Endothelins activate Ca2+-gated K+ channels via endothelin B receptors in CD-1 mouse erythrocytes.

Cell dehydration mediated by Ca2+-activated K+ channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca2+-activated K+ channel (Gardos channel) with maximal velocity ( V max) of 0.154 ± 0.02 mmol ⋅ l cells-1 ⋅ min-1and an affinity constant ( K 0.5) for Ca2+ of 286 ± 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased their V max by 88 ± 9% ( n = 8) and decreased their K 0.5 for Ca2+ to 139 ± 63 nM ( P < 0.05; n = 4). Activation of the Gardos channel resulted in an EC50 of 75 ± 20 nM for ET-1 and 374 ± 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 ± 51 and 785 ± 143 nM and with capacity of binding sites of 298 ± 38 and 1,568 ± 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ETB) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ETB receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ETB receptors and a calphostin-sensitive mechanism.