CIS-ALLOSTERIC EFFECTS OF CYTOPLASMIC NA+/K+ DISCRIMINATION AT VARYING PH - LOW-AFFINITY MULTISITE INHIBITION OF CYTOPLASMIC K+ IN RECONSTITUTED NA+/K+-ATPASE ENGAGED IN UNCOUPLED NA+-EFFLUX

In liposomes with reconstituted shark Na+/K+-ATPase the effect of cytoplasmic K+ was investigated in the absence of extracellular alkali ions. During such conditions the Na+/K+-ATPase is engaged in the so called uncoupled Na+ efflux mode in which cytoplasmic Na+ activates and binds to the enzyme and becomes translocated without countertransport of K+ as in the physiological Na+/K+ exchange mode. In this uncoupled flux mode only low-affinity inhibition by K(cyt)+ is found to be present, The inhibition pattern is consistent with a model in which cytoplasmic K+ exhibit mixed inhibition of Na+ activation, probably by binding at the three cytoplasmic loading sites on E1ATP (E1A). With determined intrinsic binding constants for cytoplasmic Na+ to this form of K(S1), K(S2), K(S3) = 40 mM, 2 mM, 2 mM the inhibition pattern can be simulated assuming three K(cyt)+ sites with equal affinity for K(i) = 40 mM, similar to K(S1) for the first Na(cyt)+ site. The discrimination between cytoplasmic Na+ and K+ is therefore enhanced by allosteric interaction initiated from the cis-side due to binding of the first Na+, as opposed to K+, which induces the positive cooperativity in the successive Na+ bindings. pH is found to influence the pattern of K(cyt)+ inhibition: A lowering of the pH potentiates the K(cyt)+ inhibition, whereas at increased pH the inhibition is decreased and transformed into a pure competitive competition.