Proc. Natl. Acad. Sci. USAVol. 75, No. 4, pp. 1703-1707, April 1978Biochemistry

Acetylcholine-receptor-mediated ion flux in electroplax membranemicrosacs (vesicles): Change in mechanism produced byasymmetrical distribution of sodium and potassium ions

(asymmetrical physiological distribution of ions/ion flux mechanism/excitability/"desensitization")

GEORGE P. HESS, STANLEY LIPKOWITZ, AND GARY E. STRUVESection of Biochemistry, 270 Clark Hall, Cornell University, Ithaca, New York 14853

Communicated by David Nachmansohn, January 25, 1978

ABSTRACT The kinetics of acetylcholine-receptor-me-diated efflux of inorganic ions from electroplax microsacs ofElectrophorus electricus in the presence of varying alkali metalion concentrations on both sides of the membrane have beeninvestigated. The efflux, a monophasic process when the iondistribution is symmetrical (the same concentrations and typesof ions on both sides of the membrane), becomes a biphasicprocess, consisting of a very rapid initial release of ions followedby a slower first-order process, under conditions that resemblethe physiological state of the neural membrane (potassium ionsinside the microsacs and sodium ions on the outside) The initialphase of the efflux discriminates between calcium and sodiumions and is inhibited by potassium ions in the external solution.The rate constant associated with this phase is at least 40 timeslarger than the rate constant associated with the slower efflux.Both phases depend on the concentration of acetylcholine orcarbamoylcholine, and are inhibited by receptor inhibitors(d-tubocurarine and a-bungarotoxin).A simple model is proposed which relates the kinetics of the

flux to ligand-induced conformational changes in the receptor.We also indicate the relationship between the biphasic kineticsof the flux observed in microsacs to "desensitization," thephenomenon in which, on addition of acetylcholine, the trans-membrane voltage of muscle and nerve cells first increases andthen decreases to its resting value within a few seconds.

The acetylcholine receptor is a membrane-bound protein ofnerve and muscle cells which, upon binding of appropriate li-gands, controls the flow of inorganic ions through the mem-brane (1, 2). The rates with which specific ions move across themembrane play a major role in determining the transmembranevoltage of these cells and whether a signal is propagated. Al-though the acetylcholine receptor has been isolated and is beingcharacterized in many laboratories (for recent reviews see refs.3 and 4), little is known about the relationship between theconcentration of ligands and the rates with which specific in-organic ions move through the membrane. Attempts to makethese measurements were made by Kasai and Changeux (5) onmicrosac preparations and they calculated (6) that 5 X 103 ionsare transferred per receptor site per min in the presence of li-gand. However, the value obtained with microsac preparationswas less, by a factor of about 105, than the value determined byKatz and Miledi (7) and others (8-10) with cells by electro-physiological methods. We (11-12) have shown that one reasonfor the apparently low efficiency of the receptor-mediatedprocess in the experiments of Kasai and Changeux is that thereceptor-mediated flux of inorganic ions is from only a smallpercentage of the microsacs, and that the flux from microsacsthat do not respond to acetylcholine dominates the kinetic

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1703

measurements. Quantitative investigations of receptor-me-diated fluxes have recently become possible through the de-velopment of microsac preparations (13) and kinetic techniques(11), which allow one to measure such fluxes without interfer-ence by fluxes that do not depend on acetylcholine. When thecomposition of the internal and external solutions is identical(90 mM KCl/10 mM NaCl/0.4 M sucrose) the receptor-me-diated efflux of 22Na+ and 86Rb+ follows a single exponentialrate law (11). The observed rate constant increases with ligandconcentration, finally reaching a ligand concentration-inde-pendent value J (11). The number of inorganic ions that movethrough the membrane in the receptor-mediated process permin was calculated to be 106 (13). This is still lower, by a factorof about 100, than the value obtained with cells. The experi-ments reported in this paper were initiated to determinewhether this discrepancy was due to potassium ions, since earlierexperiments (11) suggested that they might inhibit the recep-tor-mediated flux of inorganic ions in microsac preparations.

