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Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns.

ABSTRACT: The activity of voltage-gated K(+) (K(V)) channels plays an important role in regulating pulmonary artery smooth muscle cell (PASMC) contraction, proliferation, and apoptosis. The highly conserved NH(2)-terminal tetramerization domain (T1) of K(V) channels is important for proper channel assembly, association with regulatory K(V) beta-subunits, and localization of the channel to the plasma membrane. We recently reported two nonsynonymous mutations (G182R and E211D) in the KCNA5 gene of patients with idiopathic pulmonary arterial hypertension, which localize to the T1 domain of KCNA5. To study the electrophysiological properties and expression patterns of the mutants compared with the wild-type (WT) channel in vitro, we transfected HEK-293 cells with WT KCNA5, G182R, E211D, or the double mutant G182R/E211D channel. The mutants form functional channels; however, whole cell current kinetic differences between WT and mutant channels exist. Steady-state inactivation curves of the G182R and G182R/E211D channels reveal accelerated inactivation; the mutant channels inactivated at more hyperpolarized potentials compared with the WT channel. Channel protein expression was also decreased by the mutations. Compared with the WT channel, which was present in its mature glycosylated form, the mutant channels are present in greater proportion in their immature form in HEK-293 cells. Furthermore, G182R protein level is greatly reduced in COS-1 cells compared with WT. Immunostaining data support the hypothesis that, while WT protein localizes to the plasma membrane, mutant protein is mainly retained in intracellular packets. Overall, these data support a role for the T1 domain in channel kinetics as well as in KCNA5 channel subcellular localization.

SUBMITTER: Burg ED

PROVIDER: S-EPMC2838577 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns.

Burg Elyssa D ED Platoshyn Oleksandr O Tsigelny Igor F IF Lozano-Ruiz Beatriz B Rana Brinda K BK Yuan Jason X-J JX

American journal of physiology. Cell physiology 20091216 3

The activity of voltage-gated K(+) (K(V)) channels plays an important role in regulating pulmonary artery smooth muscle cell (PASMC) contraction, proliferation, and apoptosis. The highly conserved NH(2)-terminal tetramerization domain (T1) of K(V) channels is important for proper channel assembly, association with regulatory K(V) beta-subunits, and localization of the channel to the plasma membrane. We recently reported two nonsynonymous mutations (G182R and E211D) in the KCNA5 gene of patients ...[more]

PMID: 20018952

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Project description:Background & aimsAlcohol intake with circadian rhythm disruption (CRD) increases colon cancer risk. We hypothesized that eating during or around physiologic rest time, a common habit in modern society, causes CRD and investigated the mechanisms by which it promotes alcohol-associated colon carcinogenesis.MethodsThe effect of feeding time on CRD was assessed using B6 mice expressing a fusion protein of PERIOD2 and LUCIFERASE (PER2::LUC) were used to model colon polyposis and to assess the effects of feeding schedules, alcohol consumption, and prebiotic treatment on microbiota composition, short-chain fatty acid levels, colon inflammation, and cancer risk. The relationship between butyrate signaling and a proinflammatory profile was assessed by inactivating the butyrate receptor GPR109A.ResultsEating at rest (wrong-time eating [WTE]) shifted the phase of the colon rhythm in PER2::LUC mice. In TS4Cre × APClox468 mice, a combination of WTE and alcohol exposure (WTE + alcohol) decreased the levels of short-chain fatty acid-producing bacteria and of butyrate, reduced colonic densities of regulatory T cells, induced a proinflammatory profile characterized by hyperpermeability and an increased mucosal T-helper cell 17/regulatory T cell ratio, and promoted colorectal cancer. Prebiotic treatment improved the mucosal inflammatory profile and attenuated inflammation and cancer. WTE + alcohol-induced polyposis was associated with increased signal transducer and activator of transcription 3 expression. Decreased butyrate signaling activated the epithelial signal transducer and activator of transcription 3 in vitro. The relationship between butyrate signaling and a proinflammatory profile was confirmed in human colorectal cancers using The Cancer Genome Atlas.ConclusionsAbnormal timing of food intake caused CRD and interacts with alcohol consumption to promote colon carcinogenesis by inducing a protumorigenic inflammatory profile driven by changes in the colon microbiota and butyrate signaling. Accession number of repository for microbiota sequence data: raw FASTQ data were deposited in the NCBI Sequence Read Archive under project PRJNA523141.

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Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns.

Publications

Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns.

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