Mutations in the pore region of romk enhance ba block.
Zhou, Hao, Sergei Chepilko, Wolfgang Schutt, Han Choe, Lawrence G.
Palmer, and Henry Sackin.
Dept. of Physiology and Biophysics, Cornell University Medical
College, 1300 York Ave., New York, NY 10021
APStracts 3:0223C, 1996.
The sequence of the hydrophobic "P-region" of a K-selective channel
from the kidney (ROMK2) was altered to match that of the closely
related inward rectifier (IRK1) channel by changing two amino acids:
L117 and V121 to: I and T, respectively. The mutant channel expressed
in Xenopus oocytes had an apparent inhibition constant for barium
(Ba+2) block at zero voltage of Ki(0) = 0.07 0.01 mM, more than fifty
times lower than the Ki(0) of the wild-type channel (4.7 1.0 mM). The
increased sensitivity to Ba+2 was accounted for by the point
mutation: V121T. Single-channel measurements indicated that the
increased affinity involved an increase in the on-rate for Ba+2 block
and a decrease in the off-rate. Block by cesium (Cs+) was also
enhanced. The single-channel conductance of the L117I/V121T mutant
was increased by 50%, while the degree of inward rectification, ion
selectivity and apparent affinity for K+ ions were essentially
unchanged. When the neutral asparagine (N) residue within the second
putative membrane-spanning domain of the ROMK channel was substituted
with aspartic acid (D), the corresponding amino acid in IRK1, the
degree of inward rectification was enhanced but Ba+2 block and
single-channel inward conductance were unaffected. Thus, the site of
Ba+2 binding appears to be distinct from the locus of internal Mg+2
block and from at least one of the sites that determine K+
conductivity.
Received 8 March 1996; accepted in final form 26 June 1996.
APS Manuscript Number C132-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 25 July 1996