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(1C)/beta(2)-subunit interaction and that the ahnak-derived proteins relieve the beta(2)-subunit from this inhibition.
Highlights
Ca2ϩ influx through the L-type Ca2ϩ channel (ICaL) initiates and modulates cardiac contraction
In an attempt to define the molecular details of channel subunit phosphorylation, we have identified the 700-kDa ahnak protein as the prominent protein kinase A (PKA) target in mammalian cardiomyocytes that is recovered in anti2-subunit immunoprecipitates [9]
This study demonstrates that carboxyl-terminal ahnak protein fragments modulate Ca2ϩ channel properties
Summary
Ca2ϩ influx through the L-type Ca2ϩ channel (ICaL) initiates and modulates cardiac contraction. A protein of 5643 amino acids, has been implicated in different cell type-specific functions as diverse as cell differentiation [10, 11], signal transduction [9, 12,13,14], Ca2ϩ homeostasis [15], and regulated exocytosis [16]. By using truncated ahnak fragments, we demonstrated that the high affinity interaction (Kd Ϸ50 nM) between 2-subunit and ahnak is mediated by the most carboxyl-terminal 382 amino acids of ahnak, designated as the ahnak-C2 domain. This ahnak-C2 domain was defined to be responsible for F-actin binding [19].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
