title: Cloning and identification of tissue-specific expression of KCNN4 splice variants in rat colon.
abstract: 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.
authors.affiliation: Department of Internal Medicine, Yale University, New Haven, Connecticut, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Barmeyer
authors.forename: Christoph
authors.initials: C
authors.lastname: Rahner
authors.forename: Youshan
authors.initials: Y
authors.lastname: Yang
authors.forename: Frederick J
authors.initials: FJ
authors.lastname: Sigworth
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Binder
authors.forename: Vazhaikkurichi M
authors.initials: VM
authors.lastname: Rajendran
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D003106:Colon / Q000378:metabolism* / Q000502:physiology; D005260:Female; D005786:Gene Expression Regulation; D014644:Genetic Variation / Q000502:physiology*; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000096:biosynthesis* / Q000235:genetics; D007413:Intestinal Mucosa / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014981:Xenopus
chemical_list: D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C491575:Kcnn4 protein, rat; D020033:Protein Isoforms
references.citation: Annu Rev Physiol. 2000;62:535-72
references.pmid: 10845102
references.citation: J Membr Biol. 1996 Jul;152(1):1-11
references.pmid: 8660412
references.citation: Cell Tissue Res. 2003 Nov;314(2):179-89
references.pmid: 14513356
references.citation: Am J Respir Cell Mol Biol. 2007 Nov;37(5):525-31
references.pmid: 17585114
references.citation: Kidney Int. 2008 Sep;74(6):740-9
references.pmid: 18547995
references.citation: J Mol Biol. 2001 Jan 19;305(3):567-80
references.pmid: 11152613
references.citation: Circ Res. 1999 Oct 29;85(9):e33-43
references.pmid: 10532960
references.citation: J Cell Sci Suppl. 1993;17:13-20
references.pmid: 8144688
references.citation: J Clin Invest. 1991 Oct;88(4):1379-85
references.pmid: 1655829
references.citation: Am J Physiol Gastrointest Liver Physiol. 2003 Jul;285(1):G185-96
references.pmid: 12606302
references.citation: Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11651-6
references.pmid: 9326665
references.citation: Am J Physiol Cell Physiol. 2006 Oct;291(4):C636-48
references.pmid: 16641164
references.citation: J Biol Chem. 1998 Nov 27;273(48):32167-75
references.pmid: 9822694
references.citation: Am J Physiol Cell Physiol. 2008 Nov;295(5):C1409-16
references.pmid: 18815226
references.citation: Am J Physiol Cell Physiol. 2000 Jan;278(1):C182-9
references.pmid: 10644526
references.citation: Fed Proc. 1967 Sep;26(5):1405-14
references.pmid: 6051321
references.citation: Biochim Biophys Acta. 1983 Oct 26;735(1):1-11
references.pmid: 6626541
references.citation: Biochim Biophys Acta. 1998 Jun 10;1365(1-2):60-4
references.pmid: 9693722
references.citation: J Biol Chem. 1989 Nov 5;264(31):18638-44
references.pmid: 2553716
references.citation: J Membr Biol. 1986;91(1):19-31
references.pmid: 3016279
references.citation: Am J Physiol. 1994 Jan;266(1 Pt 1):G71-82
references.pmid: 8304460
references.citation: J Biol Chem. 2004 Feb 20;279(8):6893-904
references.pmid: 14638680
references.citation: Am J Physiol Cell Physiol. 2003 Aug;285(2):C433-45
references.pmid: 12711595
references.citation: Lancet. 1994 Jan 1;343(8888):23-5
references.pmid: 7905044
references.citation: Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):8151-6
references.pmid: 10884437
references.citation: J Membr Biol. 2003 Nov 1;196(1):15-31
references.pmid: 14724753
references.citation: Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):11013-8
references.pmid: 9380751
references.citation: J Cell Biol. 1986 Sep;103(3):755-66
references.pmid: 3528172
references.citation: J Biol Chem. 2004 Nov 12;279(46):47681-7
references.pmid: 15347667
references.citation: Am J Physiol. 1996 Nov;271(5 Pt 1):L785-95
references.pmid: 8944722
references.citation: J Biol Chem. 1998 Nov 20;273(47):30863-9
references.pmid: 9812978
references.citation: Am J Physiol Cell Physiol. 2003 Jan;284(1):C77-84
references.pmid: 12388088
references.citation: Biochemistry. 2002 Aug 6;41(31):9803-12
references.pmid: 12146946
references.citation: Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):14066-71
references.pmid: 9391153
references.citation: Am J Physiol. 1999 Feb;276(2):C350-60
references.pmid: 9950762
references.citation: Histochem Cell Biol. 1996 Jul;106(1):41-58
references.pmid: 8858366
references.citation: Pflugers Arch. 1999 Sep;438(4):437-44
references.pmid: 10519135
references.citation: Am J Physiol. 1998 Sep;275(3):C848-56
references.pmid: 9730970
references.citation: Am J Physiol. 1994 Jan;266(1 Pt 2):H156-64
references.pmid: 7508206
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: J Biol Chem. 2001 Apr 13;276(15):12249-56
references.pmid: 11278890
references.citation: Annu Rev Cell Dev Biol. 2003;19:333-66
references.pmid: 14570573
references.citation: J Clin Invest. 1996 Nov 1;98(9):2066-75
references.pmid: 8903326
references.citation: Proc Natl Acad Sci U S A. 2004 Jun 22;101(25):9479-84
references.pmid: 15194822
references.citation: J Clin Invest. 2008 Sep;118(9):3025-37
references.pmid: 18688283
references.citation: Am J Physiol Cell Physiol. 2008 Mar;294(3):C820-32
references.pmid: 18184876
references.citation: J Biol Chem. 1997 Oct 17;272(42):26479-87
references.pmid: 9334225
references.citation: J Biol Chem. 1997 Dec 26;272(52):32723-6
references.pmid: 9407042
references.citation: J Gen Physiol. 2000 Jun;115(6):719-36
references.pmid: 10828246
references.citation: J Pharmacol Exp Ther. 2001 Feb;296(2):600-11
references.pmid: 11160649
references.citation: Pflugers Arch. 1996 Oct;432(6):1011-22
references.pmid: 8781195
references.citation: Wien Klin Wochenschr. 1997 Jun 27;109(12-13):497-8
references.pmid: 9261993
references.citation: Naunyn Schmiedebergs Arch Pharmacol. 2004 Jun;369(6):602-15
references.pmid: 15127180
references.citation: J Biol Chem. 1998 Aug 21;273(34):21542-53
references.pmid: 9705284
references.citation: Biochemistry. 1995 Apr 4;34(13):4402-11
references.pmid: 7535563