1983 Cation Selectivity Characteristics of the Reconstituted Voltage-Dependent Sodium Channel...

7/30/2019 1983 Cation Selectivity Characteristics of the Reconstituted Voltage-Dependent Sodium Channel Purified From Rat Skeletal Muscle Sarcolemma

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T H E J OURNALF BIOLOGICAL CHEMISTRYVol. 258, No. 12, Issueof June25, pp.7519"7526,1983Printed in U.S.A.

Cation Selectivity Characteristicsof the Reconstituted Voltage-dependent Sodium Channel Purified from Rat SkeletalMuscleSarcolemma*(Received for publication, November18, 1982)

JacquelineC. TanakaSO, J ohn F. Ecclestonl, and Robert L . Barchi$TFrom theDewrtments of $Neurobk!y andof IlBiochemistryandBiophysics, Uniuersityof Pennsylvania School of Medicine,Philudelphia: Pennsylvania 19104

In this report, the alkalimetal cation selectivity ofthe purified, voltage-dependent sodium channeI fromrat skeletal muscleisdescribed. Isolated sodium chan-net protein (980-2840 pmol ( ofsaxitoxin bindinglmgof protein) was reconstituted intogg phosphatidylcho-line vesicles, and channels wereubsequently activatedby either batrachotoxin(5 % lo-' M) or veratridine(5x 1 0 - 4 M.Activation of the reconstituted sbdium channel bybatrachotoxin permitted rapid specific influx of cat-ions into channel-containing vesicles. Quenched flowkinetic techniques were adaptedo allow resolutionofthe kinetics of cation movement. Uptakeratesfor"K+"Rb+ and I3'Cs+ were measured directly and half-times for equilibrationat 18"C were determined to be350 ms, 2.5 s, and 10 s, respectively, in this vesiclepopulation. zzNa+equilibration occurred within themimimum quenching time of the apparatus90m) butan upper limit of 50 ms at 18"C could be assigned toits half-time. Based on this upper estimate for Na+,cation selectivity ratios of the batrachotoxin-activatedchannel wereNa+(l):K+ (0.14):Rb' (0.02):Cs' (0.005).Toxin-stimulated influx could be blocked by saxitoxin

with a K i of -5 X I O-@M at 18"C. Rates of cationmovement through eratridine-activated channelswere much slower, withhalf-timesof 1.0,1.2,2.0,and2.6 min at 36 O C for Na+, K +, Rb', and Cs+, respec-tively.The temperature dependences of batrachotoxin andveratridine-stimulated cation uptake were markedlydifferent. The activation energies forRb+ and la7Cs+movement into batrachotoxin-activated vesicles were76 and 6.1 kcal/mol, respectively, while comparablemeasurements for these two cations in veratridine-activated vesicles yielded activation energies of 31kcal/mol. Measurements of cation exchange with ba-trachotoxin-activated channelsmay reflect character-istics of an open sodium channel while the process ofchannel opening itself may be rate-limiting when ver-atridine isused for activation.

The electrical signalsor action potentials that characterizethe surface membranesof nerve and muscle are usually pro-*This work was supported inpartby National Institutesof HealthGrants NS-18013 and GM 29603 and by a grant fromthe MuscularDystrophy Association. The costsof publication of this article weredefrayed in part by the payment of page charges. This article musttherefore he hereby marked "aduertisement" in accordance with 18U.S.C.Section 1734 solely to indicate this fact.3Recipient of a Muscular Dystrophy Association postdoctoralfellowship.

