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 counterchanges for the positively charged residues in S4, selectively modulate channel gating without modifying the permeation properties, Mutations of Glu(235) in S2 that neutralize or reverse charge increase the probability of channel opening and the apparent gating valence. In contrast, replacements of Glu(272) by Arg or Thr(268) by Asp in S3 decrease the open probability and the apparent gating valence. Residue Glu(225) in S2 tolerated replacement only by acidic residues, whereas Asp(258) 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.