Analysis of calcium channel properties in cultured leech retzius cells by internal perfusion, voltage-clamp and single-channel recording

Abstract

ABSTRACT The properties of voltage-dependent calcium channels have been measured in Retzius cells isolated from the central nervous system of the leech and maintained in tissue culture. Macroscopic divalent cation currents were isolated after blocking Na+ and K+ currents by bathing the cells with Na+-free solutions containing TEA+ and 4-AP, and internally perfusing them with Cs+ and TEA+. Depolarizing voltageclamp pulses activated inward currents that were larger for Sr2+ than for Ba2+ or Ca2+. The peak currents were observed at +15 mV for Ca2+ and Ba2+ and at +7 mV for Sr2+. Divalent cation currents were blocked by Cd2+ and Mn2+ but not by dihydropyridine blockers. The activation kinetics of Ba2+ currents was sigmoid. The inactivation was approximately 10% at the end of a 50ms depolarizing pulse. Decay of Sr2+ and Ca2+ currents was larger and showed two kinetic phases. Activation and inactivation of the calcium channels were not significantly influenced by the holding potential. Deactivation kinetics observed during tail currents consisted of two exponential components. At a closing voltage of −60 mV, the time constant was ≈200 ps for the fast component and 1.9 ms for the slow component. Both time constants of deactivation were voltage-dependent over the range from −80 to −20 mV, and increased at more depolarized closing voltages. Single-channel activity was recorded with cell-attached patches in solution containing 75 mmol l−1 Ba2+. Taken together, the results define the characteristics of a distinctive type of calcium channel in isolated Retzius cells.