title: 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.
authors.affiliation: Department of Anesthesiology, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Franck
authors.initials: F
authors.lastname: Potet
authors.forename: Christina I
authors.initials: CI
authors.lastname: Petersen
authors.forename: Olivier
authors.initials: O
authors.lastname: Boutaud
authors.forename: Wen
authors.initials: W
authors.lastname: Shuai
authors.forename: Svetlana Z
authors.initials: SZ
authors.lastname: Stepanovic
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Balser
authors.forename: Sabina
authors.initials: S
authors.lastname: Kupershmidt
mesh_terms: D000818:Animals; D001145:Arrhythmias, Cardiac / Q000235:genetics / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D000072237:ERG1 Potassium Channel; D004594:Electrophysiology; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008133:Long QT Syndrome / Q000235:genetics / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference
chemical_list: C109971:ARHGAP6 protein, human; D029742:Caenorhabditis elegans Proteins; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D020690:GTPase-Activating Proteins; C000606913:KCNH2 protein, human; D015221:Potassium Channels; C474006:UNC-103 protein, C elegans; C085379:rho GTPase-activating protein; D011188:Potassium