Spontaneous openings of the acetylcholine receptor channel.

Proc. Nati. Acad. Sci. USA Vol. 81, pp. 3901-3904, June 1984

Neurobiology

Spontaneous openings of the acetylcholine receptor channel (patch clamp/neuromuscular junction/fluctuations/ionic currents/mammalian muscle)

MEYER B. JACKSON Department of Biology and Mental Retardation Research Center, University of California, Los Angeles, CA 90024

Communicated by Everett C. Olson, March 1, 1984

ABSTRACT Patch clamp recordings from embryonic mouse muscle cells in culture revealed spontaneous openings of the acetylcholine receptor channel in the absence of exogenously applied cholinergic agent. The conductance of the spontaneous channel currents was, within experimental error, identical with the conductance of suberyldicholine-activated channel currents. The comparison of channel conductance was made with sodium and with cesium, each at two concentrations, with the same result. Treatment of the cells with a-bungarotoxin blocked the spontaneous channel currents. To determine whether the spontaneous openings were caused by an endogenous agent with cholinergic activity a reactive disulfide bond near the receptor binding site was reduced with dithiothreitol and alkylated with N-ethylmaleimide. This chemical modification reduced the effectiveness with which suberyldicholine and curare activated channel currents but did not reduce the frequency of spontaneous openings. These experiments indicate that the acetylcholine receptor briefly and infrequently fluctuates into an active state in the absence of agonist. Agonist activation of the receptor presumably accelerates this spontaneously occurring process. The binding of acetylcholine (AcCho) to the nicotinic receptor of skeletal muscle induces the opening of an associated ion channel in the cell membrane (1, 2). When the binding site of the receptor is vacant, the closed conformation of the channel is favored, but thermodynamic fluctuations to the open conformation are possible. As an example, in the theory of Monod et al. (3), which has been adapted to receptor activation by Karlin (4), the active and inactive (conducting and nonconducting) states are in equilibrium, and this equilibrium is altered by a ligand that binds more strongly to the

active configuration. Previous studies have not detected AcCho receptor channel opening in the absence of agonist (5-7) but have allowed the estimation of upper bounds. Observing the equilibrium between the active and inactive states of the unliganded receptor can be difficult. In the case of the AcCho receptor channel, a current due to spontaneous openings would be small compared to a large background current through other channels and through a leakage resistance. To overcome this difficulty, I have used the patch clamp technique to record spontaneous openings of the AcCho receptor channel directly. In this way the unique amplitude of AcCho receptor channel currents distinguishes them from the background membrane current.

METHODS Muscle cultures were prepared from mice at'ages that varied from 2 days prenatal to 2 days postnatal. Thigh muscle was removed, dissociated, and cultured by standard procedures (8). Fluorodeoxyuridine was added to cultures as they ap-

proached confluence to reduce the growth of background cells. Recordings were made from cells that had been cultured for 6-11 days. Membrane current was recorded from cultured mouse muscle in the cell-attached configuration with a low-noise current amplifier (9). All recordings were made at room temperature (21-23TC). Patch electrodes fabricated from aluminosilicate glass formed seals of 10 GfI or higher with the cell membrane. Electrodes were coated with polystyrene Q-dope (GC Electronics) to reduce the noise arising from source capacitance. The bandwidth of an eight-pole Bessel filter was set at 3 kHz. The pipette potential was generally maintained at voltages between 0 and 100 mV positive to ground, hyperpolarizing the patch of membrane and increasing the driving force for current through the AcCho receptor channel. The cell bathing solution and the electrode filling solution were always the same. The specific solution compositions used are given in the figure legends. A Data Translation A/D converter (DT 2782-DI/A) was used to collect data at a 20-,usec sampling frequency into the memory of an LSI-11/23 computer. To avoid storing long segments of data with no channel currents, a crossover detector circuit was used to trigger the computer to display data that contained events exceeding a preset threshold. Records containing channel currents were stored on floppy discs for further analysis. When a patch electrode was filled with a drug-free saline, inward channel currents appeared with a frequency that was highly variable (Fig. 1). A crude estimate of the frequency of channel openings was made by counting the number of events seen during randomly triggered sweeps on a storage oscilloscope (Fig. 1 C and D). At least 50 200-msec sweeps were used in any measurement of the frequency of openings in one patch of membrane. Because of the low frequency of spontaneous openings data collection was slow, and often not enough single-channel currents were obtained for a quantitative analysis. Two measures were taken that increased this frequency to a point at which data collection was convenient. First, treatment of cultures prior to recording with tetrodotoxin for 1 or more days increased the frequency of channel openings, presumably because blockade of spontaneous electrical activity in muscle cultures increases the density of receptors in the membrane (10). Second, large-diameter low-resistance patch electrodes were used (average resistance 1 Mfl) to seal off a larger membrane area containing more receptors. Some cultures were treated with 50 nM a-bungarotoxin (Sigma) for 1 hr at 360C in growth medium. Other cultures were treated with 1 mM dithiothreitol for 20 min in Hepes