duced by transient changes in membrane conductance tosodium and potassium ions (I).These time- and voltage-dependent ion conductances are controlled by intrinsic mem-brane proteins that span the bilayer and provide an aqueouspathway or channel for on movement (2).The molecularcharacterization of these sodium and potassium channels hasbecome an active topic of current neurochemical research.The past several years have seen significant progress in theisolation and biochemical characterization of the voltage-dependent sodium channel. A sodium channel protein hasbeen purified from eel electroplax (3), rat skeletal musclesarcolemma 4) , and rat brain synaptosomes(5).In each case,alarge glycoprotein has been identified that exhibits anoma-lous migratory behavior on SDS-PAGE' (5-7). In the twomammalian channel preparations, several smaller peptidesare also thought to be components of the purified sodiumchannel (5,7).A number of investigators have studied the reconstitutionof unpurified sodium channels or channel-containing mem-brane fragments into artificial liposomes ( 8- 11) . More re-cently, wereported the functional reconstitution of a purifiedsodium channel from rat sarcolemma into phosphatidylcho-line vesicles (12). This purified channel protein retained itsability to gate 22Na+ luxes in response to activation by thealkaloid neurotoxins batrachotoxin and veratridine; thesefluxes were specifically blocked by saxitoxin. Similar resultshave now been obtained with the sodium channel partiallypurified from rat brain synaptosomes(13).Cation flux through opened sodium channels occurs veryrapidly, and he ate of cation uptake into reconstitutedvesicles through batrachotoxin-activated channels could notbe resolved in our earlier studies(12). In this report, quenchedflow kinetic techniques havebeen applied to the purified,reconstituted sarcolemmal sodium channel in order to meas-ure the kinetics of uptake for various alkali metal cations.The sodium channel selectivity among Na+K +,Rb+ andCshas been determined following batrachotoxin or veratridinestimulation,and he activation energies for cation influxmeasured.

MATERIALS AND METHODSMaterials used in the purification of sarcolemmaand in the isola-tion of the sodium channel protein were reported previously (4, 7,14).Chemicals used in the reconstitution were as detailed by Weigeleand Barchi (12).Batrachotoxin was thegift of Dr. J . W . Daly of theNational Institutes of Health. The isotopes"Na+, %b+ '%+and137Cs+were purchased from New England Nuclear Co. Dowex 50-X8'The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NP-40, Nonidet P-40; EGTA,ethylene glycol bis(/3-aminoethylether)-N,N, N ,N'-tetraacetic acid.

