A novel chloride conductance activated by extracellular ATP in mouse parotid acinar cells
Abstract
Salivary gland fluid secretion is a vital physiological process driven by the transepithelial movement of chloride ions (Cl-). This movement fundamentally involves an apical Cl- channel, the precise molecular identity of which has, until now, remained unknown. Extracellular ATP (ATP(o)) has been previously demonstrated to activate a specific Cl- conductance, termed I(ATPCl), in various secretory epithelia. To gain further mechanistic insight into I(ATPCl) within mouse parotid acinar cells, we systematically investigated the effects of ATP(o) using the whole-cell patch-clamp technique, a highly sensitive method for measuring ion currents across cell membranes.
Our experiments revealed that both ATP(o) and 2′- and 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt (Bz-ATP) effectively produced concentration-dependent, time-independent Cl- currents. The half-maximal effective concentrations (EC50) were determined to be 160 micromolar for ATP(o) and a more potent 15 micromolar for Bz-ATP, indicating Bz-ATP as a potent activator. I(ATPCl) exhibited a distinct selectivity sequence for anions: SCN- > I- = NO3- > Cl- > glutamate. This selectivity sequence was notably similar to that observed for other Cl- channels in acinar cells, which are typically activated by Ca2+, cAMP, and cell swelling.
However, in stark contrast to these known Cl- channels, I(ATPCl) proved to be remarkably insensitive to a range of pharmacological agents that are known inhibitors of these latter channels. Furthermore, I(ATPCl) demonstrated independence from intracellular Ca2+ concentrations and was not regulated by changes in cell volume, setting it apart from commonly characterized Cl- channels. Moreover, the magnitude of I(ATPCl) observed in wild-type animals was directly comparable to that measured in mice carrying null mutations in the Cftr, Clcn3, and Clcn2 Cl- channel genes, unequivocally demonstrating that I(ATPCl) is not associated with these previously identified channels.
Taken together, these results strongly demonstrate that I(ATPCl) is distinct from the Cl- channels previously described in acinar cells. The activation of I(ATPCl) by Bz-ATP, a known agonist for P2 nucleotide receptors, suggested that these receptors might be involved. However, further investigation revealed that the inhibition of G-protein activation with GDP-beta-S failed to block I(ATPCl). Additionally, Cibacron Blue 3GA and 4,4′-diisothyocyanostilbene-2,2′-disulphonic disodium salt, known P2X receptor antagonists, selectively inhibited Na+ currents (presumably through P2X receptors) without altering I(ATPCl). These findings suggest that neither P2Y nor P2X receptors are likely to be involved in the activation of I(ATPCl).
We conclude that I(ATPCl) is not associated with any Cl- channels previously characterized in mouse parotid acinar cells, nor is its activation dependent on P2 nucleotide receptor stimulation. BzATP triethylammonium The I(ATPCl) expressed in acinar cells reflects the activation of a novel ATP-gated Cl- channel. This newly identified channel may play a crucial and previously unrecognized physiological role in the intricate process of salivary gland fluid secretion.