A. Decrouy et al., BIOCHEMICAL REGULATION OF SARCOPLASMIC-RETICULUM CL- CHANNEL FROM HUMAN ATRIAL MYOCYTES - INVOLVEMENT OF PHOSPHOLAMBAN, Journal of Molecular and Cellular Cardiology, 28(4), 1996, pp. 767-780
Sarcoplasmic reticulum (SR) membrane vesicles derived from human atriu
m were characterized by specific ryanodine binding assay and fused int
o planar lipid bilayers. The tritiated form of the alkaloid bound to i
ts receptor with a K-D of 2.2 nM and a B-max of 268 fmol/mg protein re
spectively. Special emphasis was placed on an anion-selective channel
present in the SR membrane, which exhibited a mean conductance value o
f 67 pS when recorded in asymmetrical 50 mM trans/250 mM cis CsCl buff
er system and a sensitivity to SITS (1 to 100 mu M). Single and multip
le channel activities displayed low voltage sensitivity and variabilit
y in its gating behavior which might result in spontaneous channel ina
ctivation, However, the majority of the recordings (60%) resulted in a
steady-state high open probability, The inactivated channel could be
transiently reactivated with depolarizing voltage steps. This behavior
is very similar, if not identical, to that observed for the SR Cl- ch
annel in ventricular cells, The inactivation process is probably not d
irectly related to a phosphorylation/dephosphorylation mechanism since
PKA and PKG in presence of an adequate phosphorylation cocktail faile
d to reactivate the SR Cl- channel, In contrast, the use of a monoclon
al anti-phospholamban antibody allowed the inhibition of the activity
of the anionic channels. These results suggest that the regulation of
the human atrial SR Cl- channel is dependent upon an interaction with
phospholamban, which was clearly identified in our atrial preparations
by Western blot analysis using monoclonal antibody. (C) 1996 Academic
Press Limited