A single aspartate residue is involved in both intrinsic gating and blockage by Mg2+ of the inward rectifier, IRK1.
1. We describe the effects on channel function of changing an aspartate residue (Asp172) in a membrane-spanning alpha-helix of the murine inward rectifier, IRK1, by site-directed mutagenesis. 2. Alteration of Asp172 to Glu (charged) or to Gln or Asn (polar but uncharged) produced functional channels showing inward rectification, though rectification was weaker with Gln and Asn. 3. Intrinsic gating around the potassium equilibrium potential, EK, was conserved only if the charge on residue 172 was conserved. Currents through channels with Gln or Asn in this position showed no time dependence under hyperpolarization. 4. The change from Asp to Gln also reduced the affinity for internal Mg2+ at least fivefold, indicating that Asp172 also forms part of the site for Mg2+ blockage. 5. The consequences for channel structure of Asp172 lining the pore are discussed.
Stanfield
P R
PR
Department of Cell Physiology and Pharmacology, University of Leicester.
Davies
N W
NW
Shelton
P A
PA
Sutcliffe
M J
MJ
Khan
I A
IA
Brammar
W J
WJ
Conley
E C
EC
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000502:physiology; D017124:Conserved Sequence; D018698:Glutamic Acid; D015640:Ion Channel Gating; D004915:Leukemia, Erythroblastic, Acute; D008274:Magnesium / Q000494:pharmacology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D015221:Potassium Channels / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D017433:Protein Structure, Secondary; D014162:Transfection; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D008274:Magnesium