Characterization of an apamin-sensitive small-conductance Ca2+-activated K+ channel in porcine coronary artery endothelium:: relevance to EDHF

1 The apamin-sensitive small-conductance Ca2+-activated K+ channel (SKCa) was characterized in porcine coronary arteries. 2 In intact arteries, 100 nM substance P and 600 muM 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial cell hyperpolarizations (27.8 +/- 0.8 mV and 24.1 +/- 1.0 mV, respectively). Charybdotoxin (100 nM) abolished the I-EBIO response but substance P continued to induce a hyperpolarization (25.8 +/- 0.3 mV). 3 In freshly-isolated endothelial cells, outside-out patch recordings revealed a unitary K, conductance of 6.8 +/- 0.04 pS. The open-probability was increased by Ca2+ and reduced by apamin (100 nM). Substance P activated an outward current under whole-cell perforated-patch conditions and a component of this current (38%) was inhibited by apamin. A second conductance of 2.7 +/- 0.03 pS inhibited by d-tubocurarine was observed infrequently. 4 Messenger RNA encoding the SK2 and SK3, but not the SKI, subunits of SKCa, was detected by RT-PCR in samples of endothelium. Western blotting indicated that SK3 protein was abundant in samples of endothelium compared to whole arteries. SK2 protein was present in whole artery nuclear fractions. 5 Immunofluorescent labelling confirmed that SK3 was highly expressed at the plasmalemma of endothelial cells and was not expressed in smooth muscle. SK2 was restricted to the peri-nuclear regions of both endothelial and smooth muscle cells. 6 In conclusion, the porcine coronary artery endothelium expresses an apamin-sensitive SKCa containing the SK3 subunit. These channels are likely to confer all or part of the apamin-sensitive component of the endothelium-derived hyperpolarizing factor (EDHF) response.