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7520 Cation Selectivity f Reconstituted Sodium Channels(200-400 mesh) was obtained from the Sigma Chemical Co. Saxitoxinwas provided by Dr. E. J . Schantz of the University of Wisconsin.The toxin was tritiated by New England Nuclear Corp. and purifiedas described by Ritchie etal. (15).Purification of the Sarcolemmal Sodium Channel-Sarcolemmalmembranes were isolated from rat skeletal muscle of mixed fiber typeusing a LiBr extraction procedure as described previously (14). Pep-statin (0.1 rg/ml), phenylmethylsulfonyl fluoride (0.1 mM), iodoace-tamide ( 1 mM), and EGTA (0.2mM) were present during membraneisolation. Sodium channel protein was purified from these membranesusing a modified form (7) of our original purification protocol (4).Briefly, 100-150 mg of sarcolemmal membrane protein were solubi-lized at a final concentration of 2 mg/ml in 1%NP-40 detergent inthe presence of 100mM choline chloride, 50 mM potassium phosphate(pH 7.4), and 0.5 mM CaC1, at0C. The protease inhibitors pepstatin(0.1pglml), leupeptin (1pglml), o-phenanthroline(1mM), andphen-ylmethylsulfonyl fluoride(0.1mM)were present during solubilization.After centrifugation, the supernatantwas diluted to 1 mg of protein/mwith buffer containing100mM choline chloride, 50 mM potassiumphosphate, 0.5 mM CaCI2,and the above protease inhibitors. Subse-quent buffers also contained 0.1% NP-lO/asolectin phosphatides ata 5:l molar ratio. The channel protein was purified first on a guani-dinium-Sepharose column and subsequently on a wheat germ agglu-tinin-Sepharose column. The column dimensions and conditions aredetailed elsewhere (7).The guanidinium-Sepharose column described previously ( 4)wassynthesized by coupling3, 3- di amnodi pr opyl amneEastman Kodak)to Affi-Gel202 (Bio-Rad) to form an immobilized support with a 19-atom extended spacer arm, and subsequently converting the terminalprimary amine of this arm to a guanidinium group by reaction with0-methylisourea. Briefly, a 2 M aqueous solution of 3.3-diaminodi-propylamine was prepared immediately prior to use and titrated topH 7.4 at 0C with HC1. Affi-Gel 202 (45 ml of packed resin) wassuspended in H20 tovolume of 75 ml and mixed with 75 m of fresh2M 3, 3- di amnodi propyl amnesolution; solid NaCl was then addedto a final concentration of 0.1 M Coupling was initiated by theaddition of l - ethyl - 3- ( 3- di met hyl amnopropyl ) carbodi i mde2g total,added in 100-mg aliquots over 20min) and the reaction was allowedto proceed with stirring at room temperature for 5 h. The pH wasmaintained at 4.7 throughout the reaction period. The resin waswashed with 1500 ml of NaCl(0.2 M) followed by 1500ml of H20 ona siliconized sintered glass funnel, and resuspended in 75 m of HzO.75 m of an 0.75 M aqueous solution of 0-methylisourea (pH 10.0)was cooled to 0C, added to the resin suspension, and stirred at 3 Cfor 24 h. The final resin product was washed extensively with 0.2Msodium chloride and H20prior to use.Proteins were determined with a micro-adaptation of the Lowrymethod (16). Specific binding of [3H]saxitoxin was measured aspreviously described (4, 17,18).Reconstitutionof the Purified Sodium Chnnel Protein-Peak frac-tions from the wheat germ column of the sodium channel purificationwere pooled and used immediately for reconstitution into egg phos-phatidylcholine (Sigma Chemical Co., type V-E, 99% purity) aspreviously described (12). Stock phosphatidylcholine (50 mg/m) wasprepared in 10%NP-40 by stirring under argon. The purified sodiumchannel solution was obtained in 100 mM NaCI, 20 mM potassiumphosphate (pH 7.4), 0.5 mM MgCI,,0.5 mM CaC12, 0.05% NP-40/phosphatidylcholine. The concentrated phosphatidylcholine/NP-40stock was then added to obtain a final concentration of 1% NP-40and 5 mg/ml phosphatidylcholine. Bio-Beads SM-2 were prepared asdescribedby Holloway (19) and 0.3 g of moist, packed beads wereadded per ml of reconstitution solution. Detergent removal was ac-complishedby gentle tumbling at 3 C for 3 h. After this period, theresidual NP-40 concentration was estimated to be(0.001% as meas-ured by the intrinsic fluorescence of the NP-40 relative to a standardsolution. The resultant vesicle suspension was filtered through glasswool to remove the Bio-Beads and used directly for flux experiments.In some cases, reconstitutions were performed in solutions containingdiffering concentrations of cations, or with the addition of saxitoxin,as specifically ndicated in the text.Cation Flux Measurements-As a standard for comparison, assaysof batrachotoxin-stimulated 22Na+uptake at 15and 45 s were doneon each preparation. These assays, as well as allassays of veratridine-stimulated uptake, were done using a manual technique as previouslydetailed(12).Briefly, channel-containing vesicles n buffer containing100mM NaCl, 20 mM potassium phosphate (pH 7.4) were preincu-bated for 45 min at 36 C n the presence of 5 X Mbatrachotoxinprepared in ethanol (final ethanol concentation in incubation was

less than 1%) orn the presence of an equivalent amount of ethanolalone. Following the preincubation, 330-~lliquots of vesicles wereequilibrated to the assay temperature (18C unless otherwise indi-cated) and uptake was initiated by the addition of Na+(usually8plof a 400 rCi/ml solution of isotope). A t 15 and 45 s, 150$1 of thevesicle suspension were removed and rapidly applied under pressureto a small (1.5 ml) Dowex cation exchange column (50-W-X8, 100-200 mesh, Tris form) in a disposable syringe barrel (20). Two 0.8-mlwashes of isotonic sucrose containing1mg/ml bovine serum albuminwere then forced through the column under pressure. The entireelution and wash required about 10 s. Using this technique, morethan 99.9% of the extravesicular cations were taken upby the resinwhile greater than 98%of the vesicles were recovered in the eluate(12). The eluate was subsequently counted in 10 m of scintillationfluid in order to quantitate the concentration of labeled cations inthe vesicles.Quenched Flow Measurements-In order to measure the rapiduptakeof cations into batrachotoxin-activated vesicles, a quenchedflow instrument was used. This instrument was similar in design tothat described by Gutfreund (21) but was modified to incorporate thepulsed flow mode of Fersht and J akes(22). Reaction times of lessthan 300 ms were obtained using a single push in which the age ofthe solution was determined by the volume of the aging tube and theflow rate. Longer times were obtained using the pulsed flow mode inwhich the mixed solutions were held in the aging tube for an elec-tronically determined time before being expelled.An important difference between this instrument and those pre-viously described is that the two reactants (in this case, batracho-toxin-activated vesicles and labeled cation) were loaded into twocapillary tubes placed between the mixing chamber and the drivesyringes while the drive syringes contained only buffer. This allowedvolumes of reactants as small as 25$1 to be used. Test reactions wereused as described by Gutfreund (21) to show that the mixing time ofthe instrument was less than 4 ms. Furthermore, results were obtainedfrom this instrument that were identical with those generated byconventional quenched flow instruments with model enzymatic re-actions. Full details of the construction and performance of theinstrument will be presented elsewhere?For either single push or pulsed flow operation, the mixed solutioncontaining vesicles and labeled cation was quenched by direct injec-tion into a slurry of Dowex 5O-W-XS (2.5 ml; 200-400 mesh) in theTris form. Under these conditions, the cation exchange resin rapidlysequestered residual cations in the extravesicular space. The vesicleswere then rapidly separated from the Dowex by positive pressure andthe resin was washed twice with 0.8-ml aliquots of isotonic sucrose-bovine serum albumin buffer. The cation content of the vesicles wasdetermined by liquid scintillation counting.The overall quenching time for the quenched flow system includingthe Dowex resin step was determined as detailed under Results bymeasurement of the early linear time course of K+uptake intobatrachotoxin-activated vesicles.A quenching time of approximately90 ms was obtained. Attempts at stopping cation influx at fastertimes by quenching the reaction solution n Mtetrodotoxin wereunsuccessful.

