Monovalent ion effects on acetylcholine receptor from Torpedo californica

ARCHIVES

OF

BIOCHEMISTRY

Monovalent

RAYMOND Section

AND

BIOPHYSICS

Ion Effects

183-188

(I9771

on Acetylcholine californical

E. GIBSON, of Neurobiology

179,

SUSAN

and Behavior, Received

JUNI, Cornell June

AND University,

Receptor

from Torpedo

RICHARD

D. O’BRIEN

Ithaca,

New

York

14853

29, 1976

The effects of the five Group I monovalent ions, Li, Na, K, Rb, and Cs, on [3H]acetylcholine binding to Triton X-100 solubilized acetylcholine receptor from Torpedo californica electroplax were examined. Acetylcholine binding was not greatly affected by Li or Na, but was inhibited by the other ions in the order Cs > Rb > K. The inhibition by K appeared to occur by a mechanism identical to that for d-tubocurarine inhibition of acetylcholine binding.

The acetylcholine receptor (AChR12 is a membrane-bound protein which, upon complexing with acetylcholine, presumably changes its conformation and so leads to a change in the ion permeability of the membrane in which it is embedded. Because the AChR is a component of a membrane across which exist considerable ionic gradients of Na+ and K+, it was considered interesting to examine the effects of the ionic environment on ACh binding to its receptor. For example, the receptor in its normal environment is presumably oriented with the ACh-recognition site located extracellularly and thus in a medium which is relatively high in Na+ and low in K+ concentrations. Recent studies on Triton X-100 solubilized AChR from Torpedo californica (5, 12) have shown that ACh binds to the AChR with a high affinity (KH = 12 nM) and positive cooperativity. Ligand binding and inhibition studies suggested that the qualitative behavior of the solubilized AChR could account for the agonist and antagonist interactions observed electro-

physiologically in whole cells (5). We therefore undertook the study of the effect of monovalent ions upon r3HlACh binding to the AChR. METHODS The receptor preparations were made from a lyophilized heavy-membrane preparation of T. californica. The Torpedo were purchased live from Pacific Biomarine Supply Company (Venice, California). The electric organs were removed, the lyophilized plax powder was prepared as previously described, and the AChR was solubilized in 1% Triton X-100 in Ringer’s solution (11). Details of the dialysis assay for ACh binding (13H1ACh, 250 mCi/mmol, Amersham-Searle) have been described elsewhere (111. Dialysis baths contained the anticholinesterase Tetram [O,O,-diethyl S-(2-diethylaminoethyl) phosphorothiolate] at 10m4 M and r3H]ACh in Ringer’s solution (115 mM NaCI, 4.6 mM KCl, 0.65 mM CaCl,, 1.15 mM MgSO,, and 15.7 mM NaZHP04 adjusted to pH 7.4 with HCl) or the monovalent ion under study in 10 mM Na,HPO, (pH 7.4). All agents were added to the dialysis baths. Although the AChR was prepared in Ringer’s solution, the ratio of the volume of the dialysis bags to baths was kept above 200, thus diluting the salts in the Ringer’s solution far below the concentration of the added ions, i.e., Na+ in Ringer’s solution (115 mM) was reduced to 0.6 mM, compared to 100 mM, as the lowest concentration of monovalent ion added for study. The ionic strength of the preparation in Ringer’s solution was 0.175 M, while dialyses were carried out in ionic strengths ranging from 0.1 to 1.0 M. Therefore, the volume of the sample in the dialysis bags would increase or decrease relative to Ringer con-

1 From the Section of Neurobiology and Behavior, Cornell University, Ithaca, New York 14853. This work was supported, in part, by Grants NS 09144, ES TO1 00098, and GM 53317 from the National Institutes of Health. 2 Abbreviations used: ACh, acetylcholine; AChR, acetylcholine receptor; dTC, d-tubocurarine; PCMB, p-chloromercuribenzoate. 183 Copyright All rights

