ABSTRACT
KVLQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of KVLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to KVLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of KVLQT1 in crypt cells and surface epithelium. Expression of rKVLQT1 in Xenopus oocytes induced a typical delayed activated K+ current, that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that KVLQT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.
Subject(s)
Colon/metabolism , Cyclic AMP/metabolism , Intestinal Mucosa/metabolism , Potassium Channels, Voltage-Gated , Potassium Channels/genetics , Potassium Channels/metabolism , 1-Methyl-3-isobutylxanthine/pharmacology , Amino Acid Sequence , Animals , Barium/pharmacology , Carbachol/pharmacology , Cardiotonic Agents/pharmacology , Cloning, Molecular , Colforsin/pharmacology , Dinoprostone/pharmacology , Humans , In Vitro Techniques , Intestinal Mucosa/cytology , Intestinal Mucosa/drug effects , KCNQ Potassium Channels , KCNQ1 Potassium Channel , Male , Molecular Sequence Data , Oocytes/physiology , Patch-Clamp Techniques , Phosphodiesterase Inhibitors/pharmacology , Potassium Channels/chemistry , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Tetraethylammonium/pharmacology , Xenopus laevisABSTRACT
Corticosteroid hormone-induced factor (CHIF) is an aldosterone-induced gene, the function of which is yet unknown. It is specifically expressed in kidney collecting duct (CD) and distal colon and is upregulated by either Na+ deprivation or K+ loading. Hence, it may play a role in epithelial electrolyte transport. Previous studies have characterized regulation and tissue distribution of CHIF mRNA but provided no information on the protein itself. The present paper addresses this issue by using Western blotting, immunochemistry, and in vitro translation. CHIF is an approximately 8-kDa membranal protein, and protease digestion experiments suggest that its COOH tail faces the cell interior. The protein is abundant in distal colon, kidney medulla, and papilla but cannot be detected in a variety of other tissues. Confocal immunocytochemistry demonstrates that CHIF is present in the basolateral membrane of CD principal cells and distal colon surface cells, with occasional intracellular staining. Dexamethasone and low Na+ intake increase the abundance of CHIF. Unlike previous Northern data, induction of CHIF protein by low-Na+ intake was apparent not only in the distal colon but also in the kidney.
Subject(s)
Colon/metabolism , Intracellular Membranes/metabolism , Kidney/metabolism , Animals , Blotting, Western , Cell Line , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Rats, Wistar , Reticulocytes/metabolism , Tissue DistributionABSTRACT
1. KvLQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential. Defects in ion channels have been demonstrated in cardiac arrhythmia. This channel is inhibited potently by the chromanol 293B. The same compound has been shown to block cAMP-dependent electrolyte secretion in rat and human colon. Therefore, it was suggested that a K+ channel similar to KvLQT1 is expressed in the colonic epithelium. 2. In the present paper, expression of KvLQT1 and its function in colonic epithelial cells is described. Reverse transcription-polymerase chain reaction analysis of rat colonic mucosa demonstrated expression of KvLQT1 in both crypt cells and surface epithelium. When expressed in Xenopus oocytes, KvLQT1 induced a typical delayed activated K+ current. 3. As demonstrated, the channel activity could be further activated by increases in intracellular cAMP. These and other data support the concept that KvLQT1 is forming a component of the basolateral cAMP-activated K+ conductance in the colonic epithelium.
