Intracellular cyclic AMP inhibits native and recombinant volume-regulated chloride channels from mammalian heart

1. ClC-3 encodes a volume-regulated Cl- channel (I-Cl,I- vol) in heart, me studied the regulation of native and recombinant cardiac I-Cl,I- vol by intracellular cyclic AMP (cAMP(1)). 2. Symmetrical high Cl- concentrations were used to effectively separate outwardly rectifying I-Cl,I- vol from other non-rectifying Cl- currents, such as the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl- currents (I-Cl,I-CFTR and I-Cl,I- Ca, respectively), which are concomitantly expressed in cardiac myocytes. 3. 8-Bromo-cyclic AMP (8-Br-cAMP) significantly inhibited I-Cl,I- vol in most guinea-pig atrial myocytes. In similar to 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 I-Cl,I-CFTR, not I-Cl,I- vol. 4. In canine atrial myocytes, isoprenaline (1 mu M) consistently reduced I-Cl,I- vol in Ca2+-free hypotonic bath solutions with strong intracellular Ca2+ (Ca-1(2+)) buffering. In Ca2+-containing hypotonic bath solutions with weak Ca-1(2+) buffering, however, isoprenaline increased net macroscopic Cl- currents. Isoprenaline-stimulated difference currents were not outwardly rectifying, consistent with activation of I-Cl,I-Ca, not I-Cl,I-vol. 5. In NIH/3T3 cells transfected with gpClC-3 (the gene encoding I-Cl,I- vol), 8-Br-cAMP consistently inhibited I-Cl,I- ClC3. 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 I-Cl,I- ClC-3. 6. We conclude that cAMP(1) causes inhibition of I-Cl,I- 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 I-Cl,I- vol by I-Cl,I- CFTR and/or I-Cl,I- Ca may account for some of the inconsistent and controversial effects of cAMP(1) on I-Cl,I- vol previously reported in native cardiac myocytes.