RESULTSIn the experiments reported in this paper, the conditionsfor sodium channel reconstitution were kept as constant as

possible (see Materials and Methods). We found previouslythat the esults obtained rom reconstitutions with our urestsodium channel preparations (2000-3000 pmol of saxitoxinbinding/mg of protein, representing the product of a three-step purification including a final sucrose gradient (7)) werecomparable to those obtained with sodium channels carriedthrough only two stepsof purification and having a slightlylower specific activity (typically 1000-2200 pmol/mg) (12).Forthe studies reportedhere, we chose to optimize the numberof experiments which couldbecarried out with a given prep-aration by using the larger quantities of channelproteinavailable after the second column (wheat germ agglutinin-Sepharose) in our purification protocol (7).The saxitoxin-binding activityof the pooled fractions fromJ .F.Eccleston andR. Messerschmidt, manuscript in Preparation.

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7522 Cation Selectivity of Reconstituted SodiumChannels

, 18QXE6-WYg 4+9[L% Z

ITIME (seconds TIME (seconds)

FIG.3. Time courseof "K' and -Rb+ uptake in batrachotoxin-activated vesicles from two typicalreconstitutions. Purified sodium channel protein (specific activity, 1220 pmol/mg of protein for A , 1960pmol/mg of protein for B ) was reconstituted as detailed under "Materials and Methods." Vesicles were preincubatedeither with5 X 10" Mbatrachotoxin (BTX) n ethanol or with an equivalent volumeof ethanol alone at36 'C for45 min, then equilibrated to 18 "C.For each data point shown, 25 p1of vesicles and 25 pl of buffer containingeither "K+ (A )or &Rb+ B )were used in the quenched flow apparatus to assay uptake at each time as describedin the text. 0, otal uptake of labeled cation in batrachotoxin-activated vesicles; A , cation uptake in vesiclesincubated with 1%ethanol alone;.,pecific batrachotoxin-stimulated cation uptake.lower than that seen using the manual technique at 15 and45 s, relatively ong times compared to the rate of cationequilibration.The kineticsof uptake for eachof the alkali metal cationsexcept *'Na+ could be resolvedunequivocallyusing thequenched flow technique. '*Na+uptake was essentially com-plete at the earliest time point, taken with 8 ms betweenmixing of vesicles and isotope and the start of the Dowexquench (Fig. 4). Theactualelapsed ime, however, mustinclude the time between the injection of the mixed solutioninto the Dowex resin and the binding of all extravesicularNa+ o heresin,since his nterval will allow additionalisotope movement ntoactivated vesicles. Thisquenchingtime for the system was determined by careful measurementof the early, linear phase of uptake for &'K+the isotope withthe fastest esolvable uptake rate. Extrapolationof correctedspecific 42K+uptake values to base-line (Fig. 4) yielded aneffective quenching time of 90 ms. Using this value for thequenching time, an upper limit f 50ms could be set for theactualhalf-time for **Na+equilibrationundercomparableconditions.The timecourse for equilibrationof 42K+,Rb+, and137C~+into batrachotoxin-activated vesicles was clearly resolved US-ing the quenchedlow technique, and the half- timesor vesicleequilibration were directly measured (Fig. 5).Half-times forthese cations were sufficiently slow that the quenching ti meof the apparatus was of significance only for 42K+; or thatcation, a smallcorrection o hemeasuredhalf-time wasnecessary. The half-time for 42K+uptake, calculated eitherfrom initial rate data ashown in Fig. 4 or from the completeinflux as in Fig. 5, was approximately 350 ms, while that for=Rb+ and 1 3 T s + were 2.5 and 10 s, respectively. T he widespread of values for these four alkali metal cations indicatessignificant cation selectivity in he purif ied, reconstitutedsodium channel. Using an upper limit or the "Na+ half- timeof 50 ms, thecalculated ionselectivity ratios were (N a+)l:(K +)0.14,:(Rb+) O.O2:(Cs+)0.005 (Table I).Reproducibili ty of time courses for uptakeof a given cationwas good from reconstitution to reconstitution. For xample,points shown or 42K +uptake on Fig. 4 were derived fromthree separate reconstitutions, yet all points fall along thesame time ourse. Similar reproducibil ity was seen with137C ~+and =Rb+, suggesting that the size distribution of vesicles

