title: Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder.
abstract: The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the Ca(V)1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553-7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the Ca(V)1.2 channel. Substitution of Ile-781, the Ca(V)1.2 residue corresponding to Ile-745 in Ca(V)1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779-782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of Ca(V)1.2. Moreover there may be a flexible center of helix bending at positions 779-782 of Ca(V)1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.
authors.affiliation: Institute for Pharmacology and Toxicology, University of Vienna, Althanstrasse 14, A-1090 Wien.
authors.forename: Annette
authors.initials: A
authors.lastname: Hohaus
authors.forename: Stanislav
authors.initials: S
authors.lastname: Beyl
authors.forename: Michaela
authors.initials: M
authors.lastname: Kudrnac
authors.forename: Stanislav
authors.initials: S
authors.lastname: Berjukow
authors.forename: Eugen N
authors.initials: EN
authors.lastname: Timin
authors.forename: Rainer
authors.initials: R
authors.lastname: Marksteiner
authors.forename: Marion A
authors.initials: MA
authors.lastname: Maw
authors.forename: Steffen
authors.initials: S
authors.lastname: Hering
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001464:Barium / Q000378:metabolism; D002118:Calcium / Q000737:chemistry; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007477:Ions; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012164:Retinal Diseases / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D020746:Calcium Channels, L-Type; D007477:Ions; C419385:L-type calcium channel alpha(1C); D007532:Isoleucine; D049452:Green Fluorescent Proteins; D001464:Barium; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D001120:Arginine; D002118:Calcium