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

1 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. 2 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. 3 The presence of 2 CIM thapsigargin, 10 muM heparin, and 10 muM ryanodine did not alter the effect of 4-AP on BKCa currents at [Ca](i) 250 nM. 4 At [Ca](i)<100 nM 4-AP did not inhibit BKCa currents. 5 Application of 4-AP to the intracellular or extracellular side of excised BKCa channels did not alter channel activity or channel amplitude. 6 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. 7 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. 8 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.