Molecular Determinants of Kv2.1 Channel U-Type Inactivation

Abstract

Kv2.1 channels exhibit a U-shaped voltage-dependent inactivation that is thought to represent preferential inactivation from pre-open closed states. However, the molecular mechanism underlying Kv2.1 closed-state inactivation is not known. Here, we have performed a cysteine scan of the S3-S4 linker and S5-P-loop linker and discovered critical sites important in U-type inactivation of Kv2.1 channels. U-type inactivation was preserved in all S5-P-loop linker mutant channels, with the exception of E352C. The E352C mutation abolished U-type inactivation, whilst the E352Q mutation had no effect. Experiments with E352C treated with the reducing agent, DTT, re-established U-type inactivation as did the double mutant E352C/C241V. This suggests that a disulfide bond formed between residues 352C and C241 (in S2) prevents closed-state inactivation. The S3-S4 mutant, R289C, also reduced closed-state inactivation. In this case, application of MTSET restored WT-like U-type inactivation properties, suggesting importance of charge at this site. Kinetic modeling based on a previous scheme (Klemic et al. 1998 Biophys J 74;1779-89) suggests that both E352C and R289C mutant phenotypes resulted from a specific reduction in transitions into closed inactivated states. These data indicate that specific residues within the S3-S4 and S5-P-loop linkers play essential roles in closed-state inactivation of Kv2.1 channels.