Ca 2+ - activated K + Channels in Murine Endothelial Cells: Block by Intracellular Calcium and Magnesium

The intermediate (IK Ca ) and small (SK Ca ) conductance Ca 2+ -sensitive K + channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK Ca and SK Ca channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 (cid:2) M free [Ca 2+ ] i and 1 mM free [Mg 2+ ] i , membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectifi cation at positive membrane potentials. Blockers of large-conductance, Ca 2+ -sensitive potassium (BK Ca ) and strong inward rectifi er potassium (K ir ) channels did not affect the membrane current. However, blockers of IK Ca channels, charybdotoxin (ChTX), and of SK Ca channels, apamin (Ap), signifi cantly reduced the whole-cell current. Although IK Ca and SK Ca channels are in-trinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg 2+ signifi cantly increased these currents. Moreover, concomitant reduction of the [Ca 2+ ] i to 1 (cid:2) M caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectifi cation. Block of IK Ca and SK Ca channels caused a signifi cant endothelial membrane potential depolarization ( ≈ 11 mV) and decrease in [Ca 2+ ] i in mesenteric arteries in the absence of an agonist. These results indicate that [Ca 2+ ] i can both activate and block IK Ca and SK Ca channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium. patch clamp technique at a sampling rate of 5 obtaining a gigaohm seal with a borosilicate micropipette (6 – 8 M Ω ) on the facing endothelium. The membrane potential recordings (perfo-rated patch) were performed without current injection (I = 0 mode). All in situ experiments were performed in the presence of nisoldipine (100 nM) to minimize the contraction of the smooth muscle to KCl and the impact of smooth muscle on endothelial membrane potential. 2+ to 1 mM. These experiments were performed at room temperature. For simultaneous membrane potential and Ca 2+ imaging experiments in intact endothelium of cut-open mesenteric arteries, a physiological salt solution was used with the following constitu-ents (in mM): NaCl 119, KCl 4.7, NaHCO 3 24, KH 2 PO 4 1.2, EDTA 0.0023, MgCl 2 1.2, glucose 11, and CaCl 2 1.6, and the following pipette solution for the simultaneous membrane potential recording (in mM): K-aspartate 110, NaCl 10, KCl 30, MgCl 2 1, HEPES 10 (pH 7.2), and EGTA 0.05. Amphotericin (200 μ g/ml) was used to achieve perforated patch. These experiments were performed at 30 ° C to minimize dye leakage. paired