MATERIALS AND METHODSElectric eels were obtained from World Wide Scientific Ani-mals, Ardsley, NY. The microsacs were prepared as describedby Kasai and Changeux (5). Microsacs in sucrose-free solutionswere prepared by pelleting the microsacs, prepared in sucrose,and resuspending them in eel Ringer's solution by the procedureof Fu et al. (14). Carbamoylcholine chloride and d-tubocu-rarine chloride were purchased from Sigma Chemical Co. andICN-K & K, respectively. Acetylcholine bromide was obtainedfrom Eastman Kodak. a-Bungarotoxin was prepared by theprocedure of Bulger et al. (15). Neutralized stock solutions (1mCi/ml) of 86RbCl, 22NaCl, and 45CaCl2, obtained from NewEngland Nuclear, were used in microsac incubation mixtures.Tetram was the generous gift of R. D. O'Brien. All otherchemicals were reagent grade and were obtained from eitherFisher Scientific or Mallinkrodt. Protein concentrations andacetylcholinesterase activities were determined by the methodsof Lowry et al. (16) and Ellman et al. (17), respectively.

Ion flux measurements were made essentially as reported byHess et al. (11). The microsac preparation, about 1.2-2.0 mgof protein per ml, was incubated overnight at 40 in appropriatesolutions containing 22Na+, 45Ca2+, or 86Rb+. The latter ion isthought to serve as an effective replacement for 42K+ (18) andhas the advantage of being a more stable isotope. At the be-ginning of each experiment the incubation mixture was dilutedto a final concentration of 140-150 ,ug of protein per ml withappropriate solutions containing 1 mM phosphate buffer pH7.0 at 4°. Before addition of activating ligand, the microsacswere allowed to remain in the dilution buffer for 120 min. Bythe end of this time the radioactive ions inside the unspecific

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Addition of ligand170 _1*---Addition of ligand

1600H

E0.U

C0

as40E

0

10mcc

1500F

1400F

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I I L I0 20 40 60

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co400

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FIG. 1. 22Na+ and 86Rb+ efflux as a function of time underphysiological conditions. For the 86Rb+ efflux, sucrose-free solutionsof microsacs were prepared by the procedure of Fu et al. (14). Themicrosacs, resuspended in a potassium Ringer's solution (150 mMKC1/9.4mM NaCl/3 mM CaClJ1.5mM MgCl2/1 mM phosphate, atpH 7.0), were incubated overnight with 86RbCl (35 MCi/ml) at 4°.Efflux was initiated by dilution into a sodium eel Ringer's solution(169mM NaCl/5mM KC1/3 mM CaCl2/1 mM phosphate, at pH 7.0)at 4°. After 120 min, ligand was added. 0, 86Rb+ efflux, 1 mM car-

bamoylcholine; A, 86Rb+ efflux, 10MuM acetylcholine (microsacs werepreincubated with 56 MAM Tetram); 0, 22Na+ efflux, 1 mM carba-moylcholine. Solid symbols represent the respective average of severalexperimental points taken before the addition of ligand. The lineswere computed according to Eq. 2 (Discussion).

microsacs had equilibrated with the external ions (11). In ex-

periments with acetylcholine, the microsacs were preincubatedfor 30 min with 56 ,M Tetram (19), an irreversible inhibitorof acetylcholinesterase. The amount of radioactive ions retainedby the microsacs as a function of time was determined by a

Millipore filtration assay at 4°.

