THE

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 12, Issue of June 25,pp. 7519"7526,1983 Printed in U.S.A.

Cation Selectivity Characteristics of the Reconstituted Voltagedependent Sodium Channel Purified from Rat SkeletalMuscle Sarcolemma*
(Received for publication, November 18, 1982)

Jacqueline C. TanakaSO, John F. Ecclestonl, and Robert L. Barchi$T

From the Dewrtments of $Neurobk!y and of IlBiochemistryand Biophysics, Uniuersity of Pennsylvania School of Medicine, Philudelphia: Pennsylvania 19104
"

In this report, the alkali metal cation selectivity of the purified, voltage-dependent sodium channeI from rat skeletal muscle is described. Isolated sodium chansaxitoxin bindinglmg net protein (980-2840 pmol (of of protein) was reconstituted into egg phosphatidylcholine vesicles, and channels were subsequently activated by either batrachotoxin (5 % lo-' M) or veratridine (5 x 10-4 M ) . Activation of the reconstituted sbdium channel by batrachotoxin permitted rapid specific influx of cations into channel-containing vesicles. Quenched flow kinetic techniques were adaptedto allow resolution of the kinetics of cation movement. Uptake rates for "K+, "Rb+, and I3'Cs+ were measured directly and halftimes for equilibrationat 18 "C were determined to be 350 ms, 2.5 s, and 10 s, respectively, in this vesicle population. zzNa+ equilibration occurred within the mimimum quenching time of the apparatus(90 ms)but an upper limit of 50 ms at 18 "C could be assigned to its half-time. Based on this upper estimate for Na+, cation selectivity ratios of the batrachotoxin-activated Na+ (l):K+ (0.14):Rb' (0.02):Cs' (0.005). channel were Toxin-stimulated influx could be blocked by saxitoxin with a K i of -5 X IO-@ M at 18 "C.Rates of cation movement through veratridine-activated channels were much slower, with half-times of 1.0,1.2,2.0, and 2.6 min at 36 O C for Na+, K+, Rb', and Cs+, respectively. The temperature dependences of batrachotoxin and veratridine-stimulated cation uptake were markedly different. The activation energies for "Rb+ and la7Cs+ movement into batrachotoxin-activated vesicles were 7 . 6 and 6.1 kcal/mol, respectively, while comparable measurements for these two cations in veratridineactivated vesicles yielded activation energies of 31 kcal/mol. Measurements of cation exchange with batrachotoxin-activated channelsmay reflect characteristics of an open sodium channel while the process of channel opening itself may be rate-limiting when veratridine is used for activation.

duced by transient changes in membrane conductance to sodium and potassium ions (I). These time- and voltagedependent ion conductances are controlled by intrinsic membrane proteins that span the bilayer and provide an aqueous pathway or channel forion movement ( 2 ) . The molecular characterization of these sodium and potassium channels has become an active topic of current neurochemical research. The past several years have seen significant progress in the isolation and biochemical characterization of the voltagedependent sodium channel. A sodium channel protein has been purified from eel electroplax (3), rat skeletal muscle sarcolemma (4), and rat brain synaptosomes (5). In each case, a large glycoprotein has been identified that exhibits anomalous migratory behavior on SDS-PAGE' (5-7). In the two mammalian channel preparations, several smaller peptides are also thought to be components of the purified sodium channel (5, 7). A number of investigators have studied the reconstitution of unpurified sodium channels or channel-containing membrane fragments into artificial liposomes (8-11). More recently, we reported the functional reconstitution of a purified sodium channel from rat sarcolemma into phosphatidylcholine vesicles (12). This purified channel protein retained its ability to gate 22Na+fluxes in response to activation by the alkaloid neurotoxins batrachotoxin and veratridine; these fluxes were specifically blocked by saxitoxin. Similar results have now been obtained with the sodium channel partially purified from rat brain synaptosomes (13). Cation flux through opened sodium channels occurs very rapidly, andtherate of cation uptake into reconstituted vesicles through batrachotoxin-activated channels could not be resolved in our earlier studies (12). In this report, quenched flow kinetic techniques havebeen applied to the purified, reconstituted sarcolemmal sodium channel in order to measure the kinetics of uptake for various alkali metal cations. The sodium channel selectivity among Na+, K+, Rb+, and Cs' has been determined following batrachotoxin or veratridine stimulation,andthe activation energies for cation influx measured.
MATERIALS AND METHODS

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The electrical signals or action potentials that characterize the surface membranes of nerve and muscle are usually pro-

* This work was supported inpart by National Institutes of Health Grants NS-18013 and GM 29603 and by a grant from the Muscular Dystrophy Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 Recipient of a Muscular Dystrophy Association postdoctoral fellowship.

Materials used in the purification of sarcolemma and in the isolation of the sodium channel protein were reported previously (4, 7, 14). Chemicals used in the reconstitution were as detailed by Weigele and Barchi (12).Batrachotoxin was the gift of Dr. J. W. Daly of the National Institutes of Health. The isotopes "Na+, %b+, '%+,and 137Cs+ were purchased from New England Nuclear Co. Dowex 50-X8
I _ _ -

' The abbreviations used are: SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; NP-40, Nonidet P-40; EGTA, ethylene glycol bis(/3-aminoethylether)-N,N, N ',N'-tetraacetic acid.

