Desensitization of the acetylcholine receptor of frog end-plates measured in a Vaseline-gap voltage clamp

Abstract

1. Desensitization of the nicotinic acetylcholine receptor of the frog end-plate was investigated in dissociated frog muscle fibres using the Vaseline-gap clamp method so that a wide range of well-defined agonist concentrations could be used without having to use alpha-bungarotoxin to reduce currents, and so that the intracellular medium could be controlled. 2. Acetylcholine (ACh) concentrations between 1 and 1000 microM were used, after inactivation of acetylcholinesterase. The intracellular calcium concentration was usually kept near zero by using 80 mM-K2EGTA as the intracellular solution. 3. When using the low intracellular calcium solution, desensitization proceeded as a biphasic process with estimates of fast and slow time constants of about 8 and 80 s at 4 degrees C and 20 microM-ACh (the rates increased with concentration). In contrast, only one (fast) component of desensitization was detected when the intracellular calcium concentration was allowed to increase during ACh application. 4. Despite rapid application of ACh the time to peak response was 0.2 s (with 400 microM-ACh) to 2 s (with 1 microM-ACh); this slow rise was shown to result from diffusion delays. Nevertheless the peak current with 200 microM-ACh corresponded to opening of most of the channels present, so there is probably not much desensitization in the millisecond time range. 5. Both fast and slow time constants for onset of desensitization showed only slight dependence on membrane potential when [Ca2+]i was buffered with 80 mM-K2EGTA. 6. Increasing the intracellular cyclic AMP concentration directly, or indirectly with forskolin and IBMX, had no effect on the time course of desensitization. 7. Intracellular application of submicromolar concentrations of phorbol-12,13-dibutyrate (PDBu) and phorbol-12-myristate-13-acetate (PMA) yielded a small but reproducible reduction of the peak response to ACh. The time course of desensitization was, however, not modified by these substances. 8. The implications of these observations for the mechanism of desensitization, and their relationship to single-channel observations, are discussed.