title: Coupled and independent contributions of residues in IS6 and IIS6 to activation gating of CaV1.2.
abstract: Voltage dependence and kinetics of Ca(V)1.2 activation are affected by structural changes in pore-lining S6 segments of the alpha(1)-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 ("bundle crossing region"), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S., Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol. Chem. 280, 38471-38477). Here we report that S435P in IS6 results in a large shift of the activation curve (-25.9 +/- 1.2 mV) and slower current kinetics. Threonine substitutions at positions Leu-429 and Leu-434 induced a similar kinetic phenotype with shifted activation curves (L429T by -6.6 +/- 1.2 and L434T by -12.1 +/- 1.7 mV). Inactivation curves of all mutants were shifted to comparable extents as the activation curves. Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant cycle analysis. Double mutations in segments IS6 and IIS6 induce either additive (L429T/I781T, -34.1 +/- 1.4 mV; L434T/I781T, -40.4 +/- 1.3 mV; L429T/L779T, -12.6 +/- 1.3 mV; and L434T/L779T, -22.4 +/- 1.3 mV) or nonadditive shifts of the activation curves along the voltage axis (S435P/I781T, -33.8 +/- 1.4 mV). Mutant cycle analysis revealed energetic coupling between residues Ser-435 and Ile-781, whereas other paired mutations in segments IS6 and IIS6 had independent effects on activation gating.
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, Althanstrasse 14, A-1090 Wien, Austria.
authors.forename: Michaela
authors.initials: M
authors.lastname: Kudrnac
authors.forename: Stanislav
authors.initials: S
authors.lastname: Beyl
authors.forename: Annette
authors.initials: A
authors.lastname: Hohaus
authors.forename: Anna
authors.initials: A
authors.lastname: Stary
authors.forename: Thomas
authors.initials: T
authors.lastname: Peterbauer
authors.forename: Eugen
authors.initials: E
authors.lastname: Timin
authors.forename: Steffen
authors.initials: S
authors.lastname: Hering
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D020125:Mutation, Missense; D011817:Rabbits
chemical_list: D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C)