single exponential function (T, 7.1 ms). Thelongest closures within the burst were shorter than 150 msand in this example, less than 0.2% of all closures lastedlonger than 100 ms.

Action of Potentiators and Inhibitors of Ca2' Release. Thesensitivity of the Ca2l channel to agonists and antagonists ofSR Ca2l release was determined by monitoring their effectson the channel slow bursting behavior. At +80 mV, additionof 50 ,M dantrolene, a potent inhibitor of muscle contraction(25) and SR Ca2+ release (26), reduced the fractional burstingtime from 0.238 ± 0.043 (n = 5) in controls to 0.085 ± 0.030(n = 9) (Fig. 4B). Analysis of the frequency and distributionof the burst durations in the presence of dantrolene showedthat this effect apparently resulted from a significant (P <0.001) decrease in the burst frequency without a change inmean burst duration. Ruthenium red (27), added at a con-centration of 50 ,uM, produced a smaller decrease infb (0.178+ 0.053) that was not significantly different from the controlvalue. At a concentration of 10 ,uM, nitrendipine [a dihydro-pyridine known to block Ca2+ channels in transverse tubularmembranes (28)] produced no change infb (0.286 ± 0.098; n

= 4), nor did it alter the burst frequency or mean burstduration. None of these agents affected the single-channelconductance for Ca2+.

In marked contrast to the effects observed with dantrolene,caffeine (29) introduced at a relatively low concentration (1.6mM) dramatically increased the activity of the Ca2+ channel(Fig. 5). Compared to the control trace obtained prior toapplication of the drug, the burst intervals were longer andmore frequent and often displayed more than one currentlevel. Activation took place within seconds without a signif-icant change in the single-channel conductance but decreasedthereafter, even during sustained application of the drug.

DISCUSSIONThese results demonstrate that membranes derived from theSR of skeletal muscle contain a spontaneously active cationicchannel that is selectively permeable to Ca2+ and Ba2+ andis blocked by Cd2+ and La3". Drugs known to affect theprocess of Ca2' release from SR in skinned muscle fiberpreparations and isolated SR vesicles were found to beeffective in modulating the activity ofthis channel. Relativelylow concentrations of caffeine increased the fractional burst-ing time (fb) by increasing the mean burst duration, whereasdantrolene (at +80 mV) produced a significant decrease infbby decreasing the burst frequency without affecting the meanburst duration. Ruthenium red produced changes similar tothose caused by dantrolene except that effects were lesspronounced, presumably because of strong competition by

1.6 mM Caffeine-------

12 pA10 s

FIG. 5. Effect of caffeine on the SR Ca2l channel. Patchrecording represents a continuous trace obtained at a pipette poten-tial of +80 mV in symmetrical 200mM CaCl2 and 10 mM Tris/Hepes,pH 7.0. The upper trace depicts a control period prior to exposure ofthe patch to 1.6 mM caffeine. The trace was blanked duringimmersion and approach of a second patch pipette containing thedrug.

Ca2l at the binding site. These results, in addition to the factthat 10 gM nitrendipine failed to alter the channel activity,strongly support the view that the unitary current fluctuationsreported here originate from Ca2l channels in the SR mem-brane.The SR Ca2+ channel has unique characteristics but it

resembles the transient T-type channel described in sensoryneurones (30) and ventricular cells (31) in its small conduc-tance, its ability to retain functional activity in isolatedmembranes and its insensitivity to dihydropyridines. Unlikethe T channel, however, the SR channel opened spontane-ously and did not show transient activation in response tovoltage pulses.

Smith et al. (32) reported that heavy SR membranesincorporated into planar lipid bilayers contain a Ca2+-selective channel with a high conductance (125 pS) that isactivated by adenine nucleotides and blocked by Mg2+.Functional characteristics that serve to distinguish this con-ductance from the 5-pS channel are its activation by negativepotentials, weak response to caffeine (33), and inhibition byhigh (>1 mM) Ca2+ concentrations, which presumably ac-counts for our failure to observe it. In addition, the 125-pSchannel seems to be present exclusively in the heavy SRfraction, implying that it is localized to the terminal cisternaeregion of the SR. In contrast, we found the 5-pS channel tobe present in all of the SR fractions, including a highlypurified terminal cisternae preparation devoid of T-tubulecontamination (unpublished observations). It is unclear at themoment whether this reflects contamination of the lightfraction of vesicles by heavy SR or implies that the 5-pSconductance has a more diffuse distribution in the membranethan the nucleotide-activated channel.

