Xenopus oocyte resting potential, muscarinic responses and the role of calcium and guanosine 3',5'‐cyclic monophosphate.

Abstract

Resting potential (r.p.) and muscarinic response mechanisms were studied in X. laevis oocytes using the voltage-clamp technique. Insertion of micro-electrodes into the oocyte produced a ''shunt'' membrane conductance which partially sealed after a few minutes. The oocyte resting potential (measured with a single intracellular electrode) ranged from -40 to -60 mV. Ouabain and low K+ solution depolarized both follicles and denuded oocytes. The electrogenic Na+-K+ pump was more active in the latter. In the presence of ouabain, the r.p. agreed with the constant field theory. .alpha. (PNa+/PK+) was 0.12 in follicles and 0.24 in denuded oocytes. .beta. (PCl-/PK+) was 0.4 in both. At [Na+]o lower than 70 mM, the r.p. deviated considerably from the constant field predictions. The relatively large value of .alpha. indicated the major role of Na+ in oocyte r.p. determination. The oocyte muscarinic response was separated into four distinct components: the fast depolarizing Cl- current, ''D1''; the slow depolarizing Cl- current, ''D2''; the slow hyperpolarizing K+ current, ''H''; and the large membrane Cl- current fluctuation, ''F''. The H response reversal potential showed a Nernst relationship to [K+] and was selectively blocked by intracellular injection of tetraethylammonium (TEA). The D1 and D2 reversal potential showed a Nernst relationship to [Cl-]. In Ca2+-deficient, EGTA[ethyleneglycoltetraacetic acid]-containing medium, D2 and F were abolished and D1 and H were reduced. Verapamil inhibited all responses. Increasing [Ca2+]o caused a significant increase in D1, D2 and F response amplitudes. Intracellular injection of 0.6-10 pmol cGMP induced a large outward K+ current, similar to the muscarinic H response.