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Genetic screening in C. elegans identifies rho-GTPase activating protein 6 as novel HERG regulator.

ABSTRACT: The human ether-a-go-go related gene (HERG) constitutes the pore forming subunit of I(Kr), a K(+) current involved in repolarization of the cardiac action potential. While mutations in HERG predispose patients to cardiac arrhythmias (Long QT syndrome; LQTS), altered function of HERG regulators are undoubtedly LQTS risk factors. We have combined RNA interference with behavioral screening in Caenorhabditis elegans to detect genes that influence function of the HERG homolog, UNC-103. One such gene encodes the worm ortholog of the rho-GTPase activating protein 6 (ARHGAP6). In addition to its GAP function, ARHGAP6 induces cytoskeletal rearrangements and activates phospholipase C (PLC). Here we show that I(Kr) recorded in cells co-expressing HERG and ARHGAP6 was decreased by 43% compared to HERG alone. Biochemical measurements of cell-surface associated HERG revealed that ARHGAP6 reduced membrane expression of HERG by 35%, which correlates well with the reduction in current. In an atrial myocyte cell line, suppression of endogenous ARHGAP6 by virally transduced shRNA led to a 53% enhancement of I(Kr). ARHGAP6 effects were maintained when we introduced a dominant negative rho-GTPase, or ARHGAP6 devoid of rhoGAP function, indicating ARHGAP6 regulation of HERG is independent of rho activation. However, ARHGAP6 lost effectiveness when PLC was inhibited. We further determined that ARHGAP6 effects are mediated by a consensus SH3 binding domain within the C-terminus of HERG, although stable ARHGAP6-HERG complexes were not observed. These data link a rhoGAP-activated PLC pathway to HERG membrane expression and implicate this family of proteins as candidate genes in disorders involving HERG.

SUBMITTER: Potet F

PROVIDER: S-EPMC2708093 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Genetic screening in C. elegans identifies rho-GTPase activating protein 6 as novel HERG regulator.

Potet Franck F Petersen Christina I CI Boutaud Olivier O Shuai Wen W Stepanovic Svetlana Z SZ Balser Jeffrey R JR Kupershmidt Sabina S

Journal of molecular and cellular cardiology 20081105 2

The human ether-a-go-go related gene (HERG) constitutes the pore forming subunit of I(Kr), a K(+) current involved in repolarization of the cardiac action potential. While mutations in HERG predispose patients to cardiac arrhythmias (Long QT syndrome; LQTS), altered function of HERG regulators are undoubtedly LQTS risk factors. We have combined RNA interference with behavioral screening in Caenorhabditis elegans to detect genes that influence function of the HERG homolog, UNC-103. One such gene ...[more]

PMID: 19038263

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Project description:BackgroundIn 2010, we described a novel immunoglobulin G (IgG) autoantibody (termed anti-Ca after the index case) targeting Rho GTPase-activating protein 26 (ARHGAP26, also termed GTPase regulator associated with focal adhesion kinase [GRAF], or oligophrenin-like protein 1 [OPHN1L]) in autoimmune cerebellar ataxia (ACA). Later, ARHGAP26-IgG/anti-Ca was reported in patients with limbic encephalitis/cognitive decline or peripheral neuropathy. In several of the reported cases, the syndrome was associated with cancer. ARHGAP10/GRAF2, which is expressed throughout the central nervous system, shares significant sequence homology with ARHGAP26/GRAF. Mutations in the ARHGAP10 gene have been linked to cognitive and psychiatric symptoms and schizophrenia.ObjectiveTo assess whether ARHGAP26-IgG/anti-Ca co-reacts with ARHGAP10.MethodsSerological testing for ARHGAP10/GRAF2 autoantibodies by recombinant cell-based assays and isotype and IgG subclass analyses.Results26/31 serum samples (84%) from 9/12 (75%) ARHGAP26-IgG/anti-Ca-positive patients and 4/6 ARHGAP26-IgG/anti-Ca-positive CSF samples from four patients were positive also for ARHGAP10-IgG. ARHGAP10-IgG (termed anti-Ca2) remained detectable in the long-term (up to 109 months) and belonged mainly to the complement-activating IgG1 subclass. Median ARHGAP26-IgG/anti-Ca and median ARHGAP10-IgG/anti-Ca2 serum titres were 1:3200 and 1:1000, respectively, with extraordinarily high titres in some samples (ARHGAP26-IgG/anti-Ca: up to 1:1000,000; ARHGAP10-IgG: up to 1:32,000). ARHGAP26/anti-Ca serum titres exceeded those of ARHGAP10-IgG in all samples but one. A subset of patients was positive also for ARHGAP10-IgM and ARHGAP10-IgA. CSF/serum ratios and antibody index calculation suggested intrathecal production of ARHGAP26-IgG/anti-Ca and anti-ARHGAP10. Of 101 control samples, 100 were completely negative for ARHGAP10-IgG; a single control sample bound weakly (1:10) to the ARHGAP10-transfected cells.ConclusionsWe demonstrate that a substantial proportion of patients with ARHGAP26-IgG/anti-Ca-positive autoimmune encephalitis co-react with ARHGAP10. Further studies on the clinical and diagnostic implications of ARHGAP10-IgG/anti-Ca2 seropositivity in patients with autoimmune encephalitis are warranted.

Dataset Information

Genetic screening in C. elegans identifies rho-GTPase activating protein 6 as novel HERG regulator.

Publications

Genetic screening in C. elegans identifies rho-GTPase activating protein 6 as novel HERG regulator.

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