Active [K.sup.+] secretion through multiple [K.sub.Ca]-type channels and regulation by I[K.sub.Ca] channels in rat proximal colon

Colonic [K.sup.+] secretion stimulated by cholinergic agents requires activation of muscarinic receptors and the release of intracellular [Ca.sup.2+]. However, the precise mechanisms by which this rise in [Ca.sup.2+] leads to [K.sup.+] efflux across the apical membrane are poorly understood. In the present study, Northern blot analysis of rat proximal colon revealed the presence of transcripts encoding rSK2 [small conductance (SK)], rSK4 [intermediate conductance (IK)], and rSlo [large conductance (BK)] [Ca.sup.2+]-activated [K.sup.+] channels. In dietary [K.sup.+]-depleted animals, only rSK4 mRNA was reduced in the colon. On the basis of this observation, a cDNA encoding the [K.sup.+] channel rSK4 was cloned from a rat colonic cDNA library. Transfection of this cDNA into Chinese hamster ovary (CHO) cells led to the expression of [Ca.sup.2+]-activated [K.sup.+] channels that were blocked by the IK channel inhibitor clotrimazole (CLT). Confocal immunofluorescence confirmed the presence of IK channels in proximal colonic crypts, and Western blotting demonstrated that IK protein sorted to both the apical and basolateral surfaces of colonic epithelia. In addition, transcellular active [K.sup.+] secretion was studied on epithelial strips of rat proximal colon using unidirectional [sup.86][Rb.sup.+] fluxes. The addition of thapsigargin or carbachol to the serosal surface enhanced net [sup.86][Rb.sup.+] secretion. The mucosal addition of CLT completely inhibited carbachol-induced net [sup.86][Rb.sup.+] secretion. In contrast, only partial inhibition was observed with the BK and SK channel inhibitors, iberiotoxin and apamin, respectively. Finally, in parallel with the reduction in SK4 message observed in animals deprived of dietary [K.sup.+], carbachol-induced [.sup.86][Rb.sup.+] secretion was abolished in dietary [K.sup.+]-depleted animals. These results suggest that the rSK4 channel mediates [K.sup.+] secretion induced by muscarinic agonists in the rat proximal colon and that transcription of the rSK4 channel is downregulated to prevent [K.sup.+] loss during dietary [K.sup.+] depletion. intermediate conductance [Ca.sup.2+]-activated [K.sup.+] channel; intermediate-conductance channel; carbachol; clotrimazole; [sup.86][RB.sup.+] flux; [K.sup.+] depletion