Intracellular cyclic AMP inhibits native and recombinant volume-regulated chloride channels from mammalian heart.
1. ClC-3 encodes a volume-regulated Cl- channel (ICl,vol) in heart. We studied the regulation of native and recombinant cardiac ICl,vol by intracellular cyclic AMP (cAMPi). 2. Symmetrical high Cl- concentrations were used to effectively separate outwardly rectifying ICl,vol from other non-rectifying Cl- currents, such as the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl- currents (ICl,CFTR and ICl,Ca, respectively), which are concomitantly expressed in cardiac myocytes. 3. 8-Bromo-cyclic AMP (8-Br-cAMP) significantly inhibited ICl,vol in most guinea-pig atrial myocytes. In approximately 30 % of the atrial myocytes examined, 8-Br-cAMP increased macroscopic Cl- currents. However, the 8-Br-cAMP-stimulated difference currents exhibited a linear current-voltage (I-V ) relation, consistent with activation of ICl,CFTR, not ICl,vol. 4. In canine atrial myocytes, isoprenaline (1 microM) consistently reduced ICl,vol in Ca2+-free hypotonic bath solutions with strong intracellular Ca2+ (Ca2+i) buffering. In Ca2+-containing hypotonic bath solutions with weak Ca2+i buffering, however, isoprenaline increased net macroscopic Cl- currents. Isoprenaline-stimulated difference currents were not outwardly rectifying, consistent with activation of ICl,Ca, not ICl, vol. 5. In NIH/3T3 cells transfected with gpClC-3 (the gene encoding ICl,vol), 8-Br-cAMP consistently inhibited ICl,ClC-3. These effects were prevented by a protein kinase A (PKA) inhibitor, KT5720, or by mutation of a single consensus protein kinase C (PKC) phosphorylation site (S51A) on the N-terminus of ClC-3, which also mediates PKC inhibition of ICl,ClC-3. 6. We conclude that cAMPi causes inhibition of ICl,vol in mammalian heart due to cross-phosphorylation of the same PKC consensus site on ClC-3 by PKA. Our results suggest that contamination of macroscopic ICl,vol by ICl,CFTR and/or ICl,Ca may account for some of the inconsistent and controversial effects of cAMPi on ICl,vol previously reported in native cardiac myocytes.
Nagasaki
M
M
Department of Physiology and Cell Biology, University of Nevada, School of Medicine, Reno, NV 89557-0046, USA.
Ye
L
L
Duan
D
D
Horowitz
B
B
Hume
J R
JR
D016475:3T3 Cells / Q000378:metabolism; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000502:physiology*; D000818:Animals; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004285:Dogs; D005260:Female; D006168:Guinea Pigs; D006325:Heart Atria; D007425:Intracellular Membranes / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D051379:Mice; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors
D018118:Chloride Channels; C115376:ClC-3 channel; D011994:Recombinant Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000249:Adenosine Monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D007545:Isoproterenol