I I T

Time (msec)FIG.4. Quenched f low measurementsof "Na+and '"K' in-flux in batrachotoxin-stimulated vesicles at short incubationperiods.Data points represent the specific batrachotoxin-stimulateduptake corrected for control uptake. Control uptake at these rapidtime points was ow, typically less than 20% of the total batracho-toxin-stimulated sodium uptake. Control vesicles formed in the ab-sence of protein showed no cation influx under these conditions for

the short ntervals being studied. Eachpoint represents the mean fS.D. of three or more separate measurements after correction forcontrol influx. The effective quenching time for the apparatus wasdetermined by extrapolation of the initial linear uptake rate for "K +into batrachotoxin-activated vesicles (O), yieldinga value of 90ms.2zNa+nflux on this time scale (0 )was very rapid and an nitial ratecould not be resolved; an upper limit of 50ms for the half-time for22Na+ould be estimated based on the measured quenching time of90ms for the system.containing active sodium channels was fairly constant frompreparation to preparation. T he arger standard deviationsseen with **Na+measurements may reflect the more signifi-cant contribution f variabil ity in quenching time to measureduptake because of the rapid nflux of this cation into activatedvesicles.Veratrdine-stimulated Cation Znflux-Veratridine-stimu-lated influx was measured for "Na+, 42K+,Rb+, and 137Cs+

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7524 Cationelectivity of Reconstitutedodium Channelsmeasurements were focused onearly imepointsateachtemperature to llow quantitation of the rateof vesicle fillingduring the early, linear phasef uptake and thesealues wereused to construct A rrhenius plotsor stimulated cation nflux.An activation energy of23.6 kcal/mol was determined forveratridine-activated *'Na+ influx. The A rrhenius plot waslinear over the temperature range of 15-36 "C in which reli -able measurements could be made. Similar studies were car-ried out with =Rb+ (Fig. 7B) and 137Cs+. n both cases, l inearA rrhenius plots were obtained, with high activation energies(31kcal/mol) (Table11).Batrachotoxin-stimulated cation nflux was much less tem-peraturedependent. ?'Na+ fluxescouldeasily be detectedthroughout the temperature range of 5-36 "C, but the rapidinitial rate precluded accurate quantitation. For comparisonwith veratridine data, initial rate measurements were there-fore made with "Rb+ and Is7Cs+in batrachotoxin-activatedvesicles using the quenched flow appratus. Uptake rates forthese cations ould be readily resolved wi thin the temperaturerange studied. A rrhenius plots of the data appeared i nearthroughout his angewithout evidence of a breakpoint,although the imited number of temperatures studied restri ctsour resolution Fig. 7B). Calculated activation energies for=Rb+and 137Cs+were much lower (7.6 and 6.1 kcal/mol,respectively) n batrachotoxin-activated than in veratridine-activated vesicles (Table11).Flux M easurements nOther Ionic Enuironments-Formost flux measurements n this study, solutions nside andoutside he vesicles contained 100 mM NaCland 20 mMpotassium phosphate (pH .4).Under these conditions, meas-urements with tracer amounts( 4 0 M ) of '*Na+,4'K+ =Rb+,or l:T!s+nitially added nly to the external solution representexchange measurements inwhich the measured ratef isotopemovement was assumed to be independent of other monova-lent cations in the solution. In a control experiment, recon-stitution of a preparation of sodium channels was carried outeither in 100 mM NaCI , or 100 mM RbCI, and the influx of%Rb+was measured in each case. Identical values for the half -time of %Rb+ equil ibration ere obtained in thewo solutions(Fig. 8A).I t has been reported that an external cation-binding sitemust be saturated in order for veratridine to activateodiumchannels i n ome excitable cells 24). In an attempt to addressthis question, vesicles containing thepurified channel from asingle preparation were reconstituted in solutions containingsodium concentrations between 20 and 100mM while totalionic strength was maintainedconstant by the reciprocaladdition of choline. Z2Na+influx measurements were thencarried outwith each set of vesicles activated with veratridine.In each case, the half -time forZ2Na+nflux was the same andshowed no dependence on sodium concentration within thisrange (Fig.8B).Saxitoxin Inhibition of Batrachotoxin-actiuated Cation Zn-flux-We havepreviously shownusingmanualmeasuring

TABLE1Temperature dependence of the initial rate of isotope fi lli ng withbatrachotoxin- and veratridine-activated vesicles

BatrachotoxinCation VeratridineE." E .kcallma1"ZNa+ 23.631.06.11.0 ffiRb' 7.6137CS+

E,, the activation energy for ion influx, calculated from Arrheniusplots of initial rates of ion uptake at various temperature between 5and 36 "C.

TIME Iseconds) TIME (m~nuterl

FIG.8. Uptake of "Rb+ (A )and*'Na+ (B ) nto activatedvesicles in the presenceof varying unlabeledmonovalentcations. In A , vesicles were reconstituted either with 100 mM NaCl(0 ) r 100 mM RbCl (A) inside and outsideof the vesicles. Solutionsalso contained 20mM potassium phosphate (pH 7.41, 0.5 mM M&12and 0.5 mM CaC12 In B, the total Na+ concentration in the internaland external buffer was varied from20to 100 mM while total ionicstrength was maintained with reciprocal additions of choline. Allsolutions contained 20 mM potassium phosphate (pH 7.4), 0.5 mMMgCI,, and0.5mM CaCI2.0 ,20mMNaCI, 130 mM choline chloride;0 ,50mM NaCl, 100mM choline chloride;0, 100 mM NaCl, 50 mMcholine chloride.I N

o w /0 IO-^ 10-6 10[ ST X ] (M I

F IG .9. Saxitoxin (STX) nhibition of "Rb+uptake into ba-trachotoxin-activatedvesicles.Vesicles were preincubated with 5X M batrachotoxin and he indicated concentration of saxitoxinat 36 "C for 45 min. ffiRb+ ptake was measured at 5s after mixingof isotope and activated vesicles, about twice the half-time for specificbatrachotoxin-activated vesicle filling with this cation in the absenceof saxitoxin. Data have been corrected for nonspecific uptake andforthe contributionsof inward-facing sodium channels not inhibited byexternal saxitoxin. The solid line indicates a theoretical inhibitioncurve for saxitoxin assuming a K , of 5 X lo-' M. All assays werecarried out at 18"C.techniques with15-s ime resolution that batrachotoxin-stim-ulated **Na+ influx could benhibited by saxitoxin (12). Underthose conditions, the concentrationof saxitoxin required forcomplete inhibition of cation flux was well above the Kd forequilibrium binding of this toxin to the channel. Thisiscrep-ancy was explained by the rapidly reversible nature of saxi-toxin binding, the very short period required for fi ll ing of agiven vesicle (typically tens of milli seconds), and theelativelylong influx period permitted by the assay method eing used.Saxitoxin inhibition of activated cation influx was re-exam-ined here under conditions inwhich the ini tial rate of cationuptake could be resolved.

