Dual role of ATP in supporting volume-regulated chloride channels in mousefibroblasts

The effects of inhibitors of protein tyrosine kinases (PTKs) on the Cl- cur rent (I-Cl(vol)) through volume-regulated anion/chloride (VRAC) channels wh ilst manipulating cellular ATP have been studied in mouse fibroblasts using the whole-cell patch clamp technique. Removal of ATP from the pipette-fill ing solution prevented activation of the current during osmotic cell swelli ng 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-h ydrolyzable analogs, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) or a denylyl-(beta,gamma-methylene)-diphosphonate (AMP-PCP), not only supported activation of the current but also maintained its amplitude. The PTK inhibi tors, tyrphostins A25, B46, 3-amino-2,4-dicyano-5-(4-hydroxyphenyl)penta-2, 4-dienonitrile and genistein (all at 100 mu M), inhibited I-Cl(vol) in a ti me-dependent manner, Tyrphostin Al, which does not inhibit PTK activity, di d not affect the current amplitude. The PTK inhibitors also inhibited I-Cl( vol) under conditions where ATP in the pipette was substituted with ATP gam ma S 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 phosphorylatio n to keep VRAC channels operational and, through non-hydrolytic binding, de termines 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. (C) 2000 Elsevier Science B.V. All rights reserved.