Ser165 in the second transmembrane region of the Kir2.1 channel determines its susceptibility to blockade by intracellular Mg2+.
The strong inward rectification of Kir2.1 currents is reportedly due to blockade of the outward current by cytoplasmic magnesium (Mg(2+)(i)) and polyamines, and is known to be determined in part by three negatively charged amino acid residues: Asp172, Glu224, and Glu299 (D172, E224, E299). Our aim was to identify additional sites contributing to the inward rectification of Kir2.1 currents. To accomplish this, we introduced into wild-type Kir2.1 and its D172N and D172N & E224G & E299S mutants various point mutations selected on the basis of a comparison of the sequences of Kir2.1 and the weak rectifier sWIRK. By analyzing macroscopic currents recorded from Xenopus oocytes using two-electrode voltage clamp, we determined that S165L mutation decreases inward rectification, especially with the triple mutant. The susceptibility to blockade by intracellular blockers was examined using HEK293 transfectants and the inside-out patch clamp configuration. The sensitivity to spermine was significantly diminished in the D172N and triple mutant, but not the S165L mutant. Both the S165L and D172N mutants were less susceptible to blockade by Mg(2+)(i) than the wild-type channel, and the susceptibility was still lower in the D172N & S165L double mutant. These results suggest that S165 is situated deeper into the pore from inside than D172, where it is accessible to Mg(2+)(i) but not to spermine. The single channel conductance of the D172N mutant was similar to that of the wild-type Kir2.1, whereas the conductance of the S165L mutant was significantly lower. Permeation by extracellular Rb+ (Rb(+)(o)) was dramatically increased by S165L mutation, but was increased only slightly by D172N mutation. By contrast, the Rb+/K+ permeability ratio was increased equally by D172N and S165L mutation. We therefore propose that S165 forms the narrowest part of the Kir2.1 pore, where both extracellular and intracellular blockers plug the permeation pathway.
Fujiwara
Yuichiro
Y
Department of Physiology and Cell Biology, Tokyo Medical and Dental University Graduate School and Faculty of Medicine, Tokyo 113-8519, Japan.
Kubo
Yoshihiro
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002412:Cations / Q000378:metabolism / Q000494:pharmacology; D015640:Ion Channel Gating / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D016297:Mutagenesis, Site-Directed / Q000502:physiology*; D009865:Oocytes; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000502:physiology; D012413:Rubidium / Q000378:metabolism / Q000493:pharmacokinetics; D012694:Serine / Q000737:chemistry* / Q000235:genetics; D013096:Spermine / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014981:Xenopus
D002412:Cations; D024661:Potassium Channels, Inwardly Rectifying; D013096:Spermine; D012694:Serine; D008274:Magnesium; D012413:Rubidium