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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK018777-27
Application #
7667917
Study Section
Clinical and Integrative Gastrointestinal Pathobiology Study Section (CIGP)
Program Officer
May, Michael K
Project Start
1976-06-01
Project End
2011-08-31
Budget Start
2009-09-01
Budget End
2010-08-31
Support Year
27
Fiscal Year
2009
Total Cost
$285,783
Indirect Cost
Name
West Virginia University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
191510239
City
Morgantown
State
WV
Country
United States
Zip Code
26506
Coon, Steven D; Rajendran, Vazhaikkurichi M; Schwartz, John H et al. (2015) Glucose-dependent insulinotropic polypeptide-mediated signaling pathways enhance apical PepT1 expression in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 308:G56-62
Rajendran, Vazhaikkurichi M; Nanda Kumar, Navalpur S; Tse, Chung M et al. (2015) Na-H Exchanger Isoform-2 (NHE2) Mediates Butyrate-dependent Na+ Absorption in Dextran Sulfate Sodium (DSS)-induced Colitis. J Biol Chem 290:25487-96
O'Hara, Bryan; Alvarez de la Rosa, Diego; Rajendran, Vazhaikkurichi M (2014) Multiple mineralocorticoid response elements localized in different introns regulate intermediate conductance K+ (Kcnn4) channel expression in the rat distal colon. PLoS One 9:e98695
Kanthesh, Basalingappa M; Sandle, Geoffrey I; Rajendran, Vazhaikkurichi M (2013) Enhanced K(+) secretion in dextran sulfate-induced colitis reflects upregulation of large conductance apical K(+) channels (BK; Kcnma1). Am J Physiol Cell Physiol 305:C972-80
Coon, Steven D; Schwartz, John H; Rajendran, Vazhaikkurichi M et al. (2013) Glucose-dependent insulinotropic polypeptide regulates dipeptide absorption in mouse jejunum. Am J Physiol Gastrointest Liver Physiol 305:G678-84
Barmeyer, Christian; Ye, Jeff Huaqing; Soroka, Carol et al. (2013) Identification of functionally distinct Na-HCO3 co-transporters in colon. PLoS One 8:e62864
Talukder, Jamilur R; Boyd, Brittney; Griffin, Ashley et al. (2013) Inflammatory cytokine TNF-? inhibits Na(+)-glutamine cotransport in intestinal epithelial cells. Can J Physiol Pharmacol 91:275-84
Singh, Satish K; O'Hara, Bryan; Talukder, Jamilur R et al. (2012) Aldosterone induces active K? secretion by enhancing mucosal expression of Kcnn4c and Kcnma1 channels in rat distal colon. Am J Physiol Cell Physiol 302:C1353-60
Sandle, Geoffrey I; Rajendran, Vazhaikkurichi M (2012) Cyclic AMP-induced K+ secretion occurs independently of Cl- secretion in rat distal colon. Am J Physiol Cell Physiol 303:C328-33
Basalingappa, Kanthesh M; Rajendran, Vazhaikkurichi M; Wonderlin, William F (2011) Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line. Am J Physiol Gastrointest Liver Physiol 301:G905-11

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