7519

7520

Cation Selectivityof Reconstituted Sodium Channels

less than 1%) or in the presence of an equivalent amount of ethanol alone. Following the preincubation, 3 3 0 - ~ aliquots l of vesicles were equilibrated to the assay temperature (18C unless otherwise indicated) and uptake was initiated by the addition of Na+ (usually 8 pl of a 400 rCi/ml solution of isotope). At 15 and 45 s, 150 $1 of the vesicle suspension were removed and rapidly applied under pressure to a small (1.5 ml) Dowex cation exchange column (50-W-X8, 100200 mesh, Tris form) in a disposable syringe barrel (20). Two 0.8-ml washes of isotonic sucrose containing 1mg/ml bovine serum albumin were then forced through the column under pressure. The entire elution and wash required about 10 s. Using this technique, more than 99.9% of the extravesicular cations were taken up by the resin while greater than 98% of the vesicles were recovered in the eluate (12). The eluate was subsequently counted in 10 ml of scintillation fluid in order to quantitate the concentration of labeled cations in the vesicles. Quenched Flow Measurements-In order to measure the rapid uptake of cations into batrachotoxin-activated vesicles, a quenched flow instrument was used. This instrument was similar in design to that described by Gutfreund (21) but was modified to incorporate the pulsed flow mode of Fersht and Jakes (22). Reaction times of less than 300 ms were obtained using a single push in which the age of the solution was determined by the volume of the aging tube and the flow rate. Longer times were obtained using the pulsed flow mode in which the mixed solutions were held in the aging tube for an electronically determined time before being expelled. An important difference between this instrument and those previously described is that the two reactants (in this case, batrachotoxin-activated vesicles and labeled cation) were loaded into two capillary tubes placed between the mixing chamber and the drive syringes while the drive syringes contained only buffer. This allowed volumes of reactants as small as 25 $1 to be used. Test reactions were used as described by Gutfreund (21) to show that the mixing time of the instrument was less than 4 ms. Furthermore, results were obtained from this instrument that were identical with those generated by conventional quenched flow instruments with model enzymatic reactions. Full details of the construction and performance of the instrument will be presented elsewhere? For either single push or pulsed flow operation, the mixed solution containing vesicles and labeled cation was quenched by direct injection into a slurry of Dowex 5O-W-XS (2.5 ml; 200-400 mesh) in the Tris form. Under these conditions, the cation exchange resin rapidly sequestered residual cations in the extravesicular space. The vesicles were then rapidly separated from the Dowex by positive pressure and the resin was washed twice with 0.8-ml aliquots of isotonic sucrosebovine serum albumin buffer. The cation content of the vesicles was determined by liquid scintillation counting. The overall quenching time for the quenched flow system including the Dowex resin step was determined as detailed under Results by measurement of the early linear time course of K+ uptake into batrachotoxin-activated vesicles. A quenching time of approximately 90 ms was obtained. Attempts a t stopping cation influx at faster M tetrodotoxin were times by quenching the reaction solution in unsuccessful.
RESULTS

(200-400 mesh) was obtained from the Sigma Chemical Co. Saxitoxin was provided by Dr. E. J. Schantz of the University of Wisconsin. The toxin was tritiated by New England Nuclear Corp. and purified as described by Ritchie et al. (15). Purification of the Sarcolemmal Sodium Channel-Sarcolemmal membranes were isolated from rat skeletal muscle of mixed fiber type using a LiBr extraction procedure as described previously (14). Pepstatin (0.1 rg/ml), phenylmethylsulfonyl fluoride (0.1 mM), iodoacetamide (1 mM), and EGTA (0.2 mM) were present during membrane isolation. Sodium channel protein was purified from these membranes using a modified form (7) of our original purification protocol (4). Briefly, 100-150 mg of sarcolemmal membrane protein were solubilized at a final concentration of 2 mg/ml in 1%NP-40 detergent in M choline chloride, 50 m M potassium phosphate the presence of 100 m (pH 7.4), and 0.5 m M CaC1, at 0 C. The protease inhibitors pepstatin (0.1 pglml), leupeptin (1pglml), o-phenanthroline(1mM), andphenylmethylsulfonyl fluoride (0.1 mM)were present during solubilization. After centrifugation, the supernatantwas diluted to 1 mg of protein/ m l with buffer containing 100 mM choline chloride, 50 m M potassium phosphate, 0.5 m M CaCI2,and the above protease inhibitors. Subsequent buffers also contained 0.1% NP-lO/asolectin phosphatides at a 5:l molar ratio. The channel protein was purified first on a guanidinium-Sepharose column and subsequently on a wheat germ agglutinin-Sepharose column. The column dimensions and conditions are detailed elsewhere (7). The guanidinium-Sepharose column described previously (4)was (Eastman Kodak) synthesized by coupling 3,3-diaminodipropylamine to Affi-Gel202 (Bio-Rad) to form an immobilized support with a 19atom extended spacer arm, and subsequently converting the terminal primary amine of this arm to a guanidinium group by reaction with 0-methylisourea. Briefly, a 2 M aqueous solution of 3.3-diaminodipropylamine was prepared immediately prior to use and titrated to pH 7.4 a t 0 C with HC1. Affi-Gel 202 (45 ml of packed resin) was suspended in H20 to a volume of 75 ml and mixed with 75 ml of fresh 2 M 3,3-diaminodipropylaminesolution; solid NaCl was then added to a final concentration of 0.1 M. Coupling was initiated by the (2 g total, addition of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide added in 100-mg aliquots over 20 min) and the reaction was allowed to proceed with stirring at room temperature for 5 h. The pH was maintained at 4.7 throughout the reaction period. The resin was washed with 1500 mlof NaCl(0.2 M) followed by 1500ml of H20 on a siliconized sintered glass funnel, and resuspended in 75 ml of HzO. 75mlof an 0.75 M aqueous solution of 0-methylisourea (pH 10.0) was cooled to 0 C, added to the resin suspension, and stirred at 3 C for 24 h. The final resin product was washed extensively with 0.2 M sodium chloride and H 2 0 prior to use. Proteins were determined with a micro-adaptation of the Lowry method (16). Specific binding of [3H]saxitoxin was measured as previously described (4, 17, 18). Reconstitution of the Purified Sodium Chnnel Protein-Peak fractions from the wheat germ column of the sodium channel purification were pooled and used immediately for reconstitution into egg phosphatidylcholine (Sigma Chemical Co., type V-E, 99% purity) as previously described (12). Stock phosphatidylcholine (50 mg/ml) was prepared in 10%NP-40 by stirring under argon. The purified sodium channel solution was obtained in 100 m M NaCI, 20 m M potassium M MgCI,,0.5 m M CaC12, 0.05% NP-40/ phosphate (pH 7.4), 0.5 m phosphatidylcholine. The concentrated phosphatidylcholine/NP-40 stock was then added to obtain a final concentration of 1% NP-40 and 5 mg/ml phosphatidylcholine. Bio-Beads SM-2 were prepared as described by Holloway (19) and 0.3 g of moist, packed beads were added per ml of reconstitution solution. Detergent removal was accomplished by gentle tumbling a t 3 C for 3 h. After this period, the residual NP-40 concentration was estimated to be (0.001% as measured by the intrinsic fluorescence of the NP-40 relative to a standard solution. The resultant vesicle suspension was filtered through glass wool to remove the Bio-Beads and used directly for flux experiments. In some cases, reconstitutions were performed in solutions containing differing concentrations of cations, or with the addition of saxitoxin, as specifically indicated in the text. Cation Flux Measurements-As a standard for comparison, assays of batrachotoxin-stimulated 22Na+ uptake at 15 and 45 s were done on each preparation. These assays, as well as allassays of veratridinestimulated uptake, were done using a manual technique as previously detailed (12).Briefly, channel-containing vesicles in buffer containing 100 m M NaCl, 20 m M potassium phosphate (pH 7.4) were preincubated for 45 min at 36 C in the presence of 5 X M batrachotoxin prepared in ethanol (final ethanol concentation in incubation was

