Phosphorylation of dihydropyridine receptor II-III loop peptide regulates skeletal muscle calcium release channel function. Evidence for an essential role of the beta-OH group of Ser687.
In vertebrate skeletal muscle, excitation-contraction coupling may occur by a mechanical coupling mechanism involving protein-protein interactions between the dihydropyridine receptor (DHPR) of the transverse tubule membrane and the ryanodine receptor (RYR)/Ca2+ release channel of the sarcoplasmic reticulum membrane. We have previously shown that the cytoplasmic II-III loop peptides of the skeletal and cardiac muscle DHPR alpha 1 subunits (SDCL and CDCL, respectively) activate the skeletal muscle RYR. We now report that cyclic AMP-dependent protein kinase-mediated phosphorylation of Ser687 of SDCL yields a peptide that fails to activate the RYR, as determined in [3H]ryanodine binding and single channel measurements. The phosphorylated SDCL bound to the skeletal muscle but not cardiac muscle RYR, and the binding could be displaced by the unphosphorylated SDCL. A mutant SDCL with a Ser687-->Ala substitution failed to activate the RYR, but was still able to bind. Similarly, a Ser813-->Ala substitution in CDCL yielded a peptide that failed to activate the skeletal RYR. Use of three smaller overlapping peptides within the SDCL region identified an amino acid region from 666 to 726 including Ser687, which bound to and activated the skeletal muscle RYR. These results suggest that cyclic AMP-dependent protein kinase-mediated phosphorylation of the DHPR alpha 1 subunit may play a role in the functional interaction of the DHPR and RYR in skeletal muscle.
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Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260, USA.
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Meissner
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004285:Dogs; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D009124:Muscle Proteins; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium