Abstract
The hypothesis whether or not 4-AP can affect vascular smooth muscle BK(Ca) 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 BK(Ca) 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 microM thapsigargin, 10 microM heparin, and 10 microM ryanodine did not alter the effect of 4-AP on BK(Ca) currents at [Ca](i) 250 nM. At [Ca](i)<100 nM 4-AP did not inhibit BK(Ca) currents. Application of 4-AP to the intracellular or extracellular side of excised BK(Ca) 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 BK(Ca) 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 BK(Ca) currents via an alteration of intracellular pH.
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