Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Cellular acidification increases NHE activity through H+ binding to a H+-modifier site. The mechanism for this activation is not known. Nevertheless, pH-dependent changes in NHE protein conformation are likely involved. We used isolated vesicles from renal brush border membranes (NHE3) to measure Na+-dependent H+ fluxes. Previously, we proposed that the pH-dependent NHE activation involved a transition from an inactive monomeric to an active oligomeric (possibly a tetramer) protein. Consistent with these earlier findings, inactive NHE undergoes a slow (t1/2 2 minutes) pH-dependent conformational transition to activate NHE. Active NHE also undergoes a slow pH-dependent conformational change to the inactive state (t1/2 20 minutes). Kinase and phosphatase inhibitors do not affect these rates. In Western blots, glutaraldehyde cross-linking caused shifts in the apparent NHE molecular weight to multiples of the monomer (2, 3, and 4 subunits) regardless of whether the cross-linking was done under activating or inactivating conditions. We conclude that NHE likely exists in the membrane as an oligomer, and that intracellular acidification leads to slow conformational changes that functionally couple and activate those subunits. Variations in activation and inactivation rates will change the apparent H+-modifier site affinity and will alter intracellular pH under both normal and pathological conditions.

Back to the top.
Presentation Number SAkinsella0254
Keywords: NHE, Oligomers, pH, Exchanger, Conformation