RESULTSEfflux of 22Na+ and 86Rb+ from membrane microsacs underphysiological conditions is depicted in Fig. 1. The microsacswere incubated in an eel Ringer's solution that approximatedthe ion composition inside an electroplax cell (20), and effluxwas initiated by dilution of the incubation mixture into an eelRinger's solution that approximated the extracellular ioncomposition (21). Unlike the single exponential efflux observedby Hess et al. (11) when the composition of the external andinternal solutions was identical (Fig. 2, curve I), physiologicalconditions (a high concentration of potassium internally andof sodium externally} produced a very rapid initial efflux fol-lowed by a slower single exponential efflux at the concentrationof ligand indicated. In the measurements shown in Fig. 1 theinitial fast phase had ended before the first measurement couldbe made, 20 sec after addition of the ligand. To facilitate thestudy of the biphasic kinetics observed under physiologicalconditions, we used simplified incubation and dilution mediacontaining only NaCl and KC1 for the experiments shown inFig. 2 and in subsequent experiments. The total ionic strengthof Na+ and K+ was held constant at 100 mM both inside andoutside the microsacs. In addition, all media contained 0.4 Msucrose, thereby eliminating the need to pellet the microsacsafter the sucrose density gradient centrifugation. The results

Time, min

FIG. 2. Effect of NaCl and KCl in the external solution on 86Rb+and 45CA2+ efflux. Microsacs incubated overnight with 100mM KCl,1 mM CaCl2, 0.4 M sucrose, and 30 MCi of 86RbCl or 10MCi of 45CaCl2per ml, were diluted into a medium containing 100mM KCl or NaCland 1 mM CaCl2/0.4 M sucrose/1 mM phosphate, at pH 7.0, at 4°.After 120 min, carbamoylcholine was added to a final concentrationof 0.2 mM. (Curve I) o, 86Rb+ efflux, KCl versus KCl; (curve II) A,45Ca2+ efflux, KCI versus NaCl; (curve I) 0, 86Rb+ efflux, KC1 versusNaCl. Solid symbols represent the respective average of several ex-perimental points taken before the addition of ligand. The solid anddashed lines were computed according to Eq. 2 (Discussion).

show that under these conditions also the type of inorganic ionin the external solution determines the flux kinetics. A singleexponential efflux was observed when the internal and externalsolutions of the microsacs were 100 mM in KCl (Fig. 2, curveI), and biphasic efflux kinetics were observed when 100 mMKC1 was inside and 100 mM NaCl outside the microsacs (Fig.2, curve III). In this experiment the initial fast phase had endedbefore the first measurements could be made. The kinetics ofthe receptor-mediated efflux of Na+ and K+ were similar tothose of Rb+, but those of Ca2+ (Fig. 2, curve II) differed. It canbe seen in Fig. 2 that the initial fast process discriminates be-tween 45Ca2+ on one hand and 86Rb+ on the other. Under thesame experimental conditions, when 86Rb+ efflux was biphasicand 30% proceeded by an initial fast phase (Fig. 2, curve III),the 45Ca2+ efflux followed a single exponential rate law and aninitial fast phase could not be detected (Fig. 2, curve II).To show that the conditions under which the fast process was

observed did not cause physical disruption of the microsacs orchanges in the amount of microsacs retained by the Milliporefilter, we equilibrated the microsacs with 295 mM KCl and 10mM sucrose. Either 86RbCl (40 ,Ci/ml of incubation mixture)or [14C]sucrose (40 ACi/ml of incubation mixture) was addedto the incubation mixture. Efflux of 86Rb+ or ['4C]sucrose wasthen measured in a solution of 295 mM NaCi and 10 mM su-crose. Addition of 1 mM carbamoylcholine produced an initialfast phase in which 70% of the internal 86Rb+ was exchangedfor Na+. No effect on the [14C]sucrose efflux was observed onaddition of carbamoylcholine. In addition, it was shown that1 MM d-tubocurarine inhibited the initial fast phase as well asthe slow phase of the ligand-induced efflux. When 0.8 MAM a-bungarotoxin was added to the microsac preparation it did notaffect the passive efflux of inorganic ions from microsacs, butcompletely abolished the effect of 1 mM carbamoylcholine.

Fig. 3 shows that K+ in the external solution inhibits the initialfast phase of carbamoylcholine-induced efflux both of 86Rb+and 22Na+. The ordinate of the graph indicates the fraction ofthe flux, a, that occurs in the initial fast phase. As the molefraction of Na+ in the external solution increases, a increases.

