Biochemical regulation of sarcoplasmic reticulum Cl- channel from human atrial myocytes: involvement of phospholamban.
Sarcoplasmic reticulum (SR) membrane vesicles derived from human atrium were characterized by specific ryanodine binding assay and fused into planar lipid bilayers. The tritiated form of the alkaloid bound to its receptor with a K(D) of 2.2 nM and a Bmax of 268 fmol/mg protein respectively. Special emphasis was placed on an anion-selective channel present in the SR membrane, which exhibited a mean conductance value of 67 pS when recorded in asymmetrical 50 mM trans/250 mM cis CsCl buffer system and a sensitivity to SITS (1 to 100 microM). Single and multiple channel activities displayed low voltage sensitivity and variability in its gating behavior which might result in spontaneous channel inactivation. 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- channel in ventricular cells. The inactivation process is probably not directly related to a phosphorylation/dephosphorylation mechanism since PKA and PKG in presence of an adequate phosphorylation cocktail failed to reactivate the SR Cl- channel. In contrast, the use of a monoclonal 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.
Decrouy
A
A
Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Quebec, Canada.
Juteau
M
M
Proteau
S
S
Teijiera
J
J
Rousseau
E
E
D000200:Action Potentials; D019138:Antibodies, Blocking / Q000276:immunology; D015153:Blotting, Western; D015220:Calcium Channels / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism*; D006325:Heart Atria / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D006801:Humans; D009124:Muscle Proteins / Q000502:physiology; D011485:Protein Binding; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013997:Time Factors; D000099211:Phospholamban
D019138:Antibodies, Blocking; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D018118:Chloride Channels; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D000099211:Phospholamban