Invited Symposium: Na-H Exchangers and Intracellular pH Regulation
In a series of papers examining NHE (Na+-H+ exchanger) activity in the pre-steady state, we measured the first turnover of the exchanger (i.e., the initial binding and translocation of Na+) in isolated renal brush border membranes and made several unique and important discoveries (1,2,3). We proposed that the molecular mechanism for NHE activation by the H+ modifier site, first described by Aronson et al. (4), involves the formation of a tetramer with one Na+ binding site per subunit. During the first turnover, there was evidence of interacting Na+ sites. Upon completion of the initial transport cycle, the four Na+ sites in the tetramer are one quarter out-of-phase with each other (flip-flop mechanism), such that in each successive cycle only one Na+ ion binds to the complex and one is released.
At alkaline pHi, where the H+ modifier site is unoccupied, only 50% of the sites estimated from the acid pHi experiments are available during the initial turnover, and these sites do not interact with each other. Continuous (steady state) NHE turnover is inhibited by alkaline pHi conditions. To account for these results, we proposed that the NHE completes a single cycle of Na+ transport as an "uncoupled oligomer" in which the Na+ sites on the subunits behave largely independent of one another.
The difference between the alkaline and the acid states likely relates to the protein conformational changes controlling the functional coupling of subunits. Significant changes in the tertiary and quaternary structure must occur in order to allow H+ binding to the H+ transport sites and recruit subunits into an functionally active tetrameric complex. Although H+ binding is assumed to be fast (microsecond time scale), there is no published evidence on the stoichiometry and apparent binding affinity of the H+ modifier site, the number or nature of subsequent reactions involved in forming the fully active complex, or their kinetics. This communication represents an initial examination of mechanisms involved in pH-dependent activation of NHE.
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|Kinsella, J; Froehlich, J; (1998). NHE Proton Modifier Site: Activation and Inactivation Are Controlled by Slow Protein Conformational Changes. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/fliegel/kinsella0254/index.html|
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