Mutation of conserved negatively charged residues in the S2 and S3 transmembrane segments of a mammalian K+ channel selectively modulates channel gating.
Voltage-gated channel proteins sense a change in the transmembrane electric field and respond with a conformational change that allows ions to diffuse across the pore-forming structure. Site-specific mutagenesis combined with electrophysiological analysis of expressed mutants in amphibian oocytes has previously established the S4 transmembrane segment as an element of the voltage sensor. Here, we show that mutations of conserved negatively charged residues in S2 and S3 of a brain K+ channel, thought of as countercharges for the positively charged residues in S4, selectively modulate channel gating without modifying the permeation properties. Mutations of Glu235 in S2 that neutralize or reverse charge increase the probability of channel opening and the apparent gating valence. In contrast, replacements of Glu272 by Arg or Thr268 by Asp in S3 decrease the open probability and the apparent gating valence. Residue Glu225 in S2 tolerated replacement only by acidic residues, whereas Asp258 in S3 was intolerant to any attempted change. These results imply that S2 and S3 are unlikely to be involved in channel lining, yet, together with S4, may be additional components of the voltage-sensing structure.
Planells-Cases
R
R
Department of Neuroscience, University of California at San Diego, La Jolla 92093-0366, USA.
Ferrer-Montiel
A V
AV
Patten
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CD
Montal
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D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001224:Aspartic Acid; D001921:Brain / Q000201:enzymology*; D017124:Conserved Sequence; D005260:Female; D018698:Glutamic Acid; D015640:Ion Channel Gating; D007700:Kinetics; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013912:Threonine; D014982:Xenopus laevis
D015221:Potassium Channels; D011994:Recombinant Proteins; D013912:Threonine; D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine