Abstract
Ahnak1 has been implicated in the beta-adrenergic regulation of the cardiac L-type Ca2+ channel current (I CaL) by its binding to the regulatory Cavβ2 subunit. In this study, we addressed the question whether ahnak1/Cavβ2 interactions are essential or redundant for beta-adrenergic stimulation of I CaL. Three naturally occurring ahnak1 variants (V5075 M, G5242R, and T5796 M) identified by genetic screening of cardiomyopathy patients did essentially not influence the in vitro Cavβ2 interaction as assessed by recombinant proteins. But, we observed a robust increase in Cavβ2 binding by mutating Ala at position 4984 to Pro which creates a PxxP consensus motif in the ahnak1 protein fragment. Surface plasmon resonance measurements revealed that this mutation introduced an additional Cavβ2 binding site. The functionality of A4984P was supported by the specific action of the Pro-containing ahnak1-derived peptide (P4984) in beta-adrenergic regulation of I CaL. Patch clamp recordings on cardiomyocytes showed that intracellular perfusion of P4984 markedly reduced I CaL response to the beta-adrenergic agonist, isoprenaline, while the Ala-containing counterpart failed to affect I CaL. Interestingly, I CaL of ahnak1-deficient cardiomyocytes was not affected by peptide application. Moreover, I CaL of ahnak1-deficient cardiomyocytes showed intact beta-adrenergic responsiveness. Similarly isolated ahnak1-deficient mouse hearts responded normally to adrenergic challenge. Our results indicate that ahnak1 is not essential for beta-adrenergic up-regulation of I CaL and cardiac contractility in mice. But, tuning ahnak1/Cavβ2 interaction provides a tool for modulating the beta-adrenergic response of I CaL.
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Acknowledgments
This study was supported by the German-Israeli Foundation and by the Stiftung für Pathobiochemie und Molekulare Diagnostik. Special thanks go to Dr. Kouno (Osaka, Japan) for providing the ahnak1-deficient mouse strain. We thank Dr. Sandro Keller for providing the spectrometer for CD spectra recordings and Dr. Andreas Perrot for critical discussions. Excellent technical assistance by Karin Karczewski and Steffen Lutter is greatly acknowledged.
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Pankonien, I., Alvarez, J.L., Doller, A. et al. Ahnak1 is a tuneable modulator of cardiac Ca(v)1.2 calcium channel activity. J Muscle Res Cell Motil 32, 281–290 (2011). https://doi.org/10.1007/s10974-011-9269-2
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DOI: https://doi.org/10.1007/s10974-011-9269-2