EFFECTS OF COMPETITIVE SODIUM-LIKE ANTAGONISTS ON NA,K-ATPASE SUGGESTTHAT CATION OCCLUSION FROM THE CYTOPLASMIC SURFACE OCCURS IN 2 STEPS

Information on cation occlusion sites of renal NA,K-ATPase has been ob tained by comparing the ability of competitive Na-like antagonists (Da vid, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S. J. D. (1992 ) J. Biol. Chem. 267, 1141-1149) with that of transported alkali metal cations to protect against covalent modification and structural pertu rbations of the protein. Sodium antagonists include p- or m-xylylenebi sguanidium, guanidinium ions, and ethylenediamine. Experiments with pr oteoliposomes reconstituted with Na,K-ATPase demonstrate that p-xylyle nebisguanidium has pronounced selectivity for the cytoplasmic surface. Tryptic digestion of Na,K-ATPase leading to ''19-kDa membranes,'' a s pecifically truncated enzyme with intact cation occlusion sites, requi res the presence of alkali metal cations. Sodium antagonists do not pr otect 19-kDa membranes against further digestion, and occlusion is des troyed. Incubation of 19-kDa membranes at 37-degrees-C, in the absence of occluded ions, leads rapidly to loss of ability to occlude rubidiu m ions. Rubidium, sodium, or other alkali metal cations protect fully, whereas sodium antagonists do not protect against this thermal inacti vation. Like the alkali metal cations, sodium antagonists protect Na,K -ATPase and, somewhat less effectively, 19-kDa membranes against inact ivation by the carboxyl reagent N,N'-dicyclohexylcarbodiimide. Cation occlusion from the cytoplasmic surface is suggested to occur in two st eps. In an initial recognition, either transported cations or sodium a ntagonists interact with carboxyl groups. The second step is selective for transported cations and involves occlusion of cations (either pot assium or sodium ions) and a conformational change to a compact struct ure, which is resistant to proteolysis and thermal inactivation. Sodiu m antagonists are sterically hindered from becoming occluded and block Na,K-ATPase activity. Implications for the structural basis of cation specificity of the Na/K pump are discussed.