title: Chloride channel activity of ClC-2 is modified by the actin cytoskeleton.
abstract: The chloride channel ClC-2 has been implicated in essential physiological functions, including cell-volume regulation and fluid secretion by specific epithelial tissues. Although ClC-2 is known to be activated by hyperpolarization and hypo-osmotic shock, the molecular basis for the regulation of this channel remains unclear. Here we show in the Xenopus oocyte expression system that the chloride-channel activity of ClC-2 is enhanced after treatment with the actin-disrupting agents cytochalasin and latrunkulin. These findings suggest that the actin cytoskeleton normally exerts an inhibitory effect on ClC-2 activity. An inhibitory domain was previously defined in the N-terminus of ClC-2, so we sought to determine whether this domain might interact directly with actin in binding assays in vitro. We found that a glutathione S-transferase fusion protein containing the inhibitory domain was capable of binding actin in overlay and co-sedimentation assays. Further, the binding of actin to this relatively basic peptide (pI 8.4) might be mediated through electrostatic interactions because binding was inhibited at high concentrations of NaCl with a half-maximal decrease in signal at 180 mM NaCl. This work suggests that electrostatic interactions between the N-terminus of ClC-2 and the actin cytoskeleton might have a role in the regulation of this channel.
authors: Programme in Cell Biology, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada.
authors: N
authors: N
authors: Ahmed
authors: M
authors: M
authors: Ramjeesingh
authors: S
authors: S
authors: Wong
authors: A
authors: A
authors: Varga
authors: E
authors: E
authors: Garami
authors: C E
authors: CE
authors: Bear
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009994:Osmolar Concentration; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D014982:Xenopus laevis
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D053778:Thiazolidines; D015638:Cytochalasin D; C037068:latrunculin B