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Am J Physiol Cell Physiol
2010 Aug 01;2992:C251-63. doi: 10.1152/ajpcell.00091.2009.
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Cloning and identification of tissue-specific expression of KCNN4 splice variants in rat colon.
Barmeyer C
,
Rahner C
,
Yang Y
,
Sigworth FJ
,
Binder HJ
,
Rajendran VM
.
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KCNN4 channels that provide the driving force for cAMP- and Ca(2+)-induced anion secretion are present in both apical and basolateral membranes of the mammalian colon. However, only a single KCNN4 has been cloned. This study was initiated to identify whether both apical and basolateral KCNN4 channels are encoded by the same or different isoforms. Reverse transcriptase-PCR (RT-PCR), real-time quantitative-PCR (RT-QPCR), and immunofluorescence studies were used to clone and identify tissue-specific expression of KCNN4 isoforms. Three distinct KCNN4 cDNAs that are designated as KCNN4a, KCNN4b, and KCNN4c encoding 425, 424, and 395 amino acid proteins, respectively, were isolated from the rat colon. KCNN4a differs from KCNN4b at both the nucleotide and the amino acid level with distinct 628 bp at the 3'-untranslated region and an additional glutamine at position 415, respectively. KCNN4c differs from KCNN4b by lacking the second exon that encodes a 29 amino acid motif. KCNN4a and KCNN4b/c are identified as smooth muscle- and epithelial cell-specific transcripts, respectively. KCNN4b and KCNN4c transcripts likely encode basolateral (40 kDa) and apical (37 kDa) membrane proteins in the distal colon, respectively. KCNN4c, which lacks the S2 transmembrane segment, requires coexpression of a large conductance K(+) channel beta-subunit for plasma membrane expression. The KCNN4 channel blocker TRAM-34 inhibits KCNN4b- and KCNN4c-mediated (86)Rb (K(+) surrogate) efflux with an apparent inhibitory constant of 0.6 +/- 0.1 and 7.8 +/- 0.4 muM, respectively. We conclude that apical and basolateral KCNN4 K(+) channels that regulate K(+) and anion secretion are encoded by distinct isoforms in colonic epithelial cells.
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