Molecular Determinants of Slow Inactivation in Voltage-Gated Potassium Channels

Potassium channels respond to prolonged depolarizations with structural rearrangements that result in inactivated, nonconducting channels through a process termed slow inactivation. Significant efforts to understand this process have been made using structural, theoretical and experimental approaches yet the molecular details of slow inactivation in eukaryotic voltage-gated potassium (Kv) channels remain poorly understood. Data gleaned from prokaryotic (KcsA) and eukaryotic (Kv1.2/2.1) channels have implicated two adjacent and highly conserved aromatic side chains near the selectivity filter as critical determinants for slow inactivation. In particular, the indole nitrogens of both side chains, Trp434 and Trp435 in Shaker potassium channels, have been proposed to contribute to a hydrogen bond network that modulates slow inactivation, yet the direct demonstration of this notion is missing in Kv channels. Here we incorporate unnatural derivatives of Trp to directly demonstrate that the indole nitrogen of Trp434 is a crucial component of the slow inactivation process through two complementary substitutions. By subtly increasing the acidity of the indole nitrogen through fluorination (and therefore strengthening its hydrogen bond donor ability), the rate of slow inactivation was substantially decreased (4-fold slower than WT). Conversely, the novel unnatural amino acid side chain Ind, which lacks the indole nitrogen but is otherwise isosteric to Trp, increased the rate of inactivation more than 10-fold when Ind-containing channels were co-expressed with WT channels, as Ind-containing channels alone did not produce ionic current. In contrast to Trp434, the indole nitrogen of Trp435 does not contribute to slow inactivation as even relatively nonconservative mutations to Phe or Tyr at this site do not affect slow inactivation. Taken together, these results directly demonstrate the functional importance of the H-bonding ability of Trp434 in open pore stability in Kv channels.