Gating of the 5-pS channel was voltage dependent asshown by the prolongation in the mean duration of thelong-lived open state that resulted from an increase in thevoltage (Fig. 4A). For a two-state system

C 0O

where C is a closed state and 0 is an open state, the decreasein kL1 implied by the potential-sensitive increase in meanburst duration should be accompanied by an equal butopposite change in k, (34) resulting in a shorter mean closedtime. The fact that the burst frequency declined as thepotential was raised leaving the fractional bursting timeunchanged (Fig. 4A) implies that the gating mechanismincludes an additional closed state whose occupancy in-creases at higher voltages, which could be an expression ofchannel inactivation. Further evidence for this may be foundin the closed duration histogram for the slow burstingbehavior (Fig. 3C) that contains both fast and slow compo-nents. A plausible arrangement of these states is

Cl k° k 2'_1 k-2

where the transitions connecting the open state to the first(C1) and second (C2) closed states are voltage-dependent.According to this scheme, decreasing kL1 by raising thevoltage will increase both the mean burst duration and themean closed time provided that k2 increases (and hence k-2decreases) at more positive potentials and that kL1 is sub-stantially larger than k2 over the experimentally tested rangeof voltages. The latter constraint follows from the relationbetween the mean open lifetime T0 and the rate constants forthe transitions leading away from the open state

O = 1/(k1 + k2).

Within the context of this linear scheme, the effects producedby dantrolene can be attributed to a decrease in k, that will

Proc. Natl. Acad. Sci. USA 83 (1986)

-A-.

Proc. Natl. Acad. Sci. USA 83 (1986) 7745

extend the interval between bursts by increasing the sojournin C1. By contrast, caffeine is expected to stabilize thechannel in the open state by reducing the frequency oftransitions from the open to the closed states. In addition,caffeine may activate quiescent channels in the patch assuggested by the appearance of multiple current levels (Fig.5) following application of the drug.The above kinetic analysis applies only to the slow bursting

behavior and does not take into consideration the very briefgaps that interrupt the large openings (Fig. lA). Becausethese events are characterized by a time constant in themillisecond range, an explanation for this behavior wouldseem to require the presence of a third nonconducting statethat intercommunicates rapidly with the open state. Addi-tional complexity in the activation mechanism is implied bythe existence of the two short-lived open state components inFig. 3D the significance of which remains unclear. Thepossibility that the brief interruptions may result from block-age of the channel by Ca2" itself is suggested by the similarityof this behavior to the rapid flickering block produced byCd2` ions on Ca2" channels in cardiac myocytes (31). This isstrengthened by the fact that in isolated SR membranevesicles Ca2" release induced by chemical depolarization (35)is blocked by the addition of high concentrations of Ca2".An important criterion that must be fulfilled to ensure the

rapid development of tension in muscle subsequent to elec-trical stimulation is a rapid release of Ca2+ from the SR to themyofibrillar proteins. In terms of charge moving capacity the125-pS channel would appear to be better suited to meet thisrequirement than the Ca2+ channel described here. It isinteresting to note, however, that a close agreement existsbetween the single current density of the 5-pS channel underphysiological conditions and estimates of the required Ca2+flux per channel reported in the literature. From an investi-gation of the myoplasmic free-[Ca2+] transient in frog skeletalmuscle, Baylor et al. (36) estimated that Ca2' release fromthe SR occurs with an initial rate of 36 MkM/ms that corre-sponds to a macroscopic current density of -100 piA/cm2 SRmembrane. On the assumption that the Ca2' release sites arecorrelated 1:1 with the electron-dense foot processes of thetransverse tubular system (37), they calculated a single-channel current density of 0.02-0.03 pA. This value is similarto that obtained by Melzer et al. (38) from an analysis of theintramembrane charge movement associated with SR Ca2'release. In the present study, extrapolation of the conduc-tance vs. concentration relationship (Fig. 2) to a physiologicCa2+ level (5 mM) gave a single-channel conductance of 0.5pS. This is equivalent to a unitary current amplitude of 0.05pA assuming that the Ca2' diffusion potential across the SRmembrane is =100 mV (39) and that the channel is highlyselective for Ca2 . Considering the various approximationsand assumptions that were used in making this comparison,there is a distinct possibility that the similarity between thesingle-channel current densities estimated from the patch-clamp and single-fiber studies is fortuitous. It is clear that theanswer to these and other questions pertaining to the role ofthe 5-pS channel in excitation-contraction coupling mustawait further studies on the gating mechanism, density, andlocalization of Ca2' release sites and on the contribution ofalternative pathways for Ca2' release.

We wish to thank Dr. Stanley I. Rapoport for his support in thisproject and Drs. Sandra E. Guggino and Eduardo Rojas for helpfuldiscussions.

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