Sarcoplasmic reticulum ca2+-pump in pig coronary artery smooth
muscle is regulated by a novel pathway.
Grover, A. K., A. Xu, S. E. Samson, and N. Narayanan.
Department of Biomedical Sciences, McMaster University, Hamilton,
Ontario, Canada L8N3Z5 and Department of Physiology, University of
Western Ontario, London, Ontario N6A 5C1, Canada
APStracts 3:0042C, 1996.
Coronary artery smooth muscle expresses an alternative splice
(SERCA2b) of the sarcoplasmic reticulum (SR) Ca2+-pump gene SERCA2
which is also expressed in cardiac muscle (SERCA2a) but how the
activity of this transporter is regulated in the coronary artery is
not known. SERCA2a in the cardiac muscle can be regulated via
phospholamban or, as recently reported, by a direct phosphorylation
of this protein by calmodulin kinase (Xu,A., Hawkins,C. and
Narayanan,N.(1993) J. Biol. Chem. 268:8394-8397). Since both SERCA2a
and b contain this calmodulin kinase phosphorylation site, we
examined the effect of endogenous calmodulin kinase phosphorylation
of the SR Ca2+-pump in the coronary artery. SR enriched membranes
were isolated from coronary artery smooth muscle and washed in EGTA
to remove bound calmodulin. When these membranes were incubated with
MgATP2- in the presence of Ca2++calmodulin, a 115 kDa protein was
phosphorylated. This phosphorylated 115 kDa protein was identified as
SERCA2b in Western blots and by immunoprecipitation using a SERCA2
selective antibody. Preincubating the membranes in MgATP2- in the
presence of Ca 2++calmodulin stimulated the subsequent Ca2+-uptake in
the presence of oxalate+MgATP2- and azide. The stimulation of the
Ca2+-uptake was inhibited by including the SR Ca 2+ pump inhibitors
thapsigargin and cyclopiazonic acid in the Ca2+-uptake medium or by
including the calmodulin antagonist W-7 or the calmodulin kinase II
peptide fragment 290-309 in the phosphorylation solution. Thus, an
endogenous calmodulin-dependent kinase phosphorylated SERCA2b and
activated it. Phospholamban could not be detected in these membranes
in Western blots. Therefore, the regulation of the SR Ca2+-pump
activity in coronary artery smooth muscle may involve a direct
phosphorylation of the pump protein by an endogenous calmodulin
-dependent kinase.
Received 1 August 1995; accepted in final form 24 January 1996.
APS Manuscript Number C472-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 8 February 96