Mutational analysis of "transmembrane" histidines in the amiloride
-sensitive na+/h+ exchanger (nhe-1).
Wang, Danher, Daniel F. Balkovetz, and David G. Warnock.
Departments of Medicine, and Physiology and Biophysics, Nephrology Research
and Training Center, Vascular Biology and Hypertension Program, University of
Alabama at Birmingham, and, Department of Veterans Affairs, Birmingham VA
Medical Center, UAB Station, Birmingham, Alabama 35294-0007
APStracts 2:0076C, 1995.
The histidine-reactive reagent, diethyl pyrocarbonate (DEPC) inhibits the
human amiloride-sensitive Na+/H+ exchanger (NHE-1) in stably transfected
fibroblasts. NHE-1 was protected by cimetidine and amiloride from DEPC, and
DEPC inhibition was reversed with hydroxylamine, suggesting a role for
critical histidine groups in NHE activity. We replaced the histidines (H) in
putative transmembrane domains (H35, H120, H349) with glycine (G) using site
-directed mutagenesis. There was no significant change in NHE activity of the
H120G; H349G; H120,349G; and H35,120,349G mutants compared to wild type. The
IC50 values for amiloride, EIPA and cimetidine of the H349G mutant were
significantly increased compared to the wild type NHE-1. We also examined the
DEPC effect on the transport activity of the triple histidine mutant
(H35,120,349G), and found that NHE-1 activity was still inhibited by DEPC
with reversal by hydroxylamine, and protected by amiloride and cimetidine.
Kinetic analysis of DEPC inhibition indicated that two "critical" histidine
residues are required for NHE transport activity.
Received 18 May 1994; accepted in final form 17 January 1995.
APS Manuscript Number C270-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 28 February 1995.