Korol S. Modification of functional activity of neuronal plasma membrane voltage-gated calcium channels by external calcium-free solution

Dissertation is devoted to the investigation of influence of calcium-free external solution on functional characteristics of voltage-gated calcium channels of rat dorsal root ganglion and hippocampal cultured neurons. In calcium-free external solution nonspecific potassium and sodium currents through voltage-gated calcium channels of rat dorsal root ganglion neurons and nonspecific potassium current via voltage-gated calcium channels of rat hippocampal cell culture were revealed with whole cell patch clamp technique. Inward current has been revealed after replacement of control solution with calcium concentration 2 mM by calcium-free one with potassium concentration 10 mM at the end of prepulse with amplitude -100 mV and duration 100 ms. Holding potential was -60 mV. After returning to control solution amplitude of current returned to initial value. In control solution calcium current was neglible small in comparison with discovered current in calcium-free solution at used prepulse. If calcium-free solution contained cesium ions instead of potassium ones any current could not be detected. Current amplitude increased with increasing of potassium concentration. Such dependence could be observed also in the case of hippocampal cell culture. It has been found that low voltage-activated calcium channel antagonist nickel in concentration 300 ?M inhibited potassium current in calcium-free solution by 69 ± 4 % (mean ± SEM) (p < 0.001, n = 5). After administration of nifedipine, the blocker of L-type calcium channel, in calcium-free solution amplitude of potassium current was 94 ± 2 % (p < 0.001, n = 6) smaller than amplitude of potassium current in calcium-free solution without nifedipine. So we concluded that potassium current passed through at least L- and T-type calcium channels in calcium-free medium. It was shown that current-voltage characteristic of voltage-gated calcium channels shifted 10 2 mV (p < 0.05, n = 9) to hyperpolarization direction in solution without calcium ions: in control external solution peak of the current-voltage relation of calcium channels was at membrane potential -10.1 2.3 mV and in calcium-free medium maximum of the current-voltage characteristic has been observed at -19.9 2 mV (p < 0.05, n = 9). Steady-state activation curve obtained in calcium-free medium also underwent some changes. Half-maximal potential of steady-state activation curve in control conditions was -34.3 ± 2.2 mV and in calcium-free solution this parameter acquired value of -44.3 ± 3.1 mV (р < 0.02, n = 15). Therefore steady-state activation curve shifted 10 ± 2.5 mV (р < 0.02, n = 15) to more negative membrane potentials in test conditions. Slope factor has not been changed and it was about 4.5 mV. Statistically reliable alterations in steady-state inactivation curve of calcium channels were not detected in calcium-free external solution. Half-maximal potential of steady-state inactivation had value about -76 mV and slope factor of curve was approximately 5.5 mV in both solutions (n = 15). In order to investigate sodium permeability of calcium channels of dorsal root ganglion neurons in calcium-free solution it have been used difference method. Nonselective calcium channel blocker cobalt was used in this method to separate sodium current to two components one of which passed through calcium channels and another one passed via sodium channels in calcium-free solution. Peak of current-voltage relation of sodium current through calcium channels was near -22 mV (n = 8). During comparison of current-voltage characteristics of voltage-gated calcium channels of dorsal root ganglion neurons in external solutions with different ion compositions it was determined that calcium channels are less permeable for potassium than for sodium ions. Maximal value of current-voltage relation for calcium current was taken as 100 %. According to such scale maximum of current-voltage characteristic for sodium current was 57 %, and maximal value of current-voltage curve for potassium current was 39 % in calcium-free solution. It was concluded that such distribution of current amplitudes is due to different crystal radii of taken monovalent cations. Crystal radius of potassium ion is bigger than that one of sodium ion, so we could observe smaller potassium current than sodium one. Described shifts in voltage dependent characteristics can be explained by changes in surface potential after calcium ion removal. Keywords: voltage-gated calcium channel, dorsal root ganglion neuron, hippocampal cell culture, calcium-free external solution, nonspecific potassium current, current-voltage characteristic, steady-state activation curve.