Abstract
The mechanisms by which external Ca ions block sodium channels were studied by a gigaohm seal patch clamp method using membranes excised from N1E-115 neuroblastoma cells. Tetramethrin was used to prolong the open time of single channels so that the current-voltage relationship could be readily determined over a wide range of membrane potentials. Comparable experiments were performed in the absence of tetramethrin. Increasing external Ca ions from 0.18 to 9.0 mM reduced the single channel conductance without causing flickering. From the dose-response relation the dissociation constant for Ca block at 0 mV was estimated to be 32.4 +/- 1.05 mM. The block was intensified by hyperpolarization. The voltage dependence indicates that Ca ions bind to sodium channels at a site located 37 +/- 2% of the electrical distance from the outside. The current increased with increasing external Na concentrations but showed a saturation; the concentration for half-maximal saturation was estimated to be 185 mM at -50 mV and 204 mM at 0 mV. A model consisting of a one-ion pore with four barriers and three wells can account for the observations that deviate from the independence principle, namely, the saturation of current, block by Ca ions, and rectification in current-voltage relationship. The results suggest that the Ca-induced decrease of the macroscopic sodium current results from a reduced single sodium channel conductance.