title: Characterization of the gating brake in the I-II loop of Ca(v)3.2 T-type Ca(2+) channels.
abstract: Mutations in the I-II loop of Ca(v)3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high alpha-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials.
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Imilla I
authors.initials: II
authors.lastname: Arias-Olguín
authors.forename: Iuliia
authors.initials: I
authors.lastname: Vitko
authors.forename: Michal
authors.initials: M
authors.lastname: Fortuna
authors.forename: Joel P
authors.initials: JP
authors.lastname: Baumgart
authors.forename: Svetlana
authors.initials: S
authors.lastname: Sokolova
authors.forename: Igor A
authors.initials: IA
authors.lastname: Shumilin
authors.forename: Amy
authors.initials: A
authors.lastname: Van Deusen
authors.forename: Manuel
authors.initials: M
authors.lastname: Soriano-García
authors.forename: Juan C
authors.initials: JC
authors.lastname: Gomora
authors.forename: Edward
authors.initials: E
authors.lastname: Perez-Reyes
mesh_terms: D000595:Amino Acid Sequence; D020747:Calcium Channels, T-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017124:Conserved Sequence; D004594:Electrophysiology; D006801:Humans; D015640:Ion Channel Gating; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
chemical_list: C479952:CACNA1H protein, human; D020747:Calcium Channels, T-Type