4‐Aminopyridine affects rat arterial smooth muscle BKCa currents by changing intracellular pH

The hypothesis whether or not 4‐AP can affect vascular smooth muscle BKCa currents was tested using the patch‐clamp technique, pH‐ and calcium‐fluorimetry, and freshly isolated rat arterial smooth muscle cells. Application of 4‐AP reversibly inhibited BKCa currents at an intracellular calcium ([Ca]i) of 250 nM with a half‐block of 2.5 mM at +50 mV. The presence of 2 μM thapsigargin, 10 μM heparin, and 10 μM ryanodine did not alter the effect of 4‐AP on BKCa currents at [Ca]i 250 nM. At [Ca]i<100 nM 4‐AP did not inhibit BKCa currents. Application of 4‐AP to the intracellular or extracellular side of excised BKCa channels did not alter channel activity or channel amplitude. Replacement of the pH‐sensitive calcium buffer EGTA by the pH‐insensitive calcium buffer BAPTA in the intracellular solution turned the 4‐AP‐induced inhibition of BKCa currents into a stimulation at [Ca]i 250 nM. Application of 4‐AP to single cells increased intracellular pH, which was accompanied by a reduction of [Ca]i in EGTA‐loaded cells and a stable [Ca]i in BAPTA‐loaded cells. Thus, these results suggest that in isolated vascular smooth muscle cells at [Ca]i>100 nM 4‐AP affects BKCa currents via an alteration of intracellular pH.