Dataset Information

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.

SUBMITTER: Barmeyer C

PROVIDER: S-EPMC2928625 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Cloning and identification of tissue-specific expression of KCNN4 splice variants in rat colon.

Barmeyer Christian C Rahner Christoph C Yang Youshan Y Sigworth Frederick J FJ Binder Henry J HJ Rajendran Vazhaikkurichi M VM

American journal of physiology. Cell physiology 20100505 2

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-specifi ...[more]

PMID: 20445171

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Project description:BackgroundVoltage-gated Na+ channel beta1 (Scn1b) subunits are multi-functional proteins that play roles in current modulation, channel cell surface expression, cell adhesion, cell migration, and neurite outgrowth. We have shown previously that beta1 modulates electrical excitability in vivo using a mouse model. Scn1b null mice exhibit spontaneous seizures and ataxia, slowed action potential conduction, decreased numbers of nodes of Ranvier in myelinated axons, alterations in nodal architecture, and differences in Na+ channel alpha subunit localization. The early death of these mice at postnatal day 19, however, make them a challenging model system to study. As a first step toward development of an alternative model to investigate the physiological roles of beta1 subunits in vivo we cloned two beta1-like subunit cDNAs from D. rerio.ResultsTwo beta1-like subunit mRNAs from zebrafish, scn1ba_tv1 and scn1ba_tv2, arise from alternative splicing of scn1ba. The deduced amino acid sequences of Scn1ba_tv1 and Scn1ba_tv2 are identical except for their C-terminal domains. The C-terminus of Scn1ba_tv1 contains a tyrosine residue similar to that found to be critical for ankyrin association and Na+ channel modulation in mammalian beta1. In contrast, Scn1ba_tv2 contains a unique, species-specific C-terminal domain that does not contain a tyrosine. Immunohistochemical analysis shows that, while the expression patterns of Scn1ba_tv1 and Scn1ba_tv2 overlap in some areas of the brain, retina, spinal cord, and skeletal muscle, only Scn1ba_tv1 is expressed in optic nerve where its staining pattern suggests nodal expression. Both scn1ba splice forms modulate Na+ currents expressed by zebrafish scn8aa, resulting in shifts in channel gating mode, increased current amplitude, negative shifts in the voltage dependence of current activation and inactivation, and increases in the rate of recovery from inactivation, similar to the function of mammalian beta1 subunits. In contrast to mammalian beta1, however, neither zebrafish subunit produces a complete shift to the fast gating mode and neither subunit produces complete channel inactivation or recovery from inactivation.ConclusionThese data add to our understanding of structure-function relationships in Na+ channel beta1 subunits and establish zebrafish as an ideal system in which to determine the contribution of scn1ba to electrical excitability in vivo.

Dataset Information

Cloning and identification of tissue-specific expression of KCNN4 splice variants in rat colon.

Publications

Cloning and identification of tissue-specific expression of KCNN4 splice variants in rat colon.

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