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J. Biol. Chem., Vol. 284, Issue 2, 1242-1251, January 9, 2009

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Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor β₁-null Zebrafish Relaxed Is an Exclusive Function of the β_1a Subunit^*

Johann Schredelseker¹, Anamika Dayal¹, Thorsten Schwerte, Clara Franzini-Armstrong^¶, and Manfred Grabner²

From the Department of Medical Genetics, Clinical and Molecular Pharmacology, Division of Biochemical Pharmacology, Innsbruck Medical University, A-6020 Innsbruck, Austria, the Institute of Zoology, University of Innsbruck, Innsbruck, A-6020 Austria, and the ^¶Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β_1a subunit. Lack of β_1a results in (i) reduced membrane expression of the pore forming DHPR _1S subunit, (ii) elimination of _1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β_1a from rather general functions of β isoforms. Zebrafish and mammalian β_1a subunits quantitatively restored _1S triad targeting and charge movement as well as intracellular Ca²⁺ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β_2a as the phylogenetically closest, and the ancestral housefly β_M as the most distant isoform to β_1a also completely recovered _1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca²⁺ transients and very limited tetrad formation compared with β_1a. Consequently, larval motility was either only partially restored (β_2a-injected larvae) or not restored at all (β_M). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β_1a isoform. Consequently, we postulate a model that presents β_1a as an allosteric modifier of _1S conformation enabling skeletal muscle-type EC coupling.

Received for publication, October 8, 2008

^* This work was supported, in whole or in part, by National Institutes of Health Grant HL-48093 (to C. F.-A.). This work was also supported by Austrian Fonds zur Förderung der wissenschaftlichen Forschung Research Grant FWF-16098-B04 (to M. G.) and Medizinische Forschungsförderung Innsbruck Grant MFI-6180 (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Sektion für Biochemische Pharmakologie, Medizinische Universität Innsbruck, A-6020 Innsbruck, Austria. Tel.: 43-0-512-9003-70407; Fax: 43-0-512-9003-73407; E-mail: manfred.grabner{at}i-med.ac.at.

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