Calcium current in rat cardiomyocytes is modulated by the carboxyl-terminal ahnak domain

Item Type:	Article
Title:	Calcium current in rat cardiomyocytes is modulated by the carboxyl-terminal ahnak domain
Creators Name:	Alvarez, J. and Hamplova, J. and Hohaus, A. and Morano, I. and Haase, H. and Vassort, G.
Abstract:	Ahnak, a protein of 5643 amino acids, interacts with the regulatory {beta}-subunit of cardiac calcium channels and with F-actin. Recently, we defined the binding sites among the protein partners in the carboxyl-terminal domain of ahnak. Here we further narrowed down the {beta} 2-interaction sites to the carboxyl-terminal 188 amino acids of ahnak by the recombinant ahnak protein fragments P3 (amino acids 5456-5556) and P4 (amino acids 5556-5643). The effects of these P3 and P4 fragments on the calcium current were investigated under whole-cell patch clamp conditions on rat ventricular cardiomyocytes. P4 but not P3 increased significantly the current amplitude by 22.7 ± 5% without affecting its voltage dependence. The slow component of calcium current inactivation was slowed down by both P3 and P4, whereas only P3 slowed significantly the fast one. The composite recombinant protein fragment P3-P4 induced similar modifications to the ones induced by each of the ahnak fragments. In the presence of carboxyl-terminal ahnak protein fragments, isoprenaline induced a similar relative increase in current amplitude and shift in current kinetics. The actin-stabilizing agents, phalloidin and jasplakinolide, did not modify the effects of these ahnak protein fragments on calcium current in control conditions nor in the presence of isoprenaline. Hence, our results suggest that the functional effects of P3, P4, and P3-P4 on calcium current are mediated by targeting the ahnak-{beta} 2-subunit interaction rather than by targeting the ahnak-F-actin interaction. More specifically they suggest that binding of the {beta} 2-subunit to the endogenous subsarcolemmal giant ahnak protein re-primes the {alpha} aC/{beta} 2-subunit interaction and that the ahnak-derived proteins relieve the {beta} 2-subunit from this inhibition.
Keywords:	Actins, Binding Sites, Calcium, Cardiac Myocytes, Cyclic Peptides, Depsipeptides, Electrophysiology, Heart Ventricles, Kinetics, Membrane Proteins, Neoplasm Proteins, Patch-Clamp Techniques, Phalloidine, Phosphorylation, Plasmids, Protein Binding, Protein Conformation, Recombinant Proteins, Tertiary Protein Structure, Time Factors, Wistar Rats, Animals, Rats
Source:	Journal of Biological Chemistry
ISSN:	0021-9258
Publisher:	American Society for Biochemistry and Molecular Biology
Volume:	279
Number:	13
Page Range:	12456-12461
Date:	26 March 2004
Official Publication:	https://doi.org/10.1074/jbc.M312177200
PubMed:	View item in PubMed

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