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In the experiments reported in this paper, the conditions for sodium channel reconstitution were kept as constant as possible (see Materials and Methods). We found previously that theresults obtainedfrom reconstitutions with our purest sodium channel preparations (2000-3000 pmol of saxitoxin binding/mg of protein, representing the product of a threestep purification including a final sucrose gradient (7)) were comparable to those obtained with sodium channels carried through only two steps of purification and having a slightly lower specific activity (typically 1000-2200 pmol/mg) (12). For the studies reported here, we chose to optimize the number of experiments which could be carried out with a given preparation by using the larger quantities of channelprotein available after the second column (wheat germ agglutininSepharose) in our purification protocol (7). The saxitoxin-binding activityof the pooled fractions from
J. F. Eccleston and R. Messerschmidt, manuscript in Preparation.

Selectivity Cation

of Reconstituted Sodium Channels

7521

the wheat germ agglutinin-Sepharosecolumnranged from 980 to 2,840 pmol/mg of protein in the 40 preparations used to generate the results reported below. The mean specific activity of the preparations used was1,245 pmol/mg of protein ("40% purity based on theoretical maximal binding of 3,180 pmol/mg for a protein of M, = 314,000 (23)). In spite of the variability in specific activity, the major protein components of the material used for reconstitution were the same and included a large glycoprotein which ran anomalously in the high molecular weight region of our 7-20% SDS-PAGE and two smaller peptides of M, "38,000 and 45,000 (Fig. 1) (7). The M, = 38,000 component often appeared as two closely spacedbands. Despite the wide range of specific activities obtained in different preparations, SDS-PAGE showed very similar gel patterns, suggesting that the variability resulted from the loss of high affinity saxitoxin binding rather than the presenceof variable amounts of contaminating proteins. We have previously shown thatpurified sodium channel protein loses its capacityfor high affinity saxitoxin binding with time although its apparent subunit composition shows no change (7). The average concentration of sodium channels in preparations prior to reconstitution 38.6 was f 12.2 pmol of saxitoxinbinding sites/ml and the average protein concentration was 0.031 & 0.013 mg/ml. All reconstitutions were carried out with

.
20

.
50

.
60

I O
SPECIFIC STX

30 40

70

BINDING ACTIVITY (prol/mg proleon)

Rotem I rq/ml I

FIG. 2. Batrachotoxin activationratio and control "Na* influx at 15 s in representative sodium channel reconstitutions. A, the ratio of batrachotoxin(BTX)-stimulated uptake to control uptake of 22Naa t 15 s in 20 preparations is plotted against binding measured in that the specific activity of ['H]saxitoxin (STX) preparation prior to reconstitution. The stimulation ratiowas variable and little correlation was found between stimulated uptake activity and specific activity over the range encountered in this study. The solid line indicates the least squares linear regression to the data (r= 0.10). B, the absolute level of control (unstimulated) **Na' uptake at 15 s for 21 preparations versus the total concentration of protein present at thetime of reconstitution. A weak correlation was found, suggesting that increasing nonspecific influx was associated with increasing protein concentration. The line indicates the least squares linear regression to the data points ( r = 0.56).

