Hammond Energy Shifts Reveal Sequence of Conformational Changes in N- and C-Type Inactivation of Kv1.4

Abstract

C-type inactivation is sensitive to mutation on extra- and intracellular side of the channel, indicating it may involve conformational changes at both extracellular and intracellular mouth of the channel. This movement is critical in determining the allosteric coupling between N-type and C-type inactivation. X-ray crystallography has provided insights into the structure of low-energy stable states in inactivation. In order to investigate the path connecting two stable end states and the sequence of movement over the energy landscape of the critical domains involved in C-type inactivation of Kv1.4 channel, we applied ϕ value analysis to this channel. We chose the V561 at intracellular side of S6. Mutations:[V561A], [V561C], [V561Q], [V561S] and [V561T] were made in N-terminal deleted and N-terminal intact channels. In the N-terminal deleted constructs a ϕ of 0.49 was observed for C-type inactivation. With N-terminal binding, the inactivation process became more complex, but analysis of kinetic components revealed a ϕ value of 0.88 for N-terminal binding, indicating an early interaction with the open pore and a ϕ value of 0.64 was obtained C-type inactivation, indicating facilitation of this step to earlier in the total process. Extracellular function was probed by titration of H508. The slopes of Hammond energy plot for pH titration of H508 from all different mutants are consistent with an average ϕ value of 0.218 (range from 0.20 to 0.23), which indicates the movement of extracellular region near selectivity filter is after the movement of intracellular side of S6. Our results reveal that C-type inactivation can be accelerated by binding of N-terminal to the intracellular mouth of the pore, followed by conformational change at intracellular portion of S6 which is transduced to the extracellular mouth region through an allosteric mechanism.