Charybdotoxin-sensitive small conductance KCa channel activated by bradykinin and substance P in endothelial cells

1 in cultured porcine coronary artery endothelial cells, we have recently shown that substance P and bradykinin stimulated different types of Ca2+-dependent K+ (K-Ca) current. A large part of this current was insensitive to iberiotoxin and apamin. The aim of the present study was to characterize the KCa channel responsible for this current. 2 In cell-attached configuration and asymmetrical K+ concentration, 100 nM bradykinin or substance P activated a 10 pS K+ channel. In inside-out configuration, the channel was half-maximally activated by 795 nm free Ca2+. 3 Apamin (1 muM) added to the pipette solution failed to inhibit the channel activity while charybdotoxin (50 nm), completely blocked it. Perfusion at the intracellular face of the cell, of an opener of intermediate conductance KCa channel, 500 muM 1-ethyl-benzimidazolinone (I-EBIO) increased the channel activity by about 4.5 fold. 4 In whole-cell mode, bradykinin and substance P stimulated an outward K+ current of similar amplitude. Charybdotoxin inhibited by 75% the bradykinin-induced current and by 80% the substance P-induced current. Charybdotoxin plus iberiotoxin (50 nm each) inhibited by 97% the bradykinin-response. Charybdotoxin plus apamin did not increase the inhibition of the substance P-response obtained in the presence of charybdotoxin alone. 5 I-EBIO activated a transient outward K+ current and hyperpolarized the membrane potential by about 13 mV. Charybdotoxin reduced the hyperpolarization to about 3 mV. 6 Taken together these results show that bradykinin and substance P activate a 10 pS KCa channel, which largely contributes to the total K+ current activated by these agonists. Despite its small conductance, this channel shares pharmacological characteristics with intermediate conductance K-Ca channels.