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egg phosphatidylcholine at a concentration of 5 mg/ml; the average protein:lipid ratio for the studies reported here was therefore 0.006 mg of protein/mg of phosphatidylcholine. X 10-3 Forty to50% of both the saxitoxin-binding sites and the total protein was typically recovered in the reconstitutedvesicles. The efficiency of each reconstitution was evaluated with a standard assay measuring the batrachotoxin-stimulated influx of "Na+ at 15 and 45 s as well as the control or leakage of batrachinflux of "Na+ at these time points in the absence *0 116 otoxin. The total batrachotoxin-stimulated sodium uptake a t 15 s rangedbetween 1.3 and 5.9 times the nonspecific or 94 h control uptake at this time point (mean = 2.8 f 1.2). Consid68 erable random scatter in this ratio was seen among preparations although the value obtained in any given preparation Yg showed little variability over a 24-h period. We found little correlation between the magnitude of the stimulated/control 43 uptake ratio and either the specific activity of saxitoxin binding (Fig. 2 . 4 ) or the concentration of binding sites/ml in the i reconstitution solution over the narrow range of values used 30 for this study,suggesting that other undefined factors were of more importance in determining the final activityof a given reconstitution. No systematic attempt was made to vary the 21.5 sodium channelproteinconcentration overa wider range a correlation with channel concentration might where such 14.4 be expected to be resolved. The nonactivatedor leakage to both sodium channel concentrauptake was also compared FRONT tion and total protein concentration. Again, no relationship was seen between the magnitude of the leak uptake and the FIG. 1. 7-20s SDS-PAGE of a sodium channel preparation concentration of saxitoxin-binding sites, but there was a weak prior to reconstitution (A) and following reconstitution into correlation between increasing concentation of total protein phosphatidylcholine vesicles (B). The specific activity of this and increasing nonspecific leakage flux (Fig. 2B). preparation was-1,200pmol of saxitoxin bound/mg of protein. Quenched Flow Measurements-Quenched flow techniques Three major components are seen which are also present in the of cation influx into batrachhighest specific activity preparations analyzed a large glycoprotein were used to study the kinetics that runs anomalously in the high molecular weight region ( I ) , a otoxin-activated vesicles. Typically, 8-10 time points were smaller band with M, = 45,000 (2), and an additional component used to define an uptake curve and 2-3 measurements were often resolved as a doublet a t M,= 38,000 (3A and 3B). For this gel, made at each time point with activated and control vesicles. 200 ng of protein were labeled with 1251-Bolton-Hunter reagent followControl values were subtracted from the total uptake to give ing denaturation in SDS using the method of Shing and Ruoho (37). the specific uptake. Examples of uptake curves for 4'K+ and Aliquots were subjected to electrophoresis as detailed elsewhere (7) x6 Rb+ are shownin Fig. 3. In general, the control (nonstimuand bands were visualized by autoradiography using a DuPont Cronex enhancing screen. lated) influx measured with this rapid technique was much

MW

ill

i - *2;

7522

Cation Selectivity of Reconstituted Sodium Channels

,18

Q
X

W Y

E6

g 4

+ 9
[L

% Z

I
TI ME (seconds1 TIME (seconds) FIG. 3. Time course of "K' and -Rb+ uptake in batrachotoxin-activated vesicles from two typical reconstitutions. Purified sodium channel protein (specific activity, 1220 pmol/mg of protein for A , 1960 pmol/ mg of protein for B ) was reconstituted as detailed under "Materials and Methods." Vesicles were preincubated 0 " M batrachotoxin (BTX) in ethanol or with an equivalent volume of ethanol alone at 36 'C for either with 5 X 1 45 min, then equilibrated to 18 "C. For each data point shown, 25 p1 of vesicles and 25 pl of buffer containing either "K+ ( A ) or &Rb+ ( B ) were used in the quenched flow apparatus to assay uptake at each time as described in the text. 0, total uptake of labeled cation in batrachotoxin-activated vesicles; A, cation uptake in vesicles incubated with 1%ethanol alone; specific batrachotoxin-stimulated cation uptake. Downloaded from www.jbc.org at BENEMRITA UNIV AUTNOMA DE PUEBLA, on April 16, 2013

lower than that seen using the manual technique at 15 and I I T of cation 45 s, relativelylong times compared to the rate equilibration. The kineticsof uptake for eachof the alkali metal cations except *'Na+ could be resolved unequivocally using the quenched flow technique. '*Na+ uptake was essentially complete at the earliest time point, taken with 8 ms between mixing of vesicles and isotope and the start of the Dowex ) . Theactualelapsedtime, however, must quench (Fig. 4 include the time between the injection of the mixed solution into the Dowex resin and the binding of all extravesicular Na+ to the resin, since this interval will allow additional isotope movementintoactivated vesicles. Thisquenching time for the system was determined by careful measurement of the early, linear phase of uptake for &'K+, the isotope with the fastestresolvable uptake rate. Extrapolationof corrected specific 42K+uptake values to base-line (Fig. 4) yielded an effective quenching time of 90 ms. Using this value for the quenching time, an upper limit of 50 ms could be set for the Time (msec) actualhalf-time for **Na+equilibrationundercomparable conditions. FIG. 4. Quenched flow measurements of "Na+ and '"K' influx in batrachotoxin-stimulated vesicles at short incubation =Rb+, and137C~+ The timecourse for equilibrationof 42K+, into batrachotoxin-activated vesicles was clearly resolved US- periods. Data points represent the specific batrachotoxin-stimulated ing the quenched flow technique, and the half-times for vesicle uptake corrected for control uptake. Control uptake at these rapid time points was low, typically less than 20% of the total batrachoequilibration were directly measured (Fig. 5). Half-times for toxin-stimulated sodium uptake. Control vesicles formed in the abthese cations were sufficiently slow that the quenching time sence of protein showed no cation influx under these conditions for of the apparatus was of significance only for 42K+;for that the short intervals being studied. Each point represents the mean f was S.D. of three or more separate measurements after correction for cation, a smallcorrectiontothemeasuredhalf-time necessary. The half-time for 42K+uptake, calculated either control influx. The effective quenching time for the apparatus was from initial rate data as shown in Fig. 4 or from the complete determined by extrapolation of the initial linear uptake rate for "K+ into batrachotoxin-activated vesicles (O), yielding a value of 90 ms. influx as in Fig. 5, was approximately 350 ms, while that for 2zNa+ influx on this time scale (0) was very rapid and an initial rate =Rb+ and 1 3 T s + were 2.5 and 10 s, respectively. The wide could not be resolved; an upper limit of 50 ms for the half-time for spread of values for these four alkali metal cations indicates 22Na+could be estimated based on the measured quenching time of significant cation selectivity inthe purified, reconstituted 90 ms for the system. sodium channel. Using an upper limitfor the "Na+ half-time of 50 ms, thecalculated ionselectivity ratios were (Na+) containing active sodium channels was fairly constant from preparation to preparation. The larger standard deviations l:(K+) 0.14,:(Rb+) O.O2:(Cs+) 0.005 (Table I). Reproducibility of time courses for uptakeof a given cation seen with **Na+measurements may reflect the more signifiof variability in quenching time to measured was good from reconstitution to reconstitution. For example, cant contribution influx of this cation into activated points shown for 42K+uptake on Fig. 4 were derived from uptake because of the rapid three separate reconstitutions, yet all points fall along the vesicles. same timecourse. Similar reproducibility was seen with1 3 7 C ~ + Veratrdine-stimulated Cation Znflux-Veratridine-stimu=Rb+, and 137Cs+ and =Rb+, suggesting that the size distribution of vesicles lated influx was measured for "Na+, 42K+,