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04r

0.3k

a 0.2

0.1

1.01Q9

0

I I I10 0.2 0.4 0.6 0.8 1.0

[Na+]

[Nat] + [K+]FIG. 3. Fraction of ligand-induced process due to fast process,

a, plotted against mole fraction of Na+ in the external medium. Mi-crosacs, incubated overnight with 100mM KCl/0.4M sucrose/35 ,Ciof 86RbCl per ml, were diluted into a medium containing variableratios of NaCl and KCl with a total concentration of 100 mM, 0.4 Msucrose, and 1 mM phosphate, at pH 7.0, at 40, to initiate efflux.Conditions for 22Na+ efflux were the same except that the microsacscontained NaCl and 22Na+ instead of KCl and 86Rb+, respectively.Carbamoylcholine was added after 120 min to a final concentrationof 0.2 mM. a, 86Rb efflux; 0, 22Na+ efflux, average oftwo experimentsdone on the same preparation on the same day.

The inhibitory effect of external K+ on the initial fast phasedepends also on the carbamoylcholine concentration, beinggreater at low than at high concentrations.The effect of ligand concentration can be seen in Fig. 4 (open

symbols). At low concentrations of carbamoylcholine (less than1.5 times K2, see Eq. 1), one observes, within the uncertaintyof the measurements of -15%, a slow efflux which obeys asingle exponential rate law. At the highest carbamoylcholineconcentrations used, 14 times K2, about 65% of the efflux is dueto the initial rapid process. The dashed line is defined by ex-perimental measurements (15) of the reaction of[125I]monoiodo-a-bungarotoxin with the receptor. This reactionis also biphasic, and a also indicates the fraction of the reactionthat proceeds by an initial rapid process.

DISCUSSIONAsymmetry of functional membrane-bound proteins such asNa+-K+ ATPase and cytochrome oxidase has been well docu-mented (22, 23). The binding of ligands to the opiate receptoris markedly affected by sodium ions (24), and the affinity ofligands for the solubilized acetylcholine receptor varies withthe ionic composition of the environment (25, 26). In this paper,we present evidence that the ionic composition of the mediumbathing the extracellular portion of the membrane-boundacetylcholine receptor exerts major effects on the ion-translo-cating properties of the receptor (Figs. 1-3). The natural stateof the membrane, with its asymmetrical distribution of Na+ andK+, gives rise to a biphasic receptor-mediated efflux with aninitial very fast flux rate followed by a slower first-order rateprocess. A high concentration of Na+ surrounding the externalportion of the receptor enhances the fraction of efflux due tothe initial rapid process (Figs. 1-3). Conversely, increases in theexternal K+ concentration inhibit the fast process (Fig. 3).

There are at least two circumstances that would give rise toan inital fast efflux followed by a single exponential efflux:

(i) Two types of microsac exist, one responding to low con-centrations of ligand and characterized by slow efflux in inor-ganic ions, the other responding only to high concentrations ofligand and from which the efflux is fast. If this were the true

Q8-0.7-0.6

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[Carbamoylcholine], mM[a-Bungarotoxin], ,uM

FIG. 4. Fraction of ligand-induced efflux due to the fast processplotted against concentration of carbamoylcholine. Microsacs incu-bated overnight with 100mM KCl/0.4M sucrose/35 pCi of 86RbCl perml were diluted into a medium containing 100 mM NaCl/0.4 M su-crose/1 mM phosphate at pH 7.0 at 4° to initiate efflux. Conditionsfor 22Na+ efflux were the same except that NaCl was substituted forKCl, and 22NaCl was substituted for 86RbCl. After 120 min carba-moylcholine was added. 0, 86Rb efflux; 0, 22Na+ efflux. The dashedline indicates the fraction of the fast phase of the reaction of [125IJ-monoiodo-a-bungarotoxin with the receptor of the microsacs. Theexperimental conditions are those that lead to biphasic efflux of in-organic ions from microsacs in the experiments shown in Fig. 1. Theprocedure for making the measurements has been described (15).

situation, the total amount of radioactive ions involved in theefflux would depend on carbamoylcholine concentration. Wefound, however, that the amount of radioactive ions involvedis the same whether the efflux is slow and monophasic or is bi-phasic.

