Role of IK channels in rat distal colon

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DESCRIPTION (provided by applicant): Intermediate conductance Ca2+-activated K+ (IK) channels are present in both apical (AP) and basolateral (BL) membranes of mammalian colonic epithelial cells. Although IK cDNA (that we have designated as IK1) has previously been cloned, we have made the following preliminary observations: 1) Isolation of a splice variant in which the entire 2nd transmembrane domain of 29 amino acids is deleted that we have designated as IK2; 2) Immunolocalization of IK1 to AP membrane and IK2 to BL membrane with antibodies that we have raised against the C-terminal end and the missing 29 aa motif; 3) Carbachol (CCH), a muscarinic-receptor agonist that increases intracellular free Ca2+, stimulates both K+ secretion and Cl- secretion that are inhibited by ctotrimazol (CLT), an IK channel inhibitor; and 4) Dietary K+ depletion down-regulates AP membrane IK1 protein, mRNA but not BL membrane IK2 protein and mRNA. We propose that IK2 is critical for activation of Cl- secretion by cell hyperpolarization while IK1 is closely associated with activation of K+ secretion and may also contribute to Cl- secretion. We propose: 1) To establish whether these IK isoforms when expressed in distinct membranes manifest the same and/or different characteristics we propose to characterize in vitro expressed IK1 and IK2 isoform channels compared to those of native IK channels of AP and BL membranes. 2) To identify the specific role of the 29 amino acid motif (deleted in IK2), we propose to establish whether a) entire motif or specific amino acid residue(s) is responsible for AP membrane delivery of IK1 proteins and b) whether deletion of the novel amino acid motif or other accessory proteins are responsible for BL membrane delivery of IK2 proteins. 3) To determine the role of these two IK channel proteins in K+ absorption, K+ secretion and Cl- secretion, physiological studies that include Isc determination and 86Rb fluxes will be performed in normal rats and in three models of selective 'knock-down' of IK function: dietary K+ depletion which represents a functional IK1 knockout; and IK knock-out mice in which we have 'knocked-in' either IK1 or IK2 by a previously published adenoviral transfection method that will result in IK2 or IK1 knockout mice, respectively. Therefore, the apparent distinct roles of CCH-induced K+ channels will be investigated and correlated with functional studies.