Surface potential reflected in both gating and permeation mechanisms of sodium and calcium channels of the tunicate egg cell membrane
Authors: Ohmori Harunori , Yoshii Mitsunobu 
Published: December 2014

1. Threshold changes of Na and Ca currents due to various polyvalent cations (stabilizing cations) or H+ ions were studied in the egg cell membrane of a tunicate, Halocynthia roretzi, by using the voltage‐clamp technique.2. With an increase in [Ca]o or a decrease in pH in the external solution, the current—voltage (I—V) relations for the peak of the Na and Ca currents shifted along the voltage axis in the positive direction. These voltage shifts in the I—V relations, measured at a potential of V½ where inward current attains its half‐maximum, were shown to be identical to shifts in voltage‐dependence of the time courses of Na and Ca currents, and also identical to shifts in the inactivation curves of Na current along the voltage axis.3. The shifts in V½ produced by various polyvalent cations or H+ ions were analysed by the Gouy‐Chapman equation for the diffuse double layer, by assuming that a change in V½ directly corresponds to a change in the surface double layer potential.4. The V½‐divalent cation concentration relations of Na current were exactly described by the predictions of the theory with a constant value of the surface charge density of 1e−/(9 Å)2. The weak stabilizing effects of Mg2+, Sr2+ and Ba2+ were quite similar to each other and were explained in terms of a ‘screening’ effect. Other divalent cations, such as Ca2+, Mn2+ and Ni2+, showed various different stabilizing effects which were explained in terms of a ‘binding’ effect. The binding constants (K1's) for Ca2+, Mn2+ and Ni2+ were 0·21, 0·45 and 0·94
M−1, respectively.5. H+ ions showed a powerful stabilizing effect upon the Na current with a KH of 6 × 104
M−1. This value indicates that the acidic sites around Na channels have a pKa of 4·78. La3+ ions also acted as a strong stabilizer upon the Na current with a KLa of 15
M−1. For both H+ and La3+, the V½‐concentration relations were also exactly described by the Gouy‐Chapman equation with the same charge density of 1e−/(9 Å)2 as estimated by varying divalent cations.6. The stabilizing effect of permeant cations such as Ca2+, Sr2+ and Ba2+ on Ca channel currents was analysed. The effect of lowering pH was also studied. It was found that the surface charge density of 1e−/(9 Å)2 estimated by Na current is also applicable to the explanation for the V½‐divalent cation concentration or — pH relationships. The estimated binding constants for H+, Ca2+ and Sr2+ were 1·2×105, 0·58 and 0·035
M−1, respectively. Ba2+ does not bind to charged sites near to the Ca channels.7. It was noticed that a considerable reduction in the conductances of Na and Ca currents occurred in parallel with a stabilizing effect. This reduction was ascribed to a decrease in the concentration of permeant cations at the external surface of the cell membrane, as predicted by the theory of the diffuse double layer. The Goldman, Hodgkin—Katz equation for ionic currents was applied to explain the conductance suppression.8. The conductance suppressions of Na and Ca channel currents due to Ca2+, Sr2+ and Ba2+ were found to be apparent ones, only reflecting decreases in the surface concentration of permeant cations without any changes in the permeability. After correction for the apparent suppression, the real permeability ratio among Ca2+, Sr2+ and Ba2+ for Ca channels was determined as 1·00, 0·56 and 0·21 respectively.9. The conductance suppression of Na current by lowering pH was explained in terms of a real suppression or blocking which is superimposed on the apparent suppression. Considering the surface [Na]o, the plot of P̄Na against the surface pH yielded a blocking curve of Na channel by H+ ions, which implies that two H+ ions are necessary to block each Na channel. For Ca channels no real blockage was observed in acidic pH.10. It was concluded from the present experiment that there exists a surface potential capable of affecting both gating and permeation mechanisms of ionic channels in this tunicate egg cell membrane.