A Novel Interaction in the Selectivity Filter of Voltage-Gated Potassium Channels Crucial for Slow Inactivation

Voltage-gated potassium (Kv) channels undergo a process termed slow inactivation in response to prolonged depolarizations. This process is thought to occur in response to structural rearrangements near the selectivity filter, which ultimately result in nonconducting (inactivated) channels. Although substantial efforts have been made to understand this process, the details of slow inactivation at the atomic level have yet to be unraveled in eukaryotic Kv channels. Based on information gleaned from crystal structures, the indole nitrogen of a highly conserved Trp, Trp435 in Shaker potassium channels, had been proposed to form a hydrogen bond with Tyr445 in the GYGD selectivity filter signature sequence, thus contributing to open pore stability and likely slow inactivation. Functionally, however, we find that the indole nitrogen of Trp435 does not contribute to slow inactivation as mutations to Phe or Tyr at this site do not affect slow inactivation. In contrast, removal of the hydroxyl group from Tyr445 by Phe substitution results in a rapidly inactivating phenotype, and this could be slowed by application of external TEA, thus suggesting a role for Tyr445 in slow inactivation. A closer examination of the available structural information of side chains in close physical proximity of Tyr445 pointed towards a possible role of Thr439, a highly conserved side chain in the pore helix. Indeed, Thr439Val channels were non-conducting and behaved similar to the permanently inactivated Trp434Phe phenotype. Re-introduction of a hydroxyl moiety in position 439 via the Thr439Ser mutation led to a WT-like conductance-voltage relationship and only a modestly increased inactivation rate compared to WT. Together, these results suggest a novel and energetically significant hydroxyl-hydroxyl interaction between Tyr445 and Thr439 that regulates slow inactivation in Kv channels.