title: Intracellular Ca2+ inactivates L-type Ca2+ channels with a Hill coefficient of approximately 1 and an inhibition constant of approximately 4 microM by reducing channel's open probability.
abstract: The patch-clamp technique was used to characterize the mechanism of Ca2+-induced inactivation of cardiac L-type Ca2+ channel alpha(1C-a) + beta3 subunits stably expressed in CHO cells. Single Ca2+ channel activity was monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)BAYK 8644 and calpastatin plus ATP. This enabled stabilization of channel activity in the inside-out patch and allowed for application of steady-state Ca2+ concentrations to the intracellular face of excised membrane patches in an attempt to provoke Ca2+-induced inactivation. Inactivation was found to occur specifically with Ca2+ since it was not observed upon application of Ba2+. Ca2+-dependent inhibition of mean Ca2+ channel activity was characterized by a Hill coefficient close to 1. Ca2+ binding to open and closed states of the channel obtained during depolarization apparently occurred with similar affinity yielding half-maximal inhibition of Ca2+ channel activity at approximately 4 microM. This inhibition manifested predominantly in a reduction of the channel's open probability whereas availability remained almost unchanged. The reduction in open probability was achieved by an increase in first latencies and a decrease in channel opening frequency as well as channel open times. At high (12-28 microM) Ca2+ concentrations, 72% of inhibition occurred due to a stabilization of the closed state and the remaining 28% by a destabilization of the open state. Our results suggest that binding of one calcium ion to a regulatory domain induces a complex alteration in the kinetic properties of the Ca2+ channel and support the idea of a single EF hand motif as the relevant Ca2+ binding site on the alpha1 subunit.
authors.affiliation: Institute for Biophysics, University of Linz, Austria.
authors.forename: G F
authors.initials: GF
authors.lastname: Höfer
authors.forename: K
authors.initials: K
authors.lastname: Hohenthanner
authors.forename: W
authors.initials: W
authors.lastname: Baumgartner
authors.forename: K
authors.initials: K
authors.lastname: Groschner
authors.forename: N
authors.initials: N
authors.lastname: Klugbauer
authors.forename: F
authors.initials: F
authors.lastname: Hofmann
authors.forename: C
authors.initials: C
authors.lastname: Romanin
mesh_terms: D000818:Animals; D001464:Barium / Q000494:pharmacology; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials; D009206:Myocardium / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D011994:Recombinant Proteins; D001464:Barium; D002118:Calcium