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Volume-sensitive K-Cl cotransport occurs in red blood cells of many species. In intact cells, activation of K-Cl cotransport by swelling requires dephosphorylation of some cell protein, but maximal activity requires the presence of intracellular ATP. We have examined the relation between K-Cl cotransport activity and ATP in ghosts prepared from human red blood cells. K-Cl cotransport activity in swollen ghosts increased by ATP, and the increase requires Mg so that it almost certainly results from the phosphorylation of some membrane component. However, even in ATP-free ghosts residual volume-sensitive K-Cl cotransport can be demonstrated. This residual cotransport in ATP-free ghosts is greater in the presence of vanadate, a tyrosyl phosphatase inhibitor, and in ghosts that contain ATP cotransport is reduced by genistein, a tyrosyl kinase inhibitor. Okadaic acid, an inhibitor of serine and threonine phosphatases, inhibits K-Cl cotransport in ghosts as it does in intact cells. Experiments in which ghosts were preexposed to okadaic acid showed that the protein dephosphorylation that permits K-Cl cotransport can proceed to completion before the ghosts are swollen and K transport measured and therefore dephosphorylation is not a response to ghost swelling. In experiments with ATP-free ghosts we found that phosphorylation is not necessary to increase the cotransport rate when shrunken ghosts are swollen, nor is rephosphorylation necessary to decrease the cotransport rate when swollen ghosts are shrunken. Cotransport is greater in swollen than in shrunken ghosts even when the swollen and shrunken ghosts have the same concentration of cytoplasmic solutes. We conclude that, although phosphorylation and dephosphorylation modify the activity of the cotransporter in swollen and in shrunken ghosts, neither of these processes nor any other known messenger is involved in signal transduction between the cell volume sensor and the cotransporter as originally proposed by Jennings and Al-Rohil (Jennings, M. L., and N. Al-Rohil. 1990. Journal of General Physiology. 95: 1021-1040).

the journal of general physiology/the journal of general physiology

The role of ATP in swelling-stimulated K-Cl cotransport in human red cell ghosts. Phosphorylation-dephosphorylation events are not in the signal transduction pathway.