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buff-

ered Earle's saline at room temperature. The sulfhydryl groups exposed by this treatment were then alkylated by treatment with 1 mM N-ethylmaleimide for 20 min under the same conditions.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviation: AcCho, acetylcholine.

3901

3902

Neurobiology: Jackson

Proc. NatL. Acad. Sci. USA 81 (1984)

B

A

20mV

-

6OmV '^-;^rw.

5pA

Si>~~e9^;.x~P

100 mV

:

5 msec

C

D

50 pA

100 msec

FIG. 1. Single-channel currents were recorded in mouse muscle cultures. (A and B) Computer plots of digitized data (40 Asec per point). The patch electrode was held at potentials positive to the bath ground as indicated in the figure. The cells were bathed in 137 mM NaCl/2 mM MgCl2/1 mM CaCI2/2.7 mM KCl/1 AM tetrodotoxin/1 mM Na2HPO4, pH 7.4 (A), or 137 mM CsCl/2 mM MgCl2/1 mM CaCl2/1 mM Na2HPO4, pH 7.4 (B). Patch electrodes were filled with the same solution used for bathing the cells. No cholinergic drugs were added to any of these solutions. (C and D) Oscilloscope sweeps of 200 msec were triggered manually. The patch electrode was held at a potential of +50 mV. (C) During five successive sweeps from one cell two spontaneous channel openings occurred. (D) In five successive sweeps in a recording from another cell five spontaneous channel openings were evident. Oscilloscope traces such as these were used to estimate the frequency of spontaneous openings.

ion was either sodium or cesium (Fig. 1 A and B). Plots of channel current versus voltage are linear, with the slope of the best-fitting line taken as the single-channel conductance (Fig. 2). Zero current intercepts were also calculated; however, since no attempt was made to control the intracellular ionic composition or resting potential, these extrapolated reversal potentials are less significant. For the hyperpolarizing patch electrode potentials used here, the conductance of a cation-selective channel is determined primarily by the solution filling the electrode. For two concentrations of NaCl and two concentrations of CsCl (120 and 137 mM) the current-voltage curves and single-channel conductances for spontaneous and agonist-induced openings are essentially indistinguishable (see Fig. 2 legend for values). The channel conductance was higher for solutions with cesium as the principal cation than for solutions with sodium as the principal cation, in agreement with a report by Hamill and Sakmann (12) for the AcCho receptor channel in cultured rat muscle. Treatment of cultures for 1 hr with 50 nM a-bungarotoxin at 360C dramatically reduced the frequency of spontaneous openings in seven recordings from two separate cultures. Spontaneous events were almost never seen on the oscilloscope; the frequency of openings determined in this way was 0.012/sec. The highest frequency was 0.033/sec, which is 1/6 of the lowest frequency of spontaneous openings in untreated sibling cultures. The average frequency for eight recordings from sibling control cultures was 1.1/sec. The average resistance of the electrodes used for measurements of openA

