Comparison of the effects of the membrane-associated Ca2+/calmodulin-dependent protein kinase on Ca(2+)-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum.
In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca(2+)-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca(2+)-ATPase. Studies comparing the effects of PKA and CaM kinase on cardiac Ca(2+)-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca(2+)-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 microM) directly to the Ca2+ transport assay medium results in minimal (approximately 112-130% of control) stimulation of Ca2+ uptake activity when the Ca2+ uptake reaction is initiated by the addition or either ATP or Ca2+/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca2+ transport assay medium results in stimulation of Ca2+ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca2+ uptake markedly (215% of control) when the Ca2+ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca2+/EGTA but minimally (130% of control) when the Ca2+ uptake reaction is initiated by the addition of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Hawkins
C
C
Department of Physiology, University of Western Ontario, London, Canada.
Xu
A
A
Narayanan
N
N
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D066298:In Vitro Techniques; D018657:Muscle Fibers, Slow-Twitch / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000252:Calcium-Transporting ATPases; D002147:Calmodulin; D017868:Cyclic AMP-Dependent Protein Kinases; D000099211:Phospholamban