Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na+ influx on large-conductance Ca2+-activated K+ (BKCa) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na+ replacement by NMDG+ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na+ concentration ([Na+]i) and vice versa. In addition, extracellular Na+ depletion decreased [Na+]i. In voltage-clamped cells, BKCa currents were markedly increased by extracellular Na+ depletion. In inside-out patches, increasing [Na+]i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BKCa channels and decreasing intracellular K+ concentration ([K+]i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na+]i gradually from 0 to 70 mM, by replacing K+, markedly reduced single channel conductance and NPo. The Na+–Ca2+ exchange blocker Ni2+ or KB-R7943 decreased [Na+]i and increased BKCa currents simultaneously, and the Na+ ionophore monensin completely inhibited BKCa currents. BKCa currents were significantly augmented by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM and significantly reduced by decreasing [K+]o from 12 or 6 to 0 mM or applying the Na+–K+ pump inhibitor ouabain. These results suggest that intracellular Na+ inhibit single channel conductance of BKCa channels and that intracellular K+ increases single channel conductance and NPo.