B

5-

0

0

44

~~~8

0

~0

0 0

M

0

0

0~~~~~~~~~~~~~~~ 0 ~~~~~0

&-

u

4

0~~~~~~~~~~~~~~ 0

RESULTS With a pipette holding potential of +50 mV the frequency of spontaneous openings was 1.3 + 1.4/sec (mean of 15 measurements ± standard deviation) in tetrodotoxin-treated cells. Frequencies ranged from 0.2 to 4.7/sec. In addition to the randomness in the number of events that can occur in a 10-sec interval, there are at least two obvious sources of variation in the frequency of openings: (i) the density of AcCho receptor channels in the cell membrane is not uniform (11); (ii) different electrodes seal off different areas of membrane. Each of these factors would lead to a variation in the number of receptors from one patch of membrane to another.

Without an estimate of the number of receptors in a patch of membrane from which a recording is made, an absolute rate constant for the opening process in the absence of agonist cannot be calculated. Since electrode resistance varies approximately with the reciprocal of the area of the electrode tip, a linear regression analysis of the frequency of openings versus the reciprocal of the electrode resistance was used to assess the correlation. A linear correlation coefficient of 0.51 was obtained for 15 data points. The probability that the two variables are independent was calculated to be 0.05. Channel currents were recorded at different patch electrode holding potentials, in solutions in which the major cat-

*

0~~~~~~~ 0 IIII

0

20 40

I

60

I

80 100

lII

III

0

20 40 60 80 100 Holding potential, mV

FIG. 2. Single-channel currents were recorded at various electrode holding potentials. The channel current amplitudes were averaged and plotted versus electrode potential. *, Spontaneous openings when the patch electrode contained no cholinergic drug. o, Channel currents seen with a patch electrode filled with 20 nM suberyldicholine. The slopes and intercepts for the best-fitting lines were taken as the conductance, y, and extrapolated reversal potential, E, respectively. (A) The solution in the bath and the electrode was 120 mM NaCl/2 mM MgC12/1 mM CaCl2/1 mM Na2HPO4, pH 7.4. For the spontaneous channel currents y = 24.6 pS and E = -83 mV (two measurements). For suberyldicholine-activated channel currents y = 23.1 pS and E = -106 mV. (B) The solution was the same as for A except that it had 120 mM CsCl instead of 120 mM NaCl. For spontaneous channel currents y = 27.8 pS and E = -85 mV (four measurements). For suberyldicholine-activated channel currents y = 28.6 pS and E = -72 mV (two measurements). (C) The solution was the same as that given for Fig. 1A. For spontaneous channel currents y = 32.1 pS and E = -85 mV (three measurements). For suberyldicholine-activated channel currents y = 33.7 pS and E = -89 mV (two measurements). (D) The solution was the same as that given for Fig. 1B. For spontaneous channel currents y = 35.8 pS and E = -63 mV (four measurements). For suberyldicholine-activated channel currents y = 37.2 pS and E = -59 mV (four measurements).

Proc. Natl. Acad. Sci. USA 81 (1984)

Neurobiology: Jackson

A

B.

\a

D a

urn channel and most potassium channels but produces

p --

E

50

pA

-V

-

100 msec

FIG. 3. Comparison between control cells (A, C, and E) and cells treated with dithiothreitol and N-ethylmaleimide (B, D, and F). All recordings were made with solutions identical to the solution given

for Fig. 1B. The patch electrode

was held at a potential of +50 mV. (A) Recording with 100 nM suberyldicholine from control cells. (B) Recording with 100 nM suberyldicholine from treated cells. (C) Recording with 20 AM curare from control cells. (D) Recording with 20 AM curare from treated cells. (E) Spontaneous channel openings in control cells. (F) Spontaneous channel openings in treated cells.

ing frequency in a-bungarotoxin-treated cells and control cells was equal, indicating that these frequency of opening measurements were made from comparable areas of membrane. Cells treated with dithiothreitol and N-ethylmaleimide much less responsive to cholinergic agents, in agreewith many previous studies (13). Recordings made with a patch electrode filled with 100 nM suberyldicholine normally show the noisy multilevel activity of more than one channel open at a time (Fig. 3A). This type of behavior was were

ment

found in eight recordings from control cells in which

no

chemical reagents were used to modify the receptor binding site. Eight recordings from cells treated with dithiothreitol and N-ethylmaleimide, using the same concentration of suberyldicholine, revealed only infrequent channel openings (Fig. 3B). Fig. 3 C and D shows a similar effect of chemical modification on the response to 20 /LM curare. Spontaneous channel openings were still seen in recordings from cell cultures in which the AcCho receptor was rendered unresponsive by chemical modification (Fig. 3F). Two determinations using 137 mM CsCl showed that the channel conductance for spontaneous openings in dithiothreitol/N-ethylmaleimidetreated cells is 36 pS and is thus unchanged by chemical modification of the binding site. The frequency of spontaneous openings in treated cells was 4.8 + 7.3/sec (11 determinations), and was higher than the frequency of 1.5 + 1.6/sec (11 determinations) found in untreated sibling cultures (Fig. 3E). Average electrode resistances in the two sets of measurements were identical (1.0MW in both cases), so the higher frequency of openings in treated cells may be significant.

DISCUSSION Together, these observations strongly support the hypothesis that these channel openings in the absence of exogenousare openings of the AcCho rechannel. Cesium blocks the voltage-dependent sodi-

ly applied cholinergic agent ceptor

3903

slightly larger currents through the AcCho receptor channel. The calcium-activated inward channel has a much lower conductance than the AcCho receptor channel (14, 15), and its conductance is reduced by replacement with cesium (15). A stretch-activated channel in chick muscle has a different conductance and ion selectivity (16). Guharay and Sachs (16) also noted that the nicotinic receptor channel of chick is not stretch activated. The single channel conductance and the ionic dependence of the spontaneous channel currents reported here are not consistent with any other well-studied channel, but they match closely the behavior of the suberyldicholine-activated channels studied in the same cultures. In addition, the specificity of a-bungarotoxin for the nicotinic receptor (11) and its blockade of the spontaneous events support the hypothesis that they are openings of the AcCho receptor channel. Although it is clear that these channel currents are openings of the AcCho receptor channel, such openings could be produced by an endogenous substance in muscle, or an impurity in the recording solutions that is capable of interacting with the AcCho binding site and activating the channel. Although these channel currents in the absence of exogenous agonist arise from openings of the AcCho receptor channel, it is still possible that they are not spontaneous in the sense of being independent of receptor binding. Most of the work reported here was done with phosphate-buffered solution rather than the commonly used buffer Hepes, to avoid a tertiary nitrogen atom, which might interact weakly with a quaternary ammonium binding site. Tetrodotoxin was used to block the sodium channel only in experiments with sodiumcontaining saline, but not in experiments with cesium-containing saline. It is still conceivable that muscle cells secrete some cholinergic agent or that the choline groups of the phospholipids surrounding the receptor can, with low probability, extend to the AcCho binding site and induce channel opening. To eliminate these possibilities it is necessary to selectively block the receptor without blocking the channel. The classical antagonist curare has the undesirable action of weakly activating the receptor in cultured muscle (17, 18) and is therefore unsuitable as a blocking agent. It is known that the AcCho binding site can be modified covalently by attacking a reactive disulfide bond, with diverse effects on AcCho responses in different preparations (13). The reactive disulfide bond is near the AcCho binding site, since affinity alkylating reagents react with a much faster rate than nonaffinity reagents (19, 20). In addition, linking bromoacetylcholine to a sulfhydryl group exposed by reduction of the disulfide bond results in a chronically activated channel (20). Finally, cholinergic agents can protect the reduced receptor from alkylating reagents (21). If the channel currents observed in the absence of exogenously applied agonist were activated by interaction of the receptor with an endogenous ligand or a contaminant in the electrode-filling solutions, the chemical modification of the receptor with dithiothreitol/N-ethylmaleimide would have reduced their frequency of occurrence. Since dithiothreitol/N-ethylmaleimide did not reduce the frequency of spontaneous openings but did substantially reduce the frequency of openings seen when suberyldicholine or curare was used, we can conclude that the channel openings seen in the absence of cholinergic agent are not a consequence of interaction of an unknown agent with the acetylcholine binding site. The difference between the effect of treatment with dithiothreitol/N-ethylmaleimide and treatment with a-bungarotoxin illustrates how spontaneous openings can be used to explore the mechanism of drug action. If a drug only prevents agonist binding, then physiological responses should be blocked, but spontaneous openings should still appear. Although a-bungarotoxin binding to the receptor is competi-

