Invited Symposium: Na-H Exchangers and Intracellular pH Regulation



Materials & Methods


Discussion & Conclusion



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NHE Proton Modifier Site: Activation and Inactivation Are Controlled by Slow Protein Conformational Changes

Contact Person: James L. Kinsella (kinsellj@grc.nia.nih.gov)

Materials and Methods

Brush border membrane vesicles (BBMV) were isolated from the rabbit kidney cortex by differential centrifugation resulting in 10- to 12-fold enrichment of membrane markers (5). A pH sensitive fluorescent dye, pyranine, was trapped inside BBMV for ratiometric determination of intravesicular pH. "Control" BBMV’s were suspended in a buffer containing 10 mM tris/HEPES (pH 7.5) and 150 mM NaCl and remained in this buffer throughout the experimental period. The addition of 30 mM MES acid to the BBMV for 30 minutes at room temperature changed the intravesicular pH to 6, where the H+-modifier site fully activates NHE. The acidified BBMV were cooled on ice and incubated with the original 10 mM tris/HEPES (pH 7.5) buffer for 1 hr. At this point the intravesicular pH returned to 7.5. We designated this condition as "Activated" BBMV.

NHE activity was measured by diluting the Na+-loaded vesicles 100-fold into a solution containing 10 mM tris/HEPES (pH 7.5), 150 mM KCl and 10 m M acridine orange (6). As intravesicular Na+ exchanges with extravesicular H+, the interior of the vesicles acidifies. Acridine orange, a weak base, accumulates inside the vesicles leading to reduced extravesicular acridine orange absorbance. Amiloride (1 mM), a NHE inhibitor, prevented the absorbance change.

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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
© 1998 Author(s) Hold Copyright