Effects of internal free calcium upon the sodium and calcium channels in the tunicate egg analysed by the internal perfusion technique.

Abstract

The unfertilized egg of the tunicate, Halocynthia roretzi, was intracellularly perfused with various solutions. The perfusion apparatus consisted of lower and upper compartments which were connected by a small glass funnel. A denuded egg cell without chorion was dropped into the funnel and brought into close contact with the glass wall of the funnel. The membrane of the egg attached to the lower compartment was ruptured by a slight difference of hydrostatic pressure and the inside of the egg was perfused with the internal solution flowing through the lower compartment. The current across the upper membrane was analyzed by the voltage-clamp technique. The egg cell in contact with 400 mM-Na external solution and perfused intracellularly with 400 mM-Na for 30 min showed a relatively low Na reversal potential, +6 mV, in comparison with +60 mV in the intact egg in standard artificial sea water. The exchange efficiency was monitored by observing the shift of Na reversal potential during perfusion with high Na interal perfusate. The internal perfusate containing F- ions stabilized the egg membrane and kept the excitability for 1-2 h during the intracellular perfusion. With the internal F- perfusate the intracellular cationic content was changed to 400 mM-Na, K, Rb or Cs (external solution of 400 mM-Na) and permeability ratios of the egg Na channel were estimated as PNa:PK:PRb:PCs = 1.0:0.14:0.05:0.04. The internal F- perfusate abolished Ca current which was consistently observed in the intact egg, while the internal Cl- perfusate kept both Na and Ca current as in the intact egg. However, with the internal Cl- perfusate the egg cell could not be kept in good condition more than 20-30 min. The effects of intracellular free Ca ions upon the egg Na and Ca channels were analyzed by using Ca ion-buffered internal Cl- and high Na perfusate. Internal Ca ions above 10-6 M apparently reduced the Ca current and enhanced the Na current at the same time. In the range between 10-5-10-4 M the Ca current became half of the control obtained with zero free Ca perfusate while the Na conductance at the zero current level doubled. The internal Ca ions above 1 mM seemed to abolish the Ca current and to reduce the Na current as well. The reciprocal effect of intracellular Ca ions upon the egg Na and Ca channels was demonstrated in the concentration range from 10-6-10-3 M.