Proper restoration of excitation-contraction coupling in the dihydropyridine receptor β 1-null zebrafish relaxed is an exclusive function of the β 1a subunit

Abstract

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 α 1s subunit, (ii) elimination of α 1scharge 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 α 1s triad targeting and charge movement as well as intracellular Ca 2+ 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 phylogeneti- cally closest, and the ancestral housefly β M as the most distant isoform to β 1a also completely recovered α 1s triad expression and charge movement. However, both revealed drastically impaired intracellular Ca 2+ 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 α 1sconformation enabling skeletal muscle-type EC coupling. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.