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

Masaaki Nagasaki, Lingyu Ye, Dayue Duan, Burton Horowitz and Joseph R. Hume

1. ClC-3 encodes a volume-regulated Cl^- channel (I_Cl,vol) in heart. We studied the regulation of native and recombinant cardiac I_Cl,vol by intracellular cyclic AMP (cAMP_i).

2. Symmetrical high Cl^- concentrations were used to effectively separate outwardly rectifying I_Cl,vol from other non-rectifying Cl^- currents, such as the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca²⁺-activated Cl^- currents (I_Cl,CFTR and I_Cl,Ca, respectively), which are concomitantly expressed in cardiac myocytes.

3. 8-Bromo-cyclic AMP (8-Br-cAMP) significantly inhibited I_Cl,vol in most guinea-pig atrial myocytes. In ~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,CFTR, not I_Cl,vol.

4. In canine atrial myocytes, isoprenaline (1 µM) consistently reduced I_Cl,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 I_Cl,Ca, not I_Cl,vol.

5. In NIH/3T3 cells transfected with gpClC-3 (the gene encoding I_Cl,vol), 8-Br-cAMP consistently inhibited I_Cl,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 I_Cl,ClC-3.

6. We conclude that cAMP_i causes inhibition of I_Cl,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,vol by I_Cl,CFTR and/or I_Cl,Ca may account for some of the inconsistent and controversial effects of cAMP_i on I_Cl,vol previously reported in native cardiac myocytes.

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