Macroscopic and Single-channel Studies of Two Ca2+ Channel Types in Oocytes of the Ascidian Ciona Intestinalis

Whole-cell and single-channel patch-clamp experiments were performed on unfertilized oocytes of the ascidian Ciona intestinalis to investigate the properties of two voltage-dependent Ca2+ currents found in this cell. The peak of the low threshold current (channel I) occurred at -20 mV, the peak of the high-threshold current (channel II) at +20 mV. The two currents could be distinguished by voltage dependence, kinetics of inactivation and ion selectivity. During large depolarizing voltage pulses, a transient outward current was recorded which appeared to be due to potassium efflux through channel II. When the external concentrations of Ca2+ and Mg2+ were reduced sufficiently, large inward Na currents flowed through both channels I and II. Using divalent-free solutions in cell-attached patch recordings, single-channel currents representing Na influx through channels I and II were recorded. The two types of unitary events could be distinguished on the basis of open time (channel I longer) and conductance (channel I smaller). Blocking events during channel I openings were recorded when micromolar concentrations of Ca2+ or Mg2+ were added to the patch pipette solutions. Slopes of the blocking rate constant vs. concentration gave binding constants of 6.4 X 10(6) M-1 sec-1 for Mg2+ and 4.5 X 10(8) M-1 sec-1 for Ca2+. The Ca2+ block was somewhat relieved at negative potentials, whereas the Mg2+ block was not, suggesting that Ca2+, but not Mg2+, can exit from the binding site toward the cell interior.