T he influx of "Rb+ was quantitated in batrachotoxin-stim-ulated vesicles, and the i nhibition of this rate was measuredas a function of saxitoxin concentration. Experiments were

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Cation Selectivity f Reconstituted Sodium Channels 7525carried out either wi th nward-facing channels blocked bysaturating concentrations of saxitoxin wi thin thevesicles, inwhich case complete block of initi al influx was obtained withtitration of external sites, or with no internal saxitoxinherea maximal inhibition of 50-70% was obtained with titrationof outward-facing channels as previously reported (12). Ineither case, the apparent K i for saxitoxin inhibition of =Rb+influx was 5-10 nM (Fig. 9) at 18"C, approximately the samevalue as the Kd for saxitoxin binding n these vesicles at thistemperature measured by equilibrium binding of [3H]saxi-toxin (12).

DISCUSSIONThe sodium channelpurif ied from rat skeletal muscle sar-colemma has been reconstituted into egg phosphatidylchol inevesicles. Thi s purif ied channel retained the abil ity topecifi-cally gate cation fluxes in response to activationby batrach-otoxin and veratridine t concentrations comparable to thoseactive on the channel i n si tu(12). These cation fluxes werespecifically blocked by saxi toxi n, and we show here that theK , for saxitoxin inhibition of cation infl ux corresponds o theKdmeasured for [3H]saxitoxin binding to the channel sing

equilibriumbinding echniques. n vesicles containing thepurif ied sarcolemmal sodium channel, cation influx stimu-lated by bactrachotoxin occurred too apidly for kinetic reso-lutionusingmanualassay echniques.Theapplication ofquenched flow methodology to this system now allows theinflux kinetics for various alkali metal cations tobe resolvedand the on selectivi ty characteristics of the purified channelto be studied. Simil ar quenched flow techniques have beenused by others n the study of rapid ion luxes in vesiclescontaining the acetylcholine receptor proteinrom eel (25).A lthough the vesicle phospholipid composition was heldconstant n heseexperiments, he ange observed in hemagnitude of specific toxin-stimulated uptake was large andnot directly correlated with the saxitoxin-binding capacityfa given preparation. We have not yet been able to define thevarious factors which govern the successful incorporation offunctional channels into these esicles, although a systematicinvestigation s n progress. Regardless, successful reconsti-tution with measurable batrachotoxin-stimulatedspecific in-flux was obtained in40 of 42 consecutive attempts using themethods given here.Leakage (control) influxdid increasewith increasing protein concentration present during the re-constitution; however, these studies do not llow us todiffer-entiate between leakage due to incompetentodium channelsand leakage contributed by the presence of a contaminantpolypeptide.Physiological studies suggest that batrachotoxin opens thesodium channel by shifting ts activation curve far towardhyperpolarizing voltages and by eliminating sodium nacti-vation (26, 27). Si nce the onic conditions used here result inzero membrane potential, e expect that functioning channelswil l be open mostof the timen the presence f batrachotoxin.Cation flux stimulated by batrachotoxin should then approx-imate cation movement through an open channel. The rapidrates measured for the alkali metal cations in our batracho-toxin-activated vesicles using the quenchedlow method sup-port his nterpretation, as do the low activation energiesdetermined for Rb' and Cs+ nflux. Cation selectivity for thebatrachotoxin-activatedchannel, based onanupper limitestimate of the half -time or "Na' influx,was 1:0.14:0.02:0.005 for Na+, K', Rb', and Cs, respectively. If the actualvalue for Na' equil ibration was in fact more rapid than thisupper imit, hechannel selectivi ty ratios would be evenhigher than those indicated ere.In voltage clamp studies, sodium channels opened by ba-

