Abstract
A peptide corresponding to residues 681-690 of the II-III loop of the skeletal muscle dihydropyridine receptor alpha(1) subunit (DHPR, alpha(1S)) has been reported to activate the skeletal muscle ryanodine receptor (RyR1) in vitro. Within this region of alpha(1S), a cluster of basic residues, Arg(681)-Lys(685), was previously reported to be indispensable for the activation of RyR1 in microsomal preparations and lipid bilayers. We have used an intact alpha(1S) subunit with scrambled sequence in this region of the II-III loop (alpha(1S)-scr) to test the importance of residues 681-690 and the basic motif for skeletal-type excitation-contraction (EC) coupling and retrograde signaling in vivo. When expressed in dysgenic myotubes (which lack endogenous alpha(1S)), alpha(1S)-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type alpha(1S). The scrambled DHPR also rescued skeletal-type EC coupling, as indicated by electrically evoked contractions in the presence of 0.5 mm Cd(2+) and 0.1 mm La(3+). Furthermore, the release of intracellular Ca(2+), as assayed by the indicator dye, Fluo-3, had similar kinetics and voltage dependence for alpha(1S) and alpha(1S)-scr. These data suggest that residues 681-690 of the alpha(1S) II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.
Highlights
From the Departments of ‡Physiology and ¶Anatomy and Neurobiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523
Within peptide A, residues 681– 690 have been identified as the “minimum essential region” of the dihydropyridine receptors (DHPRs) II-III loop for activating ryanodine binding and Ca2ϩ release [10], and it has been suggested that the integrity of a cluster of five basic residues (Arg681–Lys685) is requisite for this region to serve as the physiological trigger for skeletal-type EC coupling [10, 11]
In this paper we have shown by expression in muscle cells that wild-type ␣1S and ␣1S-scr (␣1S with scrambled sequence in residues 681– 690 of the II-III loop) do not differ in density or voltage dependence of calcium currents or in skeletal-type EC coupling, as indicated by evoked contractions in Cd2ϩ/La3ϩ and by voltage dependence of intracellular Ca2ϩ release
Summary
The release of intracellular Ca2؉, as assayed by the indicator dye, Fluo-3, had similar kinetics and voltage dependence for ␣1S and ␣1S-scr These data suggest that residues 681– 690 of the ␣1S II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling. Dysgenic myotubes expressing ␣1S or ␣1S-scr did not differ in calcium current density, voltage dependence of activation, electrically evoked contractions, or voltage dependence of intracellular calcium release These results indicate that neither the specific sequence of these residues, nor the integrity of the cluster of positive charges, is required for skeletal-type EC coupling in muscle cells
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