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

A volume-regulated chloride current (ICl,vol) is widely expressed in cardiac and many other tissues of a variety of species (Sorota, 1992; Hagiwara et al. 1992; Tseng, 1992; Vandenberg et al. 1994; Oz & Sorota, 1995; Hall et al. 1995; Duan et al. 1995; Strange et al. 1996; Vandenberg et al. 1996; Okada, 1997; Duan et al. 1997a). Several unique properties of ICl,vol distinguish these channels from other types of Cl− channels: outward rectification with symmetrical high Cl− concentrations, time-dependent inactivation at positive potentials, higher permeability to I− than Cl−, and an intermediate single-channel conductance of 28–60 pS (Strange et al. 1996; Vandenberg et al. 1996; Okada, 1997). Recent evidence suggests that ClC-3, a member of the ClC Cl− channel family, may be responsible for native ICl,vol in many mammalian cells (Kawasaki et al. 1994; Duan et al. 1997b, 1999; Yamazaki et al. 1998; von Weikersthal et al. 1999). In heart, ICl,vol plays an important role in the regulation of cell volume homeostasis and electrical activity (Vandenberg et al. 1996; Hiraoka et al. 1998; Hume et al. 2000). ICl,vol function is known to be regulated by phosphorylation and dephosphorylation (Hall et al. 1995; Okada, 1997) and we recently described the role of protein kinase C (PKC) phosphorylation in the regulation of native ICl,vol in heart and expressed ClC-3 channels by changes in cell volume (Duan et al. 1999). The present study was designed to examine the regulation of native ICl,vol and expressed ClC-3 channels by intracellular cyclic AMP (cAMPi). In many cardiac cells, elevation of cAMPi is known to activate a distinct class of Cl− channels through protein kinase A (PKA) phosphorylation (ICl,PKA) (Harvey & Hume, 1989; Bahinski et al. 1989; Gadsby et al. 1995; Hume et al. 2000). In contrast to ICl,vol, ICl,PKA exhibits a linear current-voltage relation in symmetrical high Cl−, and is generated by small (8-12 pS) unitary conductance channels encoded by the CFTR gene (Gadsby et al. 1995; Hart et al. 1996). Whether or not cardiac ICl,vol can also be regulated by cAMPi and cAMP-dependent PKA phosphorylation is controversial (Sorota, 1992; Hagiwara et al. 1992; Tseng, 1992; Oz & Sorota, 1995; Hall et al. 1995; Du & Sorota, 1997). In canine ventricular myocytes, ICl,vol has been reported to be insensitive to the PKA inhibitor H-89 (Tseng, 1992), and the stretch-induced Cl− current in rabbit heart has also been reported to be insensitive to a specific peptide inhibitor of PKA (Hagiwara et al. 1992). In contrast, in cultured chick cardiac myocytes, ICl,vol was inhibited by forskolin, and activation of ICl,vol during cell swelling was prevented when the pipette filling solution contained cAMP and isobutylmethylxanthine in the presence of okadaic acid (Hall et al. 1995). An increase in cAMPi levels by isoprenaline or forskolin was reported to stimulate ICl,vol in canine atrial myocytes (Sorota, 1992) and in human atrial and ventricular myocytes (Oz & Sorota, 1995), although these effects may be complex, with some cells responding with stimulation, inhibition, no response, or a biphasic stimulation and inhibition (Du & Sorota, 1997). Finally, in guinea-pig atrial and ventricular myocytes, no consistent effects attributable to cAMPi on ICl,vol were observed (Vandenberg et al. 1994). At this time, no clear explanation is available to account for these reported disparate effects of cAMPi on ICl,vol. It is possible, however, that some of these inconsistencies might arise due to the combined complex effects of cAMPi and PKA on different types of Cl− channels (in addition to ICl,vol) which may be concomitantly expressed in the same myocyte. For example, many cardiac myocytes that express ICl,vol also express CFTR Cl− currents (ICl,CFTR) and/or Ca2+-activated Cl− currents (ICl,Ca) (Harvey, 1996; Hiraoka et al. 1998; Hume et al. 2000), making it difficult to distinguish and interpret the effects of cAMP stimulation on channel subtypes. This problem is accentuated when membrane currents are only studied using asymmetric Cl− gradients, since ICl,vol, ICl,CFTR and ICl,Ca all exhibit indistinguishable outwardly rectifying macroscopic current-voltage (I–V) relations under these conditions (Harvey, 1996; Hume et al. 2000). In the present study, we used symmetrical high Cl− concentrations to effectively separate the outwardly rectifying ICl,vol from other possible contaminating Cl− currents like ICl,CFTR and ICl,Ca (which have linear I–V relations in symmetrical high Cl−) and re-examined the cAMPi regulation of native ICl,vol in guinea-pig and canine cardiac myocytes. We also examined cAMPi regulation of the cloned ICl,vol in gpClC-3-transfected NIH/3T3 cells. Under these conditions, we find that elevation of cAMPi consistently causes inhibition, not stimulation, of outwardly rectifying ICl,vol exhibited by both native and recombinant channels. Finally, experiments employing site-directed mutagenesis of the ClC-3 gene product, identify one primary phosphorylation site involved in the inhibitory modulation of ICl,vol by both PKC and PKA phosphorylation.