HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: 292/5/C1787    most recent 00267.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Lu, J. Articles by Boron, W. F. Search for Related Content PubMed PubMed Citation Articles by Lu, J. Articles by Boron, W. F.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Reversible and irreversible interactions of DIDS with the human electrogenic Na/HCO₃ cotransporter NBCe1-A: role of lysines in the KKMIK motif of TM5

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut

Submitted 13 May 2006 ; accepted in final form 17 January 2007

Others have shown that H₂DIDS reversibly and covalently binds to the first lysine (K) in the SKLIK motif at the extracellular end of transmembrane segment 5 of the Cl-HCO₃ exchanger AE1. Here we mutated K558, K559, and/or K562 in the homologous KKMIK motif of human NBCe1-A. We expressed constructs in Xenopus oocytes, and used a two-electrode voltage clamp to test the sensitivity of the NBC current (–160 to +20 mV) to DIDS. A 30-s DIDS exposure decreased the current at 0 mV, and a subsequent albumin wash returned the current to the initial value (less any irreversible DIDS inhibition), permitting the determination of a complete dose-response curve on a single oocyte. For all constructs, the reversible DIDS inhibition of the NBC current decreased at more negative voltages. The apparent inhibitory constant for reversible DIDS binding increased in the sequence RRMIR < KKMIK (wt, 40 µM) < NKMIK NKMIN KKMIN < KNMIN KNMIK < NNMIK < NNMIN (400 µM) < DDMID < EEMIE (800 µM). Thus the second K is the most important for reversible DIDS blockade. Nevertheless, these mutations had relatively little effect on slope conductance in the absence of DIDS. For KKMIK, RRMIR, NKMIK, KKMIN, KNMIK, and NNMIN, the rates of irreversible inhibition by DIDS roughly parallel the apparent affinities for reversible DIDS binding. The rate was extremely low for DDMID. The fitted maximal inhibitions were 80–91% for the first five constructs, and 66% for NNMIN. Thus DIDS probably reversibly binds before irreversibly reacting with NBCe1-A. Finally, tenidap blocks not only KKMIK, but also NNMIN and EEMIE.

bicarbonate; pH_i; acid-base