3904

Neurobiology: Jackson

tive with cholinergic agents (22), it blocks spontaneous openings, indicating an additional effect of either obstructing the channel or locking it in the closed configuration. An interaction of this type between a-bungarotoxin and the AcCho receptor is supported by biochemical and structural studies

(23). CONCLUSION The channel currents reported here are caused by fluctuations into the open state of the unliganded receptor channel complex. This aspect of the AcCho receptor channel may prove useful in the building of a complete model of chemically induced channel gating. With more information about unliganded receptor activation a thermodynamically consistent scheme is possible with which the Monod-Wyman-Changeux theory (3, 4), or some other theory can be tested. In addition, the AcCho receptor channel is permeable to calcium (24, 25). Hence, spontaneous openings could make the channel a weak source of membrane current and calcium in the absence of cholinergic stimulation. Increases in membrane current and calcium flux could be important consequences of denervation supersensitivity and could play a role in embryonic muscle development. A preliminary account of this work has been presented (26). Other laboratories have made preliminary reports of similar or related observations (27, 28). Sakmann et al. (29) focused on agonist-induced openings of muscarinic channels in heart, but spontaneous openings were also reported. I thank Dr. Robert Love for suggesting a chemical modification and Drs. J. Dani, R. Horn, and R. Eckert for helpful discussions and for reading this manuscript. The muscle cultures used in this study were prepared by Linda Attardo and Suzette Wright. This work was supported by U.S. Public Health Service Grant NS 19320-01 and by a grant from the Hereditary Disease Foundation and was aided by Basil O'Connor Starter Research Grant 5-366 from March of Dimes Birth Defects Foundation. 1. Del Castillo, J. & Katz, B. (1957) Proc. R. Soc. London, Ser. B 146, 362-368. 2. Neher, E. & Sakmann, B. (1976) Nature (London) 260, 799801. 3. Monod, J., Wyman, J. & Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118.

Proc. NatL. Acad. Sci. USA 81 (1984) 4. Karlin, A. J. (1967) J. Theor. Biol. 16, 306-320. 5. Katz, B. & Miledi, R. (1977) Proc. R. Soc. London, Ser. B. 196, 59-72. 6. Dionne, V. E., Steinbach, J. H. & Stevens, C. F. (1978) J. Physiol. 281, 421-444. 7. Neubig, R. R., Boyd, N. D. & Cohen, J. B. (1982) Biochemistry 21, 3460-3467. 8. Christian, C. N., Daniels, M. P., Sugiyama, H., Vogel, Z., Jacques, L. & Nelson, P. G. (1978) Proc. Natl. Acad. Sci. USA 75, 4011-4015. 9. Hamill, 0. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. (1981) Pflugers Arch. 391, 85-100. 10. Shainberg, A., Yagil, G. & Yaffe, D. (1976) Pflugers Arch. 361, 255-261. 11. Fambrough, D. (1979) Physiol. Rev. 59, 165-227. 12. Hamill, 0. P. & Sakmann, B. (1981) Nature (London) 294, 462-464. 13. Karlin, A. (1980) in The Cell Surface and Neuronal Function, eds. Cotman, C. W., Poste, G. & Nicolson, G. L. (Elsevier/ North Holland, New York), pp. 191-260. 14. Colquhoun, D., Neher, E., Reuter, H. & Stevens, C. F. (1981) Nature (London) 294, 752-754. 15. Yellin, G. (1982) Nature (London) 296, 357-359. 16. Guharay, F. & Sachs, F. (1984) J. Physiol., in press. 17. Trautmann, A. (1982) Nature (London) 298, 272-275. 18. Jackson, M. B., Lecar, H., Askanas, V. & Engel, W. K. (1982) J. Neurosci. 2, 1465-1473. 19. Karlin, A. & Winnik, M. (1968) Proc. Natl. Acad. Sci. USA 60, 668-674. 20. Silman, I. & Karlin, A. (1969) Science 164, 1420-1421. 21. Ben-Haim, D., Landau, E. M. & Silman, I. (1973) J. Physiol. 234, 305-325. 22. Colquhoun, D. & Rang, H. P. (1976) Mol. Pharmacol. 12, 519535. 23. Kistler, J., Stroud, R. M., Klymkowsky, M. W., Lalancette, R. A. & Fairclough, R. H. (1982) Biophys. J. 37, 371-383. 24. Bregestovski, P. D., Miledi, R. & Parker, I. (1979) Nature (London) 279, 638-639. 25. Takeuchi, N. (1963) J. Physiol. 167, 141-155. 26. Jackson, M. B. (1983) Soc. Neurosci. Abstr. 9, 160. 27. Sanchez, J. A., Dani, J. A., Siemen, D. & Hille, B. (1983) Biophys. J. 41, 65a (abstr.). 28. Brehm, P., Moody-Corbett, F. & Kullberg, R. (1983) Biophys. J. 41, 67a (abstr.). 29. Sakmann, B., Noma, A. & Trautwein, W. (1983) Nature (London) 303, 250-253.