.,

Selectivity Cation

of Reconstituted Sodium Channels

7523

control influx at the time of peak stimulated influx for these four cations was 1.7. Temperature Dependence of SpecificCationInflux-Batrachotoxin- and veratridine-activated cation influxes differed markedly in their temperature dependence. With veratridineactivated vesicles, specific '*Na+-activated influx was barely detectable at temperatures below 20 "C but increased sharply with increasing temperatures. A typical experiment illustrating this point is shown in Fig. 7A. In other preparations,

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12 1 6 Time (sec)

20

FIG. 5. The time course of 4aK+, "Rb+, and "'CS+ uptake into batrachotoxin-activated vesicles as determined using the quenched flow apparatus. Specific uptake data from several preparations, corrected for control uptakeand plotted asper cent maximal ffiRb+(O),and 13?Cs+(0). activated space, are shown for '*K+ (A), The maximal space accessible through activated sodium channels was the same for all cations in any given preparation. Maximal 22Na+ Time (min) uptake was already observed at the shortest interval shown on this FIG.6. Veratridine-stimulated cation uptake into vesicles graph (0). All measurements shown were made at 18 "C. containing the reconstituted sodium channel. In each case, M veratridine or an equivalent vesicleswere incubated with 5 X TABLE I volume of ethanol alone for 45 min at 36 "C.All assays were carried Rates of cation uptake in bactrachotoxin- and veratridine-activated out at 36 "C as described under "Materials and Methods." Uptake rhnnnh was initiated by the addition of the labeled cation. For the data Batrachotoxin Veratridine shown, control uptakehas been subtracted from total toyield specific Cation veratridine-stimulated uptake and each data point was plotted as t; Ratiob t.* Ratio percentage of total activated space to facilitate comparison. Data rnin from two separate reconstitutions areshown ( 0 , O ) . 22Na+ 550 ms 1.o 1.0 1.0 42K+ 0.83 3501.2 ms 0.14 ffiRb+ 0.02 2.0 0.50 2.5 s 137Cs+ 0.38 10 s 2.6 0.005 Half-time for vesicle filling. Values represent the average of influx curves on a t least two different reconstitutions,each consisting of 510 time points in triplicate and a t least three control measurements in duplicate. Time courses with batrachotoxin were carried out at 18 "C;those with veratridine were performed at 36 "C. * Selectivity ratios for the batrachotoxin-activated channel were of "Na uptake of 50 ms calculated assuming an upper limit for the tu,+ (see "Results").
2 4 6 8 1 0
I/T x 1 0 '

in reconstituted vesicles that were identical in all characteristics with those used for batrachotoxin studies (Fig. 6). In all cases, cation influx occurred much more slowly for a given cation than in parallel measurements in batrachotoxin-stimulated vesicles (Fig. 5 ) . Although measurements of veratridine-stimulated uptake could be made in the quenched flow device using a double push mode to retain the sample after mixing for longer intervals prior to quenching, time courses were so slow that most assays were carried out manually. The half-time for 22Na+ uptake at 36 "C wasapproximately 1 min, =Rb+, and 13Ts+were 1.2,2.0, while those measured for 42K+, and 2.6 min, respectively (Fig. 6). With each cation, however, the total space accessible through veratridine-activated channels was approximately the same as that accessible through batrachotoxin-activated vesicles in a given preparation. Control influx was nonlinear over these long time intervals, with the most rapid nonspecific influx occurring during the first 15 s (12). The average ratio of total stimulated influx to

Time (mm)

FIG. 7. Temperature dependence of veratridine-stimulated 22Na+influx ( A ) and Arrhenius plot of "Rb+ uptake in baand veratridine (VER)-activated vesicles trachotoxin (@TX) (B).In A, vesicles were activated with 5 X M veratridine and
equilibrated at the experimental temperature prior to addition of 22 "C (O),18 "C isotope. Influx was measured at 36 "C (O),30 "C (A), (O),and 10 "C (A). The corrected specific uptake data are plotted as per cent maximum stimulated uptake. In B, initial uptake rateswere measured for @Rb+influx in two parallel populations of vesicles stimulated with either batrachotoxin (0)or veratridine (0)at temperatures between 5 and 36 "C. In each case, activation was carried out at 36 "C for 45 min with the appropriate toxin, and vesicles were pre-equilibrated to theassay temperature priorto initiation of cation uptake. For ease of comparison, the stimulated uptake rates were normalized to the rate obtained in each preparation at 36 "C.Linear regressions of the results of the Arrhenius plots were used to calculate activation energies. The much higher temperature sensitivity exhibited by the veratridine-stimulated ffiRb+ uptake ascompared to batrachotoxin-stimulated uptake is typical for each of the cations that was studied.

