Abstract
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.
Highlights
We have previously shown that the cytoplasmic II-III loop peptides of the skeletal and cardiac muscle dihydropyridine receptor (DHPR) at subunits (SDCL and CDCL, respectively) activate the skeletal muscle ryanodine receptor (RYR)
We 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 sarcoplasmic reticulum (SR) Ca2 + release channels are thought to be linked to another Ca2 + channel (L-type), known as the dihydropyridine receptor (DHPR), which is located in infoldings of the surface membrane, the transverse (T-) tubule [3]
Summary
E-C, excitation-contraction; RYR, ryanodine receptor; DHPR, dihydropyridine receptor; SR, sarcoplasmic reticulum; PKA, cyclicAMP-dependent protein kinase; SDCL, the putative cytoplasmic loop between transmembrane repeat II and III of the skeletal muscle DHPR al subunit; CDCL,the cardiac muscle isoform of SDCL; SDCLS687A, site-directed mutant SDCL with SerB87 -> Ala substitution; CDCLS813A, site-directed mutant CDCL with Ser l 3 -> Ala substitution; CHAPS, 3-[(3-cholamidopropyl)dimethylammonioJ-lpropanesulfonic acid; BSA, bovine serum albumin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4piperazinediethanesulfonic acid. In vertebrate skeletal E-C coupling, the Ca2 + release channels are thought to be regulated by a skeletal muscle DHPR isoform through protein-protein interactions [5, 6]. In addition to the DHPR, several endogenous effector molecules were shown to regulate the RYR These include small diffusible molecules such as Ca2 + , Mg2 + , and ATP, and proteins such as calmodulin, FK506-binding protein, and triadin [2], An involvement of triadin in mediating the functional interaction between the skeletal muscle DHPR and RYR has been suggested [8]. We report that phosphorylation of Ser687 of SDCL by PKA in vitro resulted in the formation of a peptide that failed to activate the RYR This result provides novel evidence for a role of DHPR phosphorylation in regulating DHPR protein-RYR protein interactions in skeletal muscle.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
