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 ele ctric field and respond with a conformational change that allows ions to diffuse across the pore-forming structure, Site-specific mutagenesi s combined with electrophysiological analysis of expressed mutants in amphibian oocytes has previously established the S4 transmembrane segm ent 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+ ch annel, thought of as counterchanges for the positively charged residue s in S4, selectively modulate channel gating without modifying the per meation properties, Mutations of Glu(235) in S2 that neutralize or rev erse charge increase the probability of channel opening and the appare nt gating valence. In contrast, replacements of Glu(272) by Arg or Thr (268) by Asp in S3 decrease the open probability and the apparent gati ng valence. Residue Glu(225) in S2 tolerated replacement only by acidi c residues, whereas Asp(258) in S3 was intolerant to any attempted cha nge, These results imply that S2 and S3 are unlikely to be involved in channel lining, yet, together with S4, may be additional components o f the voltage-sensing structure.