7524

Selectivity Cation

of Reconstituted Sodium

Channels

measurements were focused onearlytimepointsateach temperature toallow quantitation of the rateof vesicle filling during the early, linear phase of uptake and these values were used to construct Arrhenius plots for stimulated cation influx. An activation energy of23.6 kcal/molwas determined for veratridine-activated *'Na+ influx. The Arrhenius plot was linear over the temperature range of 15-36 "C in which reliable measurements could be made. Similar studies were carried out with =Rb+ (Fig. 7B) and 137Cs+. In both cases, linear TIME Iseconds) TIME (m~nuterl Arrhenius plots were obtained, with high activation energies (31 kcal/mol) (Table 11). FIG. 8. Uptake of "Rb+ ( A ) and*'Na+ (B) into activated Batrachotoxin-stimulated cation influx was much less tem- vesicles in the presence of varying unlabeledmonovalent peraturedependent. ?'Na+ fluxescouldeasily be detected cations. In A , vesicles were reconstituted either with 100 m M NaCl throughout the temperature range of 5-36 "C, but the rapid (0)or 100 mM RbCl (A) inside and outside of the vesicles. Solutions M M&12 initial rate precluded accurate quantitation. For comparison also contained 20 mM potassium phosphate (pH 7.41, 0.5 m with veratridine data, initial rate measurements were there- and 0.5 mMCaC12. In B, the total Na+ concentration in the internal M while total ionic fore made with "Rb+ and Is7Cs+ in batrachotoxin-activated and external buffer was varied from 20 to 100 m strength was maintained with reciprocal additions of choline. All vesicles using the quenched flow appratus. Uptake rates for solutions contained 20 m M potassium phosphate (pH 7.4), 0.5 m M these cations could be readilyresolved within the temperature MgCI,, and 0.5 m M CaCI2. 0, 20 m M NaCI, 130 m M choline chloride; rangestudied. Arrhenius plots of the data appeared linear 0, 50 m M NaCl, 100 m M choline chloride; 0, 100 m M NaCl, 50 m M choline chloride. throughoutthisrangewithout evidence of a breakpoint, although thelimited number of temperatures studied restricts I our resolution(Fig. 7 B ) . Calculated activation energies for N =Rb+and 137Cs+were much lower (7.6 and 6.1 kcal/mol, respectively) in batrachotoxin-activated than in veratridineactivated vesicles (Table 11). Flux MeasurementsinOther Ionic Enuironments-For most flux measurements in this study, solutions inside and outsidethe vesicles contained 100 mM NaCland 20 mM 7.4). Under these conditions, measpotassium phosphate (pH urements with tracer amounts( 4 0 FM) of '*Na+, 4'K+, =Rb+, or l:T!s+initially added only to the external solution represent of isotope exchange measurements inwhich the measured rate movement was assumed to be independent of other monovalent cations in the solution. In a control experiment, reconstitution of a preparation of sodium channels was carried out either in 100 mM NaCI, or 100 mM RbCI, and the influx of %Rb+was measured in each case. Identical values for the halftime of %Rb+ equilibration were obtained in the two solutions o w / 0 IO-^ 10-6 10" (Fig. 8A). [S T X ] (MI It has been reported that an external cation-binding site must be saturated in order for veratridine to activate sodium FIG. 9. Saxitoxin (STX)inhibition of "Rb+ uptake into bachannels insome excitable cells(24). In an attempt to address trachotoxin-activatedvesicles. Vesicles werepreincubated with 5 M batrachotoxin andthe indicated concentration of saxitoxin this question, vesicles containing thepurified channel from a X uptake was measured at 5 s after mixing single preparation were reconstituted in solutions containing at 36 "C for 45 min. ffiRb+ sodium concentrations between 20 and 100 mM while total of isotope and activated vesicles, about twice the half-time for specific vesicle filling with this cation in the absence ionic strength was maintainedconstant by the reciprocal batrachotoxin-activated of saxitoxin. Data have been corrected for nonspecific uptake and for addition of choline. Z2Na+ influx measurements were then the contributions of inward-facing sodium channels not inhibited by carried outwith eachset of vesicles activated with veratridine. external saxitoxin. The solid line indicates a theoretical inhibition In each case, the half-time forZ2Na+influx was the same and curve for saxitoxin assuming a K, of 5 X lo-' M. All assays were showed no dependence on sodium concentration within this carried out at 18 "C. range (Fig. 8B). Saxitoxin Inhibition of Batrachotoxin-actiuated Cation Zn- techniques with15-stime resolution that batrachotoxin-stimflux-We havepreviously shownusingmanualmeasuring ulated **Na+ influx could inhibited be by saxitoxin (12). Under those conditions, the concentrationof saxitoxin required for TABLE I1 complete inhibition of cation flux was well above the Kd for Temperature dependence of the initial rate of isotope filling with equilibrium binding of this toxin to the channel. This discrepbatrachotoxin- and veratridine-activated vesicles ancy was explained by the rapidly reversible nature of saxiVeratridine Batrachotoxin toxin binding, the very short period required for filling of a Cation given vesicle (typically tens of milliseconds), and the relatively E." E. long influx period permitted by the assay method being used. kcallma1 Saxitoxin inhibition of activated cation influx was re-exam"ZNa+ 23.6 ined here under conditions inwhich the initial rate of cation 31.0 ffiRb' 7.6 137CS+ uptake could be resolved. 31.0 6.1 The influx of "Rb+ was quantitated in batrachotoxin-stimE,, the activation energy for ion influx, calculated from Arrhenius plots of initial rates of ion uptake at various temperature between 5 ulated vesicles, and the inhibition of this rate was measured as a function of saxitoxin concentration. Experiments were and 36 "C.