6 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN 0003-9861

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GIBSON,

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trols. Changes in bag volume were corrected by determining the protein concentration (7) in each dialysis bag and applying the appropriate corrections. Concentrations of monovalent ions above 0.5 na reduced the efficiency of liquid scintillation counting in a Packard Tri-Carb scintillation counter using a toluene cocktail containing Biosolve BBS-3 rated at 39.5 ml/liter. The order of the effect was K > Cs > Rb > Na > Li at 1 M salt. The greatest reduction was 26% by 1 M KCl. The reduction in counting efficiency was corrected for. It was later found that quenching in high salt concentrations does not occur in Bray’s solution (New England Nuclear). Although the studies reported herein were all performed using the toluene cocktail, the quenching corrections applied to the data have been checked using Bray’s solution and were found to be valid.

AND

O’BRIEN

.-r" 72 .G m

10 .l

1 .25

.5 b'+l

.75

, 1.0

M

FIG. 1. Effects of Group I monovalent ions upon 13HlACh binding to solubilized AChR in 10 mM phosphate buffer. Binding ratio is relative to ACh binding in 0.1 M NaCl.

RESULTS

The binding of f3HlACh to solubilized AChR in the presence of the five Group I monovalent ions in 10 mM Na,HPO, is presented in Fig. 1. The data are the average of six experiments, three using lo-’ M 13HlACh and three using 10m6M [3HlACh. Data obtained at these two concentrations did not differ and thus were averaged. As Na+ and Li+ concentrations were increased to 0.75 M, the binding of r3HlACh also increased, to a ratio of 1.2 relative to 0.1 M NaCl; at 1 M, a reduction in binding was observed. On the other hand, K+, Rb+, and Cs+ were inhibitory over the entire range of concentrations. At 1 M, the reductions (in comparison to 0.1 M NaCl) were 47% for K+, 58% for Rb+, and 63% for Cs+. The different effects may have resulted from a site-specific interaction or solely from the differences in protein solubility in the high salt concentrations. Therefore, the nature of the interactions was examined in greater detail for the electrophysiologically important monovalent ions, Na+ and K+. The effects of 1 M Na+ and 1 M K+ on ACh binding are presented in Figs. 2 and 3, respectively. The Ringer control curve in Fig. 3 showed a slight convexity in the Scatchard plot. The AChR has been shown to exhibit positive cooperativity in the binding of ACh (5) and the deviation from a linear Scatchard may reflect resid-

FIG. 2. Sodium effects as Scatchard plot of 13HlACh binding to AChR: control in Ringer’s solution (0); in 1 M NaCl in 0.01 M Na2HP0, (pH 7.4) (A); and in 1OmB M dTC, 1 M NaCl, and 0.01 M Na,HPO, (m).

ual cooperativity. The Kd for ACh estimated from the steepest part of the Scatchard plot was 2 nM. In addition, the control curve had a small low affinity component, as shown by a slight concavity at high B values, constituting less than 20% of the total ACh binding. [Acetylcholine binding curves with varying proportions of high and low affinity binding have been studied earlier (12); the low affinity form appears to result from a denaturation of the receptor (12j.l The control

ION EFFECTS

ON ACETYLCHOLINE

. . 90 %

.

B. t

;_

8 ,nrnoie*

/g f15s”eI

3. Potassium effects as Scatchard plot of r3H]ACh binding to AChR: control in Ringer’s solution (0); in 0.01 M NapHPO, (pH 7.4) only (A); in 1 M KC1 and 0.01 M Na2HP04 (+); and in 1Om6M dTC, 1 M KCI, and 0.01 M Na2HP0, (WI. FIG.

