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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Activation of Na⁺/H⁺ and K⁺/H⁺ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity

Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, California

Submitted 19 March 2008 ; accepted in final form 12 September 2008

Alteration in cell volume of vertebrates results in activation of volume-sensitive ion flux pathways. Fine control of the activity of these pathways enables cells to regulate volume following osmotic perturbation. Protein phosphorylation and dephosphorylation have been reported to play a crucial role in the control of volume-sensitive ion flux pathways. Exposing Amphiuma tridactylu red blood cells (RBCs) to phorbol esters in isotonic medium results in a simultaneous, dose-dependent activation of both Na⁺/H⁺ and K⁺/H⁺ exchangers. We tested the hypothesis that in Amphiuma RBCs, both shrinkage-induced Na⁺/H⁺ exchange and swelling-induced K⁺/H⁺ exchange are activated by phosphorylation-dependent reactions. To this end, we assessed the effect of calyculin A, a phosphatase inhibitor, on the activity of the aforementioned exchangers. We found that exposure of Amphiuma RBCs to calyculin-A in isotonic media results in simultaneous, 1–2 orders of magnitude increase in the activity of both K⁺/H⁺ and Na⁺/H⁺ exchangers. We also demonstrate that, in isotonic media, calyculin A-dependent increases in net Na⁺ uptake and K⁺ loss are a direct result of phosphatase inhibition and are not dependent on changes in cell volume. Whereas calyculin A exposure in the absence of volume changes results in stimulation of both the Na⁺/H⁺ and K⁺/H⁺ exchangers, superimposing cell swelling or shrinkage and calyculin A treatment results in selective activation of K⁺/H⁺ or Na⁺/H⁺ exchange, respectively. We conclude that kinase-dependent reactions are responsible for Na⁺/H⁺ and K⁺/H⁺ exchange activity, whereas undefined volume-dependent reactions confer specificity and coordinated control.

cell volume regulation; volume-dependent coordination of K loss and Na uptake; phosphoprotein; phosphatase inhibitors; phosphorylation