AJP - Renal Physiology, Vol 245, Issue 6 716-F725, Copyright © 1983 by American Physiological Society
Functional groups of the Na+ channel: role of carboxyl and histidyl groups
C. S. Park and D. D. Fanestil
Two titratable groups, with effect on Na+ transport and with apparent acid
dissociation constants (pKaS) of 4.2 and 6.7, were found in the apical
membrane of toad urinary bladder and are tentatively identified as a
carboxyl and an imidazole. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
(EEDQ), a reagent selective for carboxyl residues, inhibits Na+ transport
in the urinary bladder of toads. The underlying chemical reaction whereby
EEDQ produces inhibition through potential modification of carboxyl
residues was studied. The inhibitory action of EEDQ on Na+ transport was
dependent on pH of reaction media and availability of nucleophile,
indicating that formation of a covalent acyl-nucleophile bond is probably
involved in the irreversible inhibition of Na+ transport. The kinetics of
the inhibition showed a stoichiometry of formation of one acyl-nucleophile
bond per closure of one Na+ transport site, presumably the Na+ channel. The
nucleophile that appears to be involved in the formation of the
acyl-nucleophile bond was tentatively identified as having an apparent pKa
of 6.7. Amiloride and two analogues of amiloride added to the mucosal
Ringer solution (but not serosal amiloride) protected against inhibition of
Na+ transport by EEDQ--a finding consistent with the hypothesis that the
EEDQ-activated carboxyl group undergoes reaction with a nucleophile at or
near the site of specific binding of amiloride onto the apical membrane,
most likely at the Na+ channel. Our findings led us to postulate that
amiloride must interact with at least two sites on the Na+ channel in order
to block the channel. One of the two sites appears to be an ionic
interaction between the anionic carboxyl group at the Na+ channel and the
cationic guanidinium group of amiloride.
