A. Decrouy et al., EXAMINATION OF THE ROLE OF PHOSPHORYLATION AND PHOSPHOLAMBAN IN THE REGULATION OF THE CARDIAC SARCOPLASMIC-RETICULUM CL- CHANNEL, The Journal of membrane biology, 146(3), 1995, pp. 315-326
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.