Two PEST-like motifs regulate Ca2+/calpain-mediated cleavage of the CaVβ3 subunit and provide important determinants for neuronal Ca2+ channel activity

Authors

  • Alejandro Sandoval,

    1. Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico City, Mexico
    2. School of Medicine FES Iztacala and
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    • *

      A.S. and N.O. contributed equally to this work.

  • Norma Oviedo,

    1. Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico City, Mexico
    2. Department of Molecular Biology and Biotechnology, Biomedical Research Institute, National Autonomous University of Mexico, Mexico City, Mexico
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    • *

      A.S. and N.O. contributed equally to this work.

  • Abir Tadmouri,

    1. INSERM U607/DRDC, Commissariat à l'énergie atomique, Grenoble, Cedex, France
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  • Traudy Avila,

    1. Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico City, Mexico
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  • Michel De Waard,

    1. INSERM U607/DRDC, Commissariat à l'énergie atomique, Grenoble, Cedex, France
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  • Ricardo Felix

    1. Departamento de Biología Celular, Cinvestav-IPN, Avenida IPN 2508, Colonia Zacatenco, México D.F., CP 07300
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Dr Ricardo Felix, as above.
E-mail: rfelix@fisio.cinvestav.mx

Abstract

An increase in intracellular Ca2+ due to voltage-gated Ca2+ (CaV) channel opening represents an important trigger for a number of second-messenger-mediated effects ranging from neurotransmitter release to gene activation. Ca2+ entry occurs through the principal pore-forming protein but several ancillary subunits are known to more precisely tune ion influx. Among them, the CaVβ subunits are perhaps the most important, given that they largely influence the biophysical and pharmacological properties of the channel. Notably, several functional features may be associated with specific structural regions of the CaVβ subunits emphasizing the relevance of intramolecular domains in the physiology of these proteins. In the current report, we show that CaVβ3 contains two PEST motifs and undergoes Ca2+-dependent degradation which can be prevented by the specific calpain inhibitor calpeptin. Using mutant constructs lacking the PEST motifs, we present evidence that they are necessary for the cleavage of CaVβ3 by calpain. Furthermore, the deletion of the PEST sequences did not affect the binding of CaVβ3 to the ion-conducting CaV2.2 subunit and, when expressed in human embryonic kidney-293 cells, the PEST motif-deleted CaVβ3 significantly increased whole-cell current density and retarded channel inactivation. Consistent with this observation, calpeptin treatment of human embryonic kidney-293 cells expressing wild-type CaVβ3 resulted in an increase in current amplitude. Together, these findings suggest that calpain-mediated CaVβ3 proteolysis may be an essential process for Ca2+ channel functional regulation.

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