Potential-dependent calcium blockage of normal and aconitine-modified sodium channels in frog node of Ranvier.

Hydrogen and some divalent cations block Na channels in a voltage-dependent manner (Woodhull 1973; Ulbricht and Wagner 1975; Mozhayeva et al. 1981, 1982a; Begenisich and Danko 1983). The potential-dependence of this blockage suggests that the binding site for blocking ions is situated in the pore at some fractional distance (<5) from the outside (Woodhull 1973). Alkaloid aconitine has been shown to reduce ô for H blockage from about 0.4 to 0.15 (Mozhayeva et al. 1982b). The aim of the present work was to compare the potential dependence of Ca blocks in normal and aconitine-modified Na channels. Experiments were carried out on single myelinated fibres of Rana ridibunda under voltage clamp conditions (Mozhayeva et al. 1977). External solutions contained (in mmol/1): 110 NaCl, 2 or 30 CaCl2, 8 tetraethylammonium chloride, 5 tris(hydroxymethyl)aminomethane-HCl; pH 7.5. The internodes were cut in isotonic KF solution. Application of 10" mol/1 aconitine to the node was accompanied by repetitive stimulation (10 Hz, 2 ms). The "instantaneous" currents were determined in response to postpulses of different amplitudes following the test pulse of a constant amplitude opening nearly all Na channels, were recorded. These currents were extrapolated to the time of switching potential from test pulse tc postpulses. The setting time of the clamp membrane potential did not exceed 20 fis. Linear current components were subtracted using P/4 program; control pulses started from holding potential. Figs. 1 and 2 show the "instantaneous" current-voltage relations of normal and aconitine-modified Na channels in solutions with 2 or 30 mmol/1 Ca ions, and the calculated pK(Ca), respectively. pK(Ca) values were calculated by equation