RECEPTOR

185

ACh binding was examined in 0.01 M Na,HPO, (Fig. 3). The high affinity component was found to have a B$,, of 0.505, 50% smaller than the Ringer control. Thus, BE,, in 1 M K+ and 0.01 M Na,HPO, was reduced by only 20% compared to the 0.01 M Na,HPO, control. In contrast, when 1O-6 M dTC inhibition was examined in 0.01 M Na2HP0, buffer instead of Ringer’s solution, B&, was 0.22: a reduction of 55% compared to the buffer control. The binding of ACh in the presence of K+ and dTC is presented in Fig. 3. In comparison to buffer controls, lo+ M dTC reduced BE,, by 55% and 1 M K+ reduced BE,, by 20%. With both lO-‘j M dTC and 1 M K+, the reduction in B&, was 40%, not greater than that for dTC alone. In fact, dTC reduced Bi,, by 15% more than lo* M dTC plus 1 M K+. In contrast, in comparison with Ringer controls, low6 M dTC was equieffective in the presence or absence of 1 M Na+ (Fig. 2). It is interesting to note that 1 M Na+ apparently enhanced ACh binding (B,&,) by almost twofold when compared to the 0.01 M Na,HPO, buffer curve in Fig. 3. Since K+ appeared to inhibit ACh binding in a manner identical to that of dTC, the dissociation constant for K+ was determined. The concentrations of 13HlACh were chosen such that only the high affrnity binding would be observed, even in the presence of 1 M K+, i.e., 10 nM to 0.11 PM. In this concentration range, as shown in Fig. 3, the extrapolated line would suggest a reduced BE,, and it appears on a double-reciprocal plot as if it were noncompetitive inhibition (Fig. 4). The theoretical basis of dTC inhibition has been examined in detail (5): dTC appears to act allosterically in a manner which alters half of the ACh binding sites to a low affinity form. The binding of ACh in the presence of dTC or K+ may therefore be represented by Eq. [ll. P = P (1 - YJ + Y1 (Y”,,, [ll where Y is defined as BIB,,,, pH = x/(x +

curve in Fig. 2 is substantially the same but more linear than in Fig. 3 and has an apparent Kd of 5 nM; the low affinity component contributes 30% of the total ACh binding. A 1 M Na+ concentration did not alter the binding curve as compared to its control (Fig. 2). In contrast, ACh binding in the presence of 1 M K+ gave curvilinear Scatchard plots possibly reflecting twocomponent ACh binding (Fig. 3). When analyzed on the assumption of two-component binding, the high affinity component exhibited a dissociation constant of KH = 5.6 nM and a maximum binding (B&,,) of 0.42 nmol/g of original tissue. The low affinity binding exhibited a KL of 0.95 PM with B& = 0.58 nmol/g of original tissue. Thus, the low affinity binding in the presence of 1 M K+ constituted 60% of the total binding. These results are strikingly similar to those obtained with d-tubocurarine (dTC) inhibition of ACh-binding, in which, at saturating concentrations of dTC, the contribution to ACh binding by the high affinity component was reduced by 50% (5). Although 1 M K+ reduced the BE,, by KH); FHL = 0.5 [x/(x + KH) + x/(x + KJI; more than 50% of the control, Ringer’s solution contains 115 mM NaCl (as well as and Y1 = I/(1 + Ki). The high and low other salts), which may lead to enhanced affinity dissociation constants for ACh are represented by KH and KL, respecbinding of ACh, as in Fig. 1. Therefore,

