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 chan nels. A 92 +/- 5 pS anion-selective channel was recorded in asymmetric 50 mM trans/250 mM cis CsCl buffer system. Reversal potentials and th eoretical equilibrium potentials for Cl(-)ions obtained under various experimental conditions allowed us to confirm the Cl- selectivity of t his SR channel. The majority (69%) of channel recordings (n = 45) disp layed steady-state kinetics and a slight voltage dependency of the ope n probability. However, 31% of the channels inactivated after their in corporation. We now report that the channel might be reactivated by de polarizing voltage steps. Furthermore, the use of either PKA or PKG in association with adequate phosphorylating buffers lengthens the deact ivation process at the end of the voltage pulses, but does not prevent the inactivation. It was assumed that the change in gating mode was d ue to a voltage-sensitive association/dissociation mechanism with a ph osphorylated protein of the SR membrane such as phospholamban (PL). We demonstrated that a specific monoclonal antibody raised against canin e PL inhibited the activity of the channel and prevented its reactivat ion 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 m ay 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+ upt ake by the SR.