(ii) The binding of ligands to the receptor results in theconversion of one receptor conformation into another, with onebeing associated with the fast phase of the efflux and the otherwith the slow phase. The conversion of one receptor confor-mation into another was indicated by the kinetics of the reactionof a-bungarotoxin, a specific and irreversible inhibitor of thereceptor (27), with the receptor in microsacs. The reaction wasbiphasic, an initial rapid phase followed by a slow phase (15,28). The fraction of the toxin reaction that occurs in the initialfast phase depends on ligand concentration (Fig. 4, dashed line)in a similar manner and under similar experimental conditionsas the fraction of the fast phase of the receptor-mediated effluxof inorganic ions from microsacs (Fig. 4, open symbols).

Several models, in which an equilibrium exists between atleast two states of the receptor, have been proposed to explain(i) an increased permeability of the membrane to inorganic ions(29); (ii) "desensitization," a phenomenon observed in elec-trophysiological measurements with cells (30); and (imi) anumber of ligand-binding experiments and kinetic measure-ments of the reaction of neurotoxins with the receptor (3, 15,31-37). Our kinetic measurements of the receptor-mediatedflux of inorganic ions indicate a relationship between flux ratesand receptor conformations. A simple model, which relates theligand-binding mechanism first proposed by Katz and Thesleff(30) to the kinetics of the receptor-mediated flux of inorganicions through the membrane, is shown below.

Lo + R K,_ LR

[K+] || [Na+]

Lo + R/ -LKR'K2 J2

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[Na ]1|[K ]

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In this model, Lo represents the initial concentration of ligand,R and R' the concentrations of the two conformations of thereceptor, K1 and K2 the receptor-ligand dissociation constants,and the vertical arrows the rate constants for the isomerizationof receptor conformations. The initial fast efflux of inorganicions, characterized by the rate constant J1, is associated withthe receptor conformation R. The slow phase of the efflux,characterized by the rate constant J2, is associated with thereceptor conformation R'. A feature of this model is that theinterconversion between receptor conformations is in part afunction of external Na+ and K+ concentration. We considerthis a working model, which allows one to estimate the effi-ciency of the receptor-mediated translocation of inorganic ionsand which serves as a basis for further experiments.The equation for the efflux of inorganic ions, [M+], from

microsacs, based on the model, is given by:

In [M+]t=° j L j2Lo ) (1 -ea_0t)[M + at K1 + Lo K2 + LoJ J

+ J2L t [1]K2 + Lowhere ,3 contains the ligand concentration-dependent rateconstants for the isomerization of receptor forms.Some of the predictions and assumptions made in the model

and Eq. 1 are as follows. (i) The initial ligand concentration,Lo, is much larger than the concentration of the receptor andis considered to be constant, and the equilibration between li-gand and receptor is much faster than any subsequent steps. (fi)The initial phase of the efflux is fast compared to the secondphase. Therefore, J1 is much larger than J2. (ii) When theexponential term in Eq. 1 decays, the efflux follows a singleexponential rate law identical to one derived previously (11)when the composition of the solutions on either side of the mi-crosacs was identical:

[M+]t = [M+]t=o Qexp- 2+L t) [2]

