Mutations in the pore region of ROMK enhance Ba2+ block.
The sequence of the hydrophobic "P" (pore) 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, leucine (L) 117 and valine (V) 121, to isoleucine (I) and threonine (T), respectively. The mutant channel expressed in Xenopus laevis oocytes had an apparent inhibition constant at zero voltage [Ki(0)] in the presence of Ba2+ of 0.07 +/- 0.01 mM, which was more than 50 times lower than the Ki(0) of the wild-type channel (4.7 +/- 1.0 mM). The increased sensitivity to Ba2+ was accounted for by the point mutation V121T. Single-channel measurements indicated that the increased affinity involved an increase in the on-rate for Ba2+ block and a decrease in the off-rate. Block by Ca+ was also enhanced. The single-channel conductance of the L1171/ V121T mutant was increased by 50%, whereas the degree of inward rectification, ion selectivity, and apparent affinity for K+ were essentially unchanged. When the neutral asparagine residue within the second putative membrane-spanning domain of the ROMK channel was substituted with aspartic acid, the corresponding amino acid in IRK1, the degree of inward rectification was enhanced but Ba2+ block and single-channel inward conductance were unaffected. Thus the site of Ba2+ binding appears to be distinct from the locus of internal Mg2+ block and from at least one of the sites that determines K+ conjuctivity.
Zhou
H
H
Department of Physiology and Biophysics, Cornell University Medical College, New York, New York 10021, USA.
Chepilko
S
S
Schütt
W
W
Choe
H
H
Palmer
L G
LG
Sackin
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000378:metabolism*; D015870:Gene Expression; D017136:Ion Transport / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D014981:Xenopus
D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D001464:Barium