BIOCHEMICAL REGULATION OF SARCOPLASMIC-RETICULUM CL- CHANNEL FROM HUMAN ATRIAL MYOCYTES - INVOLVEMENT OF PHOSPHOLAMBAN

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