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striking contrast to the ion effects reported by Martinez-Carrion and Raftery (8), in which Na+ and K+ exhibited disso6I ciation constants of 11 and 15 mM, respectively. However, their AChR prepara5 tions exhibit only low affinity binding of ‘4 ACh; it is well established that the sensi4I tivity of high and low affinity forms is markedly different to various agents (9). In addition, the effects are different from those observed for a somewhat related protein, the axonal cholinergic binding macromolecule (2) in which all of the monovalent ions of Group I enhanced the binding of nicotine in the order Na > K > Li > Rb > Cs. FIG. 4. Double-reciprocal plot of high affinity In the case of K+, the inhibition exPHlACh binding (lo-* M ACh) to AChR in Ringer’s hibited the same pattern as that observed solution (0); in 0.2 M KC1 in Ringer’s solution (A); and in 1 M KC1 in Ringer’s solution (m). Data are for dTC (5), for which it was suggested normalized to &&, = 1.0 for control curves. that dTC binds to an allosteric site which effectively induces half-of-sites binding tively, and x is the ACh concentration. activity of ACh. When both K+ and dTC The inhibitor concentration is given by I are present in concentrations of 4 x Ki for and its dissociation constant for the recep- K+ and 29 x: Ki for dTC, the B&, value tor is Ki. Equation 111is equivalent to Eq. was not decreased with respect to dTC (viii) of Gibson (5), with the exception inhibition alone. This would be expected that cooperative effects are ignored. The if both agents acted upon the same site lines in Fig. 4 were obtained from a and dTC had saturated the site. We thereweighted regression analysis by the fore suggest that K+ binds to the dTCmethod of Wilkinson (14). The dissocia- specific allosteric site. tion constant for K+ was then determined O’Brien and Gibson (11) have demonby fitting the computed lines to Eq. [l]; strated similar conversion of high affinity the best fit values of Ki were determined binding to low affinity binding in studies by minimizing the value of x2. The lines on divalent ion interactions with the derived from this fitting to Eq. [l] coin- AChR. At concentrations of 1 mM ZnCl,, cided precisely with those derived from ACh binding appeared as predominantly the Wilkinson curve fit using KH = 7 nM low affinity. Similar results have been and K, = 1 PM. At 0.2 M K+, the dissocia- observed with mild heating (40°C) of the tion constant, Ki, was determined to be solubilized AChR or treatment with p220 mM, while, at 1 M K+ the dissociation chloromercuribenzoate, which gave prepconstant was found to be 320 mM (x2 valarations in which 90-95% of the ACh ues for the curve fits are given in the binding was low affinity (13). The heat legend to Fig. 4). Thus, the averaged Ki treatment and PCMB treatments were for K+ is approximately 270 mM. considered to produce partial denaturations (total binding was reduced by 71%) DISCUSSION and more severe treatment may elimiThe binding of 13HlACh to AChR (virnate ACh binding. In the case of dTC tually all of which was high affinity bind- inhibition, the effect giving low (KL = 2 ing) was enhanced by Na+ and Li+ at pM) and high (KH = 12 nM) affinity bindconcentrations of up to 0.75 M, particuing was reversible (5). No data were oblarly with respect to control binding in 10 tained on the reversibility of the Zn2+ inmM Na,HPO, buffer alone, but was in- hibition; however, the similarity in the hibited by K+, Rb+, and Cs+. This is in resultant binding curves in all of the

ION

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ACETYLCHOLINE

above treatments, i.e., KH = 5-10 nM and = 0.5-2.0 PM, suggests a common mechanism may be in effect. In the case of dTC, the half-of-sites binding of ACh which it induced appeared to result from a half-of-sites interaction of dTC (5), in which half of the dTC binding sites exhibited a high affinity for dTC (4 x 10e8 M) and half exhibited a lower affinity (4 x 10e7 M). We may speculate that the low affinity ACh binding is associated with the high affinity dTC binding and that the remaining high affinity ACh binding is associated with the low affinity dTC binding. Thus, for ligands such as dTC or K+, half-of-sites binding of ACh will be induced as a consequence of the half-of-sites activity of the allosteric effector. In the case of Zn2+ or PCMB, the interactions with the allosteric sites may not exhibit half-of-sites activity and, consequently, nearly all of the ACh binding is converted to low affinity. A striking feature of this study is that K+, which is in low external concentration (5 mM) for electroplax cells and high internal concentration (450 mM>, alters the ACh binding to the receptor. On the other hand, Na+, which is in high external concentration (150 mM), favors the high affinity binding of ACh (compare the buffer curve and Ringer control curve of Fig. 3; in Fig. 1, 150 mM NaCl apparently enhanced binding of 13HlACh by only 5% -however, the values in Fig. 1 are calculated relative to Ringer’s solution controls which contain 115 m&r NaCI). Since the concentration of K+ within the cell is greater than the Ki for potassium, K+ may serve a regulatory function by interacting with the allosteric site during depolarization, thus giving a change in ACh affinity and, consequently, a change in the fraction of receptors in the activated state (ri). The consequence of the K+ interaction can be estimated by calculating the fraction of receptors in the R state in 1 ,u~ ACh and the effect of 500 mM K+ on that fraction. In the two-state model for cooperativity, it can be shown that the fraction of protein in the R state will be 50% of the maximum, a,,,, when [ACh] = 1 pM, with the dissociation constant for ACh being 20 nM [for the details of the

