CHARACTERIZATION OF MEMBRANE CURRENTS IN SINGLE SMOOTH-MUSCLE CELLS FROM THE GUINEA-PIG GASTRIC ANTRUM

1. Smooth muscle cells, enzymatically isolated from the antrum of the guinea-pig stomach, were voltage clamped at room temperature using the whole-cell patch clamp technique. In physiological salt solution (PSS), step depolarization from a holding potential of -90 mV elicited inward calcium current (I(ca)) followed and superimposed by outward potassium current. 2. Outward current was divided into components depending on the presence of extracellular Ca2+ and others which were not activated as a result of Ca2+ influx. Ca2+-dependent components were (1) a fast transient component most likely representing Ca2+-activated K+ current (I(K(ca)) immediately triggered by the initial peak of I(ca) and (2) spontaneous transient outward currents (STOCs) apparently reflecting synchronized opening of I(K(ca)) channels by cyclic release of Ca2+ from intracellular stores. Ca2+ influx-independent outward current could be divided into two main components: (1) a transient component (I(to)) showing voltage-dependent activation and inactivation and (2) a non-inactivating component (I(ni)). 3. I(to) activated with a threshold around -30 mV, was fully available at -90 mV and completely inactivated at -10 mV. The time course of both activation and inactivation of I(to) at different potentials could be described by single exponential functions. Time constants of activation decreased from 35 ms at -10 mV to 10 ms at + 40 mV. The time constant of inactivation was about 2 s and only weakly voltage dependent. Time constants for exponentially developing recovery from inactivation of I(to) ranged from 0.1 s at -100 mV to 10 s at -30 mV. I(to) was insensitive to 4-aminopyridine (4-AP, 5 mmol/1), slightly sensitive to tetraethylammonium (TEA, 10 mmol/1), but substantially inhibited by caffeine (10 mmol/l) and Cd2+ (5 mmol/l). Estimates of the single-channel conductance by current fluctuation analysis indicated a small value of about 2-5 ps. 4. The action of TEA on current elicited from a holding potential of -10 mV indicated a major contribution to I(ni) of a distinct component (I(ni,K)) that was completely blocked by this substance at a concentration of 10 mmol/l. I(ni) was almost unaffected by 4-AP (5 mmol/1) and caffeine (10 mmol/l), but strongly suppressed by Cd2+ (5 mmol/l). Current fluctuation analysis Of I((ni,K) gave a value for the single-channel conductance of about 60 pS. 5. Ca2+ inward current was studied in PSS ([Ca2+]o = 2.5 mmol/1) using pipette solution in which K+ was replaced by Cs+ to suppress outward K+ currents. At a holding potential of -90 mV, I(Ca) was activated upon depolarizing step pulses with a threshold around -30 mV and maximal at about +20 mV. It was blocked by 0-5 mmol/l Cd2+. 6. The time course of inactivation of I(Ca) between -10 and +50 mV could be described as the sum of two exponentials, the time constant of the fast component increasing with membrane potential from about 30 to 50 ms, that of the slow component decreasing from 500 to 100 ms. The current-voltage relationship determined at a holding potential of -30 mV was verv similar to that obtained at -90 mV, except indicating a small degree of steady-state inactivation. 7. Bay K 8644 ((-)-enantiomer, 3 x 10(-7) mol/l) approximately doubled peak I(Ca) elicited from a holding potential of -90 mV, and shifted the current-voltage relationship and the steady-state inactivation curve in a negative direction. Nifedipine (10(-7) mol/l) produced a substantial decrease of the current amplitude and also shifted the steady-state inactivation curve in a negative direction. The results gave no convincing evidence for the existence of two distinct populations of Ca2+ channels in these cells. 8. Properties of the currents identified are discussed both in relation to those of currents described in other smooth muscles and their possible contribution to the electrical activity as observed in multicellular preparations.