Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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In our model for NHE activation, we proposed that low intracellular pH promoted the formation of an active tetrameric complex with steady-state activity (1,2,3). At alkaline pH, the subunits may exist as a oligomer, but were functionally uncoupled from each other with little or no steady-state activity. H⁺ activation may lead to tertiary and quaternary conformational changes in the protein that promote the functional coupling of NHE subunits. We also showed that the conversions between "Activated" and "Control" NHE forms are slow processes (Fig.2 and Fig. 3) compared to the rapid equilibration of H⁺ with the H⁺ modifier site. Explanations that call for changes in the NHE phosphorylation state were excluded on the grounds that kinase and phosphatase inhibitors were without effect. An alternative model where H⁺ binding to the modifier site is simultaneously linked to a change in the activity state of the protein seems unlikely based upon the results presented here.

A number of laboratories have reported shifts in NHE molecular weight when the gels were run under non-reducing conditions (7) or after chemical cross-linking (8,9). We found a similar shift to higher molecular weights regardless of the NHE activity state in cross-linking experiments. While "Activated" and "Control" NHE may represent different protein conformational states, at least some critical glutaraldehyde-sensitive reactive sites between subunits remain available. Further experiments are necessary to determine if there is identity of reactive amino acids in the two states. Regardless, these experiments support earlier findings that NHE exists in the membrane as an oligomer. We would now add that NHE exist physically in the membrane as an oligomer under both "Activated" and "Control" conditions, but that functional oligomerization occurs only with H⁺ modifier site activation.

Following the initial description of the H⁺ modifier by Aronson et al. (4), many studies have described shifts in the apparent affinity of the H⁺ modifier site in response to various factors such as serum, hormones, growth factors, and drugs (10,11,12). Until our observation about slow transition rates between active and inactive NHE forms, the simplest explanation for a shift in the pH sensitivity of the modifier site was a change in the H⁺ binding affinity. However, it can be shown that changes in the activation or the inactivation rates, without changing the H⁺ binding affinity, can reproduce the same shift in pH sensitivity. Future experiments will be necessary to distinguish if these mechanisms are involved in regulating the pH set point of the modifier site.

In summary, these results support our earlier description of the NHE as an oligomer that undergoes pH-dependent changes in protein conformation. It seems likely that the protein physically exists in the membrane as an oligomer, but the subunits are functionally uncoupled unless activated by the H⁺ modifier site. Changes in the slow transitions between the "Activated" and the "Control" states may underlie shifts in sensitivity of the H⁺ modifier site in response to physiological, pathological, or pharmacological stimuli.

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