Excitation-contraction coupling is not affected by scrambled sequence in residues 681-690 of the dihydropyridine receptor II-III loop.
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.
Proenza
C
C
Departments of Physiology and Anatomy and Neurobiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA.
Wilkens
C M
CM
Beam
K G
KG
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D009842:Oligopeptides; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium