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Ahnak1 modulates L-type Ca2+ channel inactivation of rodent cardiomyocytes

  • Cardiovascular physiology
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Ahnak1, a giant 700 kDa protein, has been implicated in Ca2+ signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavβ2 subunit plays a role in L-type Ca2+ current (I CaL) regulation. Here, we performed structure–function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavβ2 (K d ∼ 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavβ2 affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca2+ influx by enhancing I CaL density and/or increasing I CaL inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I CaL density, markedly slowed I CaL inactivation resulting in a 50–70% increase in transported Ca2+ during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba2+ as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I CaL characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I CaL inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavβ2 and/or scaffolds selectively different β-subunit isoforms in the heart.

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Acknowledgements

The authors thank Karin Karczewski, Steffen Lutter and Wolfgang Schlegel for technical assistance. J.L.A. held a master’s fellowship from the MDC (Berlin, Germany). We thank Drs. Helmut Kettenmann and Daniel Reyes-Haro (MDC) for their support with patch clamp setup. We are grateful to Dr. Nathan Dascal who provided the α1C constructs within a project funded by the German Israel Foundation (grant no.: 930.220.2/2006). I.P. receives a stipend from that GIF project.

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Authors and Affiliations

  1. Laboratorio de Electrofisiologia, Instituto de Cardiologia y Cirugia Cardiovascular, Havana, Cuba

    Julio L. Alvarez

  2. Max Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, 13092, Berlin, Germany

    Daria Petzhold, Ines Pankonien, Joachim Behlke, Ingo Morano & Hannelore Haase

  3. University Medicine Charité, Berlin, Germany

    Ines Pankonien & Ingo Morano

  4. Department of Social and Environmental Medicine, Osaka University, Osaka, Japan

    Michiyoshi Kouno

  5. Physiopathologie Cardiovasculaire, INSERM U-637, Montpellier Cedex 5, France

    Guy Vassort

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  1. Julio L. Alvarez
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Correspondence to Hannelore Haase.

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Alvarez, J.L., Petzhold, D., Pankonien, I. et al. Ahnak1 modulates L-type Ca2+ channel inactivation of rodent cardiomyocytes. Pflugers Arch - Eur J Physiol 460, 719–730 (2010). https://doi.org/10.1007/s00424-010-0853-x

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