where 4 gives the fraction of the metal ions in the microsacs thatare released in the process. The solid lines in Fig. 1 and 2 werecomputed according to Eq. 2. (iv) The second and slow phaseof the efflux can occur only if the concentration of conformationR (associated with the initial rapid phase of the flux) becomesmuch smaller during the ligand binding process than the con-centration of R' (associated with the slow phase of the flux). Thisrequires K1 to be much larger than K2. (v) When K1 is muchlarger than K2, at low ligand concentrations, conformation LR'will predominate and only a slow monophasic efflux is expected(Fig. 4). At high ligand concentrations two phases may be ob-served, depending on the effect of the inorganic ions in theexternal solutions on the equilibrium between receptor con-formations R and R' and on the value of (.. If external K+ shiftsthe equilibrium between R and R' all the way to the R' state,only the slow phase of the efflux will occur. If both conforma-tions R and R' are initially present, butI is large, the initial fastphase of the efflux may involve a concentration of ions too lowto be detected by the experimental technique used, and theefflux may appear monophasic (Fig. 4). (vi{) Under the sameexperimental conditions the efflux may be monophasic for onemetal ion, for instance, 45Ca2+ (Fig. 2, curve II), and biphasicfor another, for instance, 86Rb+ (Fig. 2, curve III), for at leasttwo reasons. First, a different receptor may control the flux of45Ca2+ The values of J2 and K2 are, however, similar for86Rb+efflux in KC1 solutions and 45Ca2+ efflux in NaCl solutions, asshown by the parallel curves I and II. A second reason is thatthe J1 values are different for different metal ions. If J1 reflects

the rate at which the metal ions move through the membraneand differs for Ca2+ and for Rb+, the efflux may appear mo-nophasic for one ion and biphasic for the other. This can seenfrom Eq. 1, which shows that when L is greater than K1, a isdetermined by J1/#. (vii) Eq. 1 can be used to calculate a lowerlimit for the value of J1. Our earliest measurement was made20 sec after the addition of ligand, by which time the initial fastphase of the efflux had ended. Accordingly, the half-time ofthe isomerization process had a maximum value of 4 sec, anda had a value of about 0.65 at the highest ligand concentrationused (Fig. 4), 1.2 mM carbamoylcholine. Assuming that thisligand concentration is sufficiently greater than K1, the half-time for the isomerization process and a can be used to calculatea lower limit for J, of 0.2 sec-1, a value that is 40 times largerthan that of J2. Our previous estimate of the number of ionstransferred per unit time in the receptor-mediated process inmicrosacs (13), about 1/100 the value obtained from electro-physiological determinations, was based on measurements ofthe slow receptor-mediated efflux of inorganic ions from mi-crosacs and the value of J1. (ix) The model does not considerrelatively slow changes in receptor inactivation observed in bothion flux experiments with microsacs (38) and ligand bindingexperiments (3, 37).Now we can consider the implications of the biphasic kinetics

of the receptor-mediated efflux of inorganic ions in microsacswith respect to studies of nerve and muscle cells. Measurementsof the flux of 22Na+ from L-6 muscle cells upon addition ofcarbamoylcholine reveals a biphasic process (D. E. Moore, T.R. Podleski, and G. P. Hess, unpublished observations). Withina few seconds the observed efflux rates become equal to therates observed in the absence of ligand. Since the transmem-brane voltage of the cell is expected to depend to a large extenton the relative rates with which inorganic ions move throughthe cell membrane in the presence and absence of ligand, theincrease in the transmembrane voltage of these cells, observedupon addition of ligand, is, therefore, expected to return to itsresting value within a few seconds. The return of the acetyl-choline-induced increase in the transmembrane voltage ofnerve, muscle, and electroplax cells of E. electrcs to the restinglevel is a well-documented phenomenon (30, 39, 40) calleddesensitization. In our view desensitization is a consequence ofthe characteristic biphasic kinetics of the receptor-mediatedtranslocation of inorganic ions that results from a ligand-bindingmechanism similar to that shown in the model, which is ob-served with many regulatory enzymes (41).Some of this work formed the thesis submitted by S.L. for Honors

in the A.B., Cornell University, 1977. G.E.S. was the recipient of U.S.Public Health Service Fellowship NS 05095. This work was supportedfinancially by Grant GM 04842 awarded by the National Institutes ofHealth and Grant PCM 75-07377 awarded by the National ScienceFoundation.

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4. Eldefrawi, M. E. & Eldefrawi, A. T. (1977) in Receptors andRecognition IV A, eds. Cuatrecasas, P., & Greaves, M. F.(Chapman and Hall, London), pp. 197-258.

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