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Exposure of Amphiuma red blood cells to millimolar concentrations of N-ethylmaleimide (NEM) resulted in net K loss. In order to determine whether net K loss was conductive or was by electroneutral K/H exchange or KCl cotransport, studies were performed evaluating K flux in terms of the thermodynamic forces to which K flux by the above pathways should couple. The direction and magnitude of the NEM-induced net K flux did not correspond with the direction and magnitude of the forces relevant to K conductance or electroneutral KCl cotransport. Both the magnitude and direction of the NEM-activated K flux responded to the driving force for K/H exchange. We therefore conclude that NEM-induced K loss, like that by osmotically swollen Amphiuma red blood cells, is by an electroneutral K/H exchanger. In addition to the above studies, we evaluated the kinetic behavior of the volume- and NEM-induced K/H exchange flux pathways in media where Cl was replaced by SCN, NO3, para-aminohippurate (PAH), or gluconate. The anion replacement studies did not permit a distinction between K/H exchange and KCl cotransport, since, depending upon the anion used as a Cl replacement, partial inhibition or stimulation of volume-activated K/H exchange fluxes was observed. In contrast, all anions used were stimulatory to the NEM-induced K loss. Since, on the basis of force-flow analysis, both volume-and NEM-induced K loss are K/H exchange, it was necessary to reevaluate assumptions (i.e., anions serve as substrates and therefore probe the translocation step) associated with the use of anion replacement as a means of flux route identification. When viewed together with the force-flow studies, the Cl replacement studies suggest that anion effects upon K/H exchange are indirect. The different anions appear to alter mechanisms that couple NEM exposure and cell swelling to the activation of K/H exchange, as opposed to exerting direct effects upon K and H translocation.

Activation of electroneutral K flux in Amphiuma red blood cells by N-ethylmaleimide. Distinction between K/H exchange and KCl cotransport.