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Cation Selectivityof Reconstituted Sodium Channels carried out either with inward-facing channels blocked by saturating concentrations of saxitoxin within thevesicles, in which case complete block of initial influx was obtained with titration of external sites, or with no internal saxitoxin where a maximal inhibition of 50-70% was obtained with titration of outward-facing channels as previously reported (12). In either case, the apparent Ki for saxitoxin inhibition of =Rb+ influx was 5-10 nM (Fig. 9) at 18 "C, approximately the same value as the Kd for saxitoxin binding in these vesicles at this temperature measured by equilibrium binding of [3H]saxitoxin (12).

7525

trachotoxin have been shown to have a lower maximal conductance (26) and a lower cation selectivity (28) than those opened by depolarization. Cation selectivity values based on isotopicflux measurementsthroughbatrachotoxin-opened channels in tissue-cultured nerve and muscle also suggest that a variable degree the ion selectivity of the this toxin alters to channel (24,29). The cation selectivity demonstrated here for the purified, reconstituted sarcolemmal sodium channel falls in the range of those reported in the literature for batrachotoxin-activated channelsin. situ;although apparentselectivity is greater than in many batrachotoxin studies, it is less than that expected for the voltage-activated sarcolemmal sodium DISCUSSION channel in its active state(30, 31). Cation influx through veratridine-activated channels ocThe sodium channelpurified from rat skeletal muscle sara much slower time scale than that through batrachcolemma has been reconstituted into egg phosphatidylcholine curred on vesicles containingthesame vesicles. This purified channel retained the ability to specifi- otoxin-activatedchannelsin cally gate cation fluxes in response to activationby batrach- purified sarcolemmal sodium channel.Sinceveratridine is otoxin and veratridine a t concentrations comparable to those known to be only a partial agonist for channel opening (32), active on the channel in situ(12). These cation fluxes were and voltage clamp studies suggest that veratridine-modified specifically blocked by saxitoxin, and we show here that the channelsactivate 1000-fold more slowly than unmodified of these slow rates would be K, for saxitoxin inhibition of cation influx corresponds to the channels (33), one interpretation is opened only for brief periods in any given Kd measured for [3H]saxitoxin binding to the channel using that the channel asequilibriumbindingtechniques.In vesicles containing the timeinterval. If batrachotoxin-activatedchannelsare purified sarcolemmal sodium channel, cation influx stimu- sumed to be opened 100% of the time, veratridine-activated lated by bactrachotoxin occurred toorapidly for kinetic reso- channels would be opened 0.5% of the time if, for example, lutionusingmanualassaytechniques.Theapplication of the relative rates of batrachotoxin- and veratridine-activated basis. For veratridinequenched flow methodology to this system now allows the K' equilibration were explained on this then chaninflux kinetics for various alkali metal cations tobe resolved activated channels, the rate-limiting step might be and theion selectivity characteristics of the purified channel nel opening rather than the rate of ion movement through an to be studied. Similar quenched flow techniques have been openchannel.Thishypothesisissupported by the much used by others in the study of rapid ion fluxes in vesicles higher activation energies measured for influx of the alkali containing the acetylcholine receptor protein from eel (25). metalcationsthroughveratridine-activated vesicles ("30 Although the vesicle phospholipid composition was held kcal/mol)ascomparedtobatrachotoxin-activated vesicles constantintheseexperiments,therange observed in the (-7 kcal/mol), corresponding to Qlo values of >3 and -1.8, magnitude of specific toxin-stimulated uptake was large and respectively. These valuesmay be comparedtothose for not directly correlated with the saxitoxin-binding capacity of sodium channel activation (Qlo "3.0) and maximal sodium a given preparation. We have not yet been able to define the channel conductance ( Qlo = 1.5) measured physiologically various factors which govern the successful incorporation of using voltage clamp techniques in intact nerve and muscle functional channels into these vesicles, although a systematic (34, 35). investigation is in progress. Regardless, successful reconstiAlthough the selectivity sequence for veratridine-activated tution with measurable batrachotoxin-stimulatedspecific in- channels is the same as for those activated with batrachoflux was obtained in 40 of 42 consecutive attempts using the toxin, the apparent relative selectivity ratio between cations methods given here.Leakage (control) influxdid increase is much lower. A similar observation has been reported for with increasing protein concentration present during the re- unpurified sodium channels inserted into soybean phosphoconstitution; however, these studies do not allow us to differ- lipid vesicles by freeze-thaw cycles and activated by grayanentiate between leakage due to incompetent sodium channels otoxin I(36). Withthatpreparation, low apparentcation and leakage contributed by the presence of a contaminant selectivity was also associated with slow rates of cation equipolypeptide. libration. These results may be due in part to veratridinePhysiological studies suggest that batrachotoxin opens the induced changes in channel structure leading to a modificasodium channel by shifting its activation curve far toward tion in selectivity, and it seems probable that at least some of hyperpolarizing voltages and by eliminating sodium inactithe difference must be ascribed to such a change. However, vation (26, 27). Since the ionic conditions used here result in other factors mustbe considered in light of the small internal zero membrane potential, we expect that functioning channels volume of the vesicles under study and the rapid equilibration will be open mostof the time in the presence of batrachotoxin. time for these vesicles through opened sodiumchannels. Thus, Cation flux stimulated by batrachotoxin should then approx- if veratridine produces infrequentchannel openings, but imate cation movement through a n open channel. The rapid channels once activatedremainopen forseveral hundred rates measured for the alkali metal cations in our batracho- milliseconds, most vesicles would fill with eitherp2Na+or 4>K+ toxin-activated vesicles using the quenched flow method sup- during a single open channel event. The very similar time port this interpretation, as do the low activation energies course for uptakeof these two cations in veratridine-activated determined for Rb' and Cs+influx. Cation selectivity for the vesicles may therefore reflect the probability of channel openbatrachotoxin-activatedchannel, based onanupper limit ing rather than the true relative cation selectivity of the open estimate of the half-time for "Na' influx,was 1:0.14:0.02: channel. These considerations could also contribute to the 0.005 for Na+, K', Rb', and Cs', respectively. If the actual veratridine-stimulated Cs' and Rb' data, but the fact that value for Na' equilibration was in fact more rapid than this the ratio of the equilibration rates for these ions to the rate upperlimit,thechannel selectivity ratios would be even for Na' is lower for batrachotoxin stimulation than for verahigher than those indicated here. tridine suggests that veratridine activationitself may cause a In voltage clamp studies, sodium channels opened by ba- further reduction in channel cation selectivity.