trachotoxin have been shown to have a lower maximal con-ductance (26) and a lower cation selectivity (28) than thoseopened by depolarization. Cation selectivity values based onisotopic lux measurements hroughbatrachotoxin-openedchannels in tissue-cultured nerve anduscle also suggest thatthis toxi n alters to variable degree the ion selectivity of thechannel (24,29). The cationelectivity demonstrated here forthe purified, reconstituted sarcolemmal sodium channel fallsin the rangeof those reported in the literature or batracho-toxin-activated channels n. si tu; although apparent electivityis greater than in many batrachotoxin studies, it isess thanthat expected for the vol tage-activated sarcolemmal sodiumchannel in its active state 30, 31).Cation influx through veratridine-activated channels oc-curred on much slower time scale than that through batrach-otoxin-activatedchannels n vesicles containing hesamepurified sarcolemmal sodium channel.Sinceveratridine isknown to be only a partial agonist for channel opening (32),and voltage clamp studies suggest that veratridine-modifiedchannelsactivate 1000-fold more slowly than unmodifiedchannels (33), one interpretationf these slow rates would bethat the channels opened only for brief periods in any giventime nterval. If batrachotoxin-activatedchannelsare as-sumed to be opened 100%of the time, veratridine-activatedchannels would be opened 0.5%of the time if, for example,the relative rates of batrachotoxin- and veratridine-activatedK' equil ibration were explained on thisasis. For veratridine-activated channels, the rate-limiting step might thene chan-nel opening rather than the ratef ion movement through anopenchannel.Thishypothesis ssupported by the muchhigher activation energies measured for influx of the alkalimetalcations hroughveratridine-activated vesicles ("30kcal/mol)ascompared obatrachotoxin-activated vesicles(-7 kcal/mol), corresponding to Qlo values of >3 and -1.8,respectively. T hese valuesmay be compared o hose forsodium channel activation (Qlo "3.0) and maximal sodiumchannel conductance (Qlo=- .5) measured physiologicallyusing voltage clamp techniques in intact nerve and muscle(34,35).A lthough the selectivity sequence for veratridine-activatedchannels s the same as for those activated with batracho-toxi n, the apparent relative selectivi ty ratio between cationsis much lower. A simil ar observation has been reported forunpurifi ed sodium channels inserted into soybean phospho-lipid vesicles by freeze-thaw cycles and activated by grayan-otoxin I 36). With hatpreparation, low apparentcationselectivity was also associated with slow rates of cation equi-libration. These results may be due in part to veratri dine-induced changes in channel structure leading to a modifica-tion in selectivity, and i t seems probable that at leastome ofthe difference must be ascribed to such a change. However,other factors mustbe considered in lightof the small internalvolume of the vesicles under study and the rapid equil ibrationtime for theseesicles through opened sodiumchannels. Thus,if veratridine produces infrequentchannel openings, butchannels once activated emainopen forseveral hundredmill iseconds, most vesicles would fill with eitherp2Na+or 4>K+during a single open channel event. The very similar timecourse for uptake f these two cations in veratridine-activatedvesicles may therefore reflect the probabil ity f channel open-ing rather than the trueelative cation selectivity of the openchannel. These considerations could also contribute to theveratridine-stimulated Cs' and Rb' data, but the fact thatthe ratio of the equilibration rates for these ions to the ratefor Na' is lower for batrachotoxin stimulation than for vera-tridine suggests that veratridine activation tself may cause afurther reduction n channel cation selectivity.

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7526 Cation Selectivity of Reconstituted Sodium ChannelsWe have previously shown that the purified, reconstituted 13. Talvenheimo, J . A., Tamkun, M. M., and Catterall, W. A. (1982)sarcolemmal sodium channel retained the capacity to gate J . Biol. Chem. 257,11868-11871so&umfluxes in responseto activation by batrachotoxin and 14. Barchi, R. L., Weigele,J . B., Chalikian, D. M., and Murphy, L.veratridine and also contained the receptor site that allowed 15. Ritchie, J . M., bgart, R.B., and Strichadz, G. R. (1976) J ,these fluxes to be specifically blockedby saxitoxin. We can Physiol. (Lond.)261,477-494now state that theurified channel exhibits selectivity among 16. Markwell, M., Haas, S., Tolbert, W., and Biever, L. (1981)four alkali metal cations which is comparable to that found Methods Enzymol. 72,296-303for the native channel in situ under similar con&tions of 17. Levinson, S. R., Curahlo, C. J ., Reed, J ., and Raftery, M. A.

activation. Demonstrationof voltage-dependent activation in 18. Barchi, R. L., andWeigele, J . B. (1979) J .physwl. 295,the purified channel remains a major goal of future research. 383-396

E. (1978) Biochim. Biophys. Acta550,59-76

(1979) Anal. Biochern.99,72-84Acknowledgments-We thank Drs. R. Furman, J . B. Weigele, andD. Jameson for their helpful discussions. The expert technical assist-ance of Lois Murphy and Steven Packard as well as theassistance ofNancy Goodman in the preparation of the manuscript are gratefullyaknowledged.

1.2.3.4.5.6.7.8.9.

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