MUTATIONAL ANALYSIS OF ION CONDUCTION AND DRUG-BINDING SITES IN THE INNER MOUTH OF VOLTAGE-GATED K+ CHANNELS

Pore properties that distinguish two cloned, voltage-gated K+ channels , Kv2.1 and Kv3.1, include single-channel conductance, block by extern al and internal tetraethylammonium, and block by LF-aminopyridine, To define the inner mouth of voltage-gated K+ channels, segmental exchang es and point mutations of nonconserved residues were used. Transplanti ng the cytoplasmic half of either transmembrane segments S5 or S6 from Kv3.1 into Kv2.1 reduced sensitivity to block by internal tetraethyla mmonium, increased sensitivity to 4-aminopyridine, and reduced single- channel conductance. In S6, changes in single-channel conductance and internal tetraethylammonium sensitivity were associated with point mut ations V400T and L403 M, respectively. Although individual residues in both S5 and S6 were found to affect 4-aminopyridine blockade, the mos t effective change was L327F in S5. Thus, both S5 and S6 contribute to the inner mouth of the pore but different residues regulate ion condu ction and blockade by internal tetraethylammonium and 4-aminopyridine.