SUMMARY1. Resting potential (r.p.) and muscarinic response mechanisms were studied in Xenopus laevis oocytes using the voltage-clamp technique.2. Insertion of micro-electrodes into the oocyte produced a 'shunt' membrane conductance which partially sealed after a few minutes;3. 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.4. In the presence of ouabain, the r.p. agreed with the constant field theory. a (PNa+/PK+) was 0-12 in follicles and 0-24 in denuded oocytes. /J (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 a indicated the major role of Na+ in oocyte r.p. determination. 5. The oocyte muscarinic response was separated into four distinct components:

SUMMARY1. Currents evoked by elevated intracellular free Ca2+ in Xenopus laevis oocytes were studied using the two-electrode voltage clamp technique. The elevation in Ca2+ concentration was achieved in three ways: by the use of the divalent cation ionophore A23187; by application of Ca2+-mobilizing neurotransmitters serotonin and acetylcholine (ACh); by the entry of Ca2+ through voltage-dependent channels.2. In most experiments, the membrane was permeabilized to Ca2+ by a 15 min pretreatment with A23187 in a Ca2+-free solution. Exposure of the ionophore-treated oocytes to external Ca2+ elicited an inward current (at holding potentials of -40 to -60 mV). At external Ca2+ concentrations ([Ca2+]) between 0.1 and 1 mm, the current had a time-to-peak of at least 10 s, and slowly decayed over tens of seconds. At [Ca2+] > 2 mm, the inward current had two distinct kinetic components, a fast and transient one (If.t) and a slow one (Islow).3. The main carrier of the Ca2+-evoked inward current was Cl-. Several data indicate the existence of a tetraethylammonium (TEA)-sensitive K+ conductance.No evidence for a Na+ current was found.4. The two components of the Ca2+-evoked inward current in ionophorepermeabilized oocytes, and the two components of the current evoked by ACh and serotonin (the latter in oocytes injected with rat brain RNA but untreated with A23187), were blocked by intracellular injection of the Ca2+ chelator, ethyleneglycolbis-(,8-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA). The two components of these currents displayed different sensitivity to Ca2+ buffering; higher doses of EGTA were necessary to inhibit the slow component than the fast one.5. One to two minutes of treatment with 2 mM-9-anthracene carboxylic acid (9-AC) fully blocked Ca2+-dependent Cl-current evoked by Ca2+ influx through voltagedependent Ca2+ channels in intact (untreated with A23187) oocytes. In ionophoretreated oocytes, block of If.t was observed at holding potentials at which the current was outward (i.e. due to Cl-influx); Islow was inhibited only partially. The block of Ca2+-evoked Cl-efflux by 9-AC developed much more slowly and was less potent. To explain these results, the existence of two sites of 9-AC action is proposed.6. Exposure of the ionophore-permeabilized oocytes to 0-1-0-2 mm [Ca2+] strongly * To whom correspondence should be sent.

SUMMARY1. The involvement of Ca ions in the mediation of muscarinic Cl-current responses in Xenopus oocytes was studied using the voltage-clamp technique and direct measurements of 45Ca efflux.2. The injection of Ca into the oocytes produced a dose-dependent transient inward (depolarizing) current carried by Cl. This current was occasionally followed by a second, long-lasting inward current. 3. The muscarinic response was evoked by the application of acetylcholine (ACh). It consisted of a transient inward current response, and a long-lasting inward current response, both inward currents carried by Cl ions. Both responses were inhibited by intracellular injection of ethyleneglycol-bis-(,f-aminoethylether)N,N'-tetraacetic acid (EGTA), the long-lasting response being inhibited faster than the transient response.4. The calmodulin inhibitor, trifluoperazine, inhibited both the Cl-current responses to ACh and to Ca injection.5. ACh (10 pM) evoked a release of 45Ca from pre-loaded oocytes. This effect was inhibited by atropine (1 ZM).6. In the absence of external Ca, the muscarinic transient and long-lasting responses were partially inhibited. The long-lasting response was more sensitive to the external Ca depletion than the transient response.

SUMMARY1. The membrane response to acetylcholine (ACh), inositol 1,4,5-trisphosphate (IP3) and intracellular Ca21 was studied in Xenopus laevis oocytes under voltageclamp conditions.2. Shallow, submembranal injections of IP3 in the animal hemisphere of the oocyte evoked a two-component response comprised of a rapid, transient component followed by a slow, sustained component.3. When the injection pipette was inserted further into the cell (to 300 ,um below the cell membrane), the fast component diminished and the slow component remained unchanged or even increased.4. The rapid component exhibited an apparent higher sensitivity to 1P3 compared to the slow component.5. The two components of the IP3 response were retained in a Ca2+-free environment.6. Injection of a single large dose (20-50 pmol) of CaCl2 into the oocyte evoked a typical two-component response, whereas repetitive threshold doses (0-1 pmol CaCl2) elicited large current fluctuations which developed into a small depolarization current.7. The delay in the peak of the slow component of the response to either IP3 or to CaC12 injections appeared too long to be accounted for by diffusion alone.8. Depletion of oocyte Ca2' by the divalent cation ionophore A23187 (>1 ,IM)inhibited the response to ACh and 'P3. Low concentrations of A23187 selectively inhibited the rapid component of the ACh response, though not the rapid component of the IP3 response. 9. Our data suggest that the two-component membrane response to ACh in Xenopus oocytes can be accounted for by ACh-induced elevation of IP3 and subsequent IP3-induced release of intracellular Ca2 .

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