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7526

Cation Selectivity of Reconstituted Sodium Channels

13. Talvenheimo, J. A., Tamkun, M. M., and Catterall, W. A. (1982) J. Biol. Chem. 257,11868-11871 14. Barchi, R. L., Weigele, J. B., Chalikian, D. M., and Murphy, L. E. (1978) Biochim. Biophys. Acta 550,59-76 15. Ritchie, J. M., bgart, R.B., and Strichadz, G. R. (1976) J , Physiol. (Lond.) 261,477-494 16. Markwell, M., Haas, S., Tolbert, W., and Biever, L. (1981) Methods Enzymol. 72,296-303 17. Levinson, S. R., Curahlo, C. J., Reed, J., and Raftery, M. A. (1979) Anal. Biochern. 99,72-84 18. Barchi, R. L., and Weigele, J. B. (1979) J. physwl. 295, 383-396 " . ." 19. Holloway, P. W. (1973) Anal. Bwchern. 53,304-308 D., Knowles, A. F., Shertzer, H. G., Suolinna, E."., 20. Gasko, 0. and Racker, E. (1976) Anal. Bwchem. 72,57-65 21. Gutfreund, H. (1969) Methods Enzymol. 16,229-249 22. Fersht, A., and Jakes, R. (1975) Biochemistry 14,3350-3356 23. Barchi, R. L., and Murphy, L. E. (1981) J. Neurochem. 36,20972100 24. Frelin, C., Vigne, P., and Lazdunski, M. (1981) Eur. J. Biochem. 119,437-442 25. Cash, D. J., and Hess, G. P. (1981) Anal. Biochem. 112,39-51 26. Huang, L. M., Moran, N., and Ehrenstein, G. (1982) Proc. Natl. Acod. Sci. U. S. A. 79,2082-2085 27. Khodorov, B. I., and Revenko, S. V. (1979) Neuroscience 4,13151330 28. Khodorov, B. I. (1978) in Membrane Transport Processes (Tosteson, D. C., Ovchennikov, Y. A., and Latorre, R., e&) Vol. 2, pp. 153-174, Raven Press, New York 29. Huang, L. M., Catterall, W. A., and Ehrenstein, G. (1979) J . Gen. Physiol. 73,839-854 30. Campbell, D. (1976) J. Gen. Physiol. 67,295-307 31. Pappone, P. (1980) J. Physiol. (Land.) 306,377-410 32. Catterall, W. A. (1977) J. Bwl. Chem. 252,8669-8676 33. Ulbricht, W. (1969) Ergeb. Physwl. Bwl. Chem. Exp.Pharmnkol. 61,18-71. 34. Schauf, C. L. (1973) J. Physwl. (Land.) 2 3 5 , 197-205 35. Hodgkin, A. L., Huxley, A.F., and Katz, B. (1952) J. Physiol. (Lond.) 116,424-448 36. Condrescu, M., and Villegas, R. (1982) Biochim. Biophys. Acta 688,660-666 37. Shing, Y., and Ruoho, A. (1981) Anal. Biochem. 110,171-174

We have previously shown that the purified, reconstituted sarcolemmal sodium channel retained the capacity to gate so&um fluxes in response to activation by batrachotoxin and veratridine and also contained the receptor site that allowed these fluxes to be specifically blocked by saxitoxin. We can now state that the purified channel exhibits selectivity among four alkali metal cations which is comparable to that found for the native channel in situ under similar con&tions of activation. Demonstration of voltage-dependent activation in the purified channel remains amajor goal of future research.
Acknowledgments-We thank Drs. R. Furman, J. B. Weigele, and D. Jameson for their helpful discussions. The expert technical assistance of Lois Murphy and Steven Packardas well as theassistance of Nancy Goodman in the preparation of the manuscript are gratefully aknowledged. REFERENCES
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