The effects of inhibitors of protein tyrosine kinases (PTKs) on the Cl(-) current (I(Cl(vol))) through volume-regulated anion/chloride (VRAC) channels whilst manipulating cellular ATP have been studied in mouse fibroblasts using the whole-cell patch clamp technique. Removal of ATP from the pipette-filling solution prevented activation of the current during osmotic cell swelling and when the volume of patched cells was increased by the application of positive pressure through the patch pipette to achieve rates exceeding 100%/min. Equimolar substitution of ATP in the pipette solution with its non-hydrolyzable analogs, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) or adenylyl-(beta,gamma-methylene)-diphosphonate (AMP-PCP), not only supported activation of the current but also maintained its amplitude. The PTK inhibitors, tyrphostins A25, B46, 3-amino-2,4-dicyano-5-(4-hydroxyphenyl)penta-2,4-dienonitrile++ + and genistein (all at 100 microM), inhibited I(Cl(vol)) in a time-dependent manner. Tyrphostin A1, which does not inhibit PTK activity, did not affect the current amplitude. The PTK inhibitors also inhibited I(Cl(vol)) under conditions where ATP in the pipette was substituted with ATPgammaS or AMP-PCP. We conclude that in mouse fibroblasts ATP has a dual role in the regulation of the current: it is required for protein phosphorylation to keep VRAC channels operational and, through non-hydrolytic binding, determines the magnitude of I(Cl(vol)). We also suggest that tyrosine-specific protein kinases and phosphatases exhibit an interdependent involvement in the regulation of VRAC channels.