Examination of the role of phosphorylation and phospholamban in the regulation of the cardiac sarcoplasmic reticulum Cl- channel.
Sarcoplasmic reticulum (SR) vesicles were prepared from either canine or sheep heart and fused into lipid bilayers to study their ionic channels. A 92 +/- 5 pS anion-selective channel was recorded in asymmetric 50 mM trans/250 mM cis CsCl buffer system. Reversal potentials and theoretical equilibrium potentials for Cl-ions obtained under various experimental conditions allowed us to confirm the Cl- selectivity of this SR channel. The majority (69%) of channel recordings (n = 45) displayed steady-state kinetics and a slight voltage dependency of the open probability. However, 31% of the channels inactivated after their incorporation. We now report that the channel might be reactivated by depolarizing voltage steps. Furthermore, the use of either PKA or PKG in association with adequate phosphorylating buffers lengthens the deactivation process at the end of the voltage pulses, but does not prevent the inactivation. It was assumed that the change in gating mode was due to a voltage-sensitive association/dissociation mechanism with a phosphorylated protein of the SR membrane such as phospholamban (PL). We demonstrated that a specific monoclonal antibody raised against canine PL inhibited the activity of the channel and prevented its reactivation by depolarizing steps. 400 to 800 ng/ml of Anti-PL Ab consistently and sequentially turned off the channel activities. In contrast, heat inactivated Anti-PL Ab had no effect. We propose that phospholamban may be a primer of the SR Cl- channel whereby Cl- anions would play the role of counter-charge carrier during rapid Ca2+ release and Ca2+ uptake by the SR.
Decrouy
A
A
Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Quebec, Canada.
Juteau
M
M
Rousseau
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000502:physiology*; D018118:Chloride Channels / Q000187:drug effects* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D004285:Dogs; D015640:Ion Channel Gating / Q000187:drug effects*; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000187:drug effects; D009124:Muscle Proteins / Q000187:drug effects* / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D012756:Sheep; D000099211:Phospholamban
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D018118:Chloride Channels; D002712:Chlorides; D008051:Lipid Bilayers; D009124:Muscle Proteins; D000099211:Phospholamban