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calculation, see Gibson (5)]. We assume that this occurs in 150 mM Na+ and 5 mM K+, the medium external to the electroplax cell. In 500 mM K+, and assuming a dissociation constant of 250 mM, the fraction of receptors in the Z? state would be reduced to 18% of Z?,,, [using Eq. (ix) of Gibson (5)]. The ACh concentration would have to be increased lo-fold (to 10 j.&M) to once again give 0.5 Iz,,,. The significance of the K+ interaction may be better assessed by a kinetic model for endplate potentials. In two other systems which are electrically excitable, K+ has been shown to exhibit interesting, and perhaps related, effects. When applied externally to squid giant axon, K+, with an apparent Ki of 20 mM, has been shown to reduce Na+ flux in a manner not accounted for by the depolarization effect (1). In studies on cultured L6 myotubes, the electrically excitable response was either greatly reduced or eliminated by 27 mM K+ (4). An interesting feature of the study of Gartner et al. (4) was the observation that the nicotinic cholinergic antagonists, d-tubocurarine and a-bungarotoxin, prevented the K+ blockade, while the agonist acetylcholine showed no effect. The presence of Ca2+ in the AChR, (3) and the implication of Ca2+ in receptor function (6, 11, 13) suggests that Ca2+ may be an alternative regulator. The Ca2+ affinity for the AChR appears to be higher than that of K+: A 5 mM concentration of Ca2+ inhibited ACh binding by 50% (11). Last, we may speculate that K+ serves the function of terminating the depolarization induced by ACh, but, when Ca2+ is released from the agonist binding site (13) by prolonged exposure to agonists, the Ca2+ binds to the allosteric site leading to desensitization by virtue of its higher affinity. REFERENCES 1. COHEN, M., POTTI, Y., ANDADELMAN, W. J., JR. (1975) J. Membr. Biol. 24, 201. 2: DENBURG, J. L. (1973) J. Membr. Biol. 11,47. J. ELDEFRAWI, M. E., ELDEFRAWI, A. T., PENFIELLI, L. A., O’BRIEN, R. D., AND VAN CAMPEN, D. (1975) Life Sci. 16, 925. 4. GARTNER, T. K., LAND, B., AND PODLESKI, T. R.

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(1976) J. Neumbiol., in press. 5. GIBSON, R. E. (1976) Biochemistry, 15, 3890. 6. KARLIN, A., AND WINNIK, M. (1968) Proc. Nat. Acad. Sci. USA 60, 668. 7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioE. Chem. 193, 265. 8. MARTINEZ-CARRION, M., AND RAFTJSRY, M. A. (1973) Biochem. Biophys. Res. Commun. 55, 1156. 9. O’BRIEN, R. D., AND GIBSON, R. E. (1974) Arch.

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O’BRIEN

Biochem. Biophys. 165, 681. 10. O’BRIEN, R. D., AND GIBSON, R. E. (1975) Biochem. Biophys. 169,458. 11. O’BRIEN, R. D., AND GIBSON, R. E. (1975) Chem. Acta 47, 409. 12. RUBSAMEN, H., HESS, G. P., ELDEFRAWI, AND ELDEFRAWI, A. T. (1976) Biochem. phys. Res. Commun. 68, 56. 13. TAYLOR, D. B. (1973) J. Pharmacol. Exp. 185, 537. 14. WILKINSON, G. N. (1961) Biochem. J. 80,

Arch. Croat. M. E., BioTher. 324.