A Random Mutagenesis Approach to Probing Electromechanical Coupling in the Hyperpolarization-Activated Channel MVP

Abstract

MVP, the hyperpolarization-activated potassium channel from the archaeon M. jannaschii, poses an interesting case study for understanding how the voltage-sensing domain (VSD) couples to the pore domain in Kv channels. Although the VSD of MVP adopts the same orientation in the membrane and senses changes in the polarity of the membrane potential in the same manner as canonical Kv channels, the pore of MVP opens in response to hyperpolarization; thus, like the eukaryotic hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, MVP opens when the S4 helix moves inwards and closes when the S4 moves outwards. Because hyperpolarization-activated channels use the same VSD for inverse gating, they offer a unique opportunity for understanding the molecular basis of coupling the VSD and pore domain. Since structural evidence in canonical Kv channels suggests that the movement of the S4 is transmitted to the pore domain via the S4-S5 linker, we have set out to identify key residues of MVP necessary for electromechanical coupling. We probed the S4-S5 linker and S6 helix with a random mutagenesis screen using the potassium uptake deficient strain LB2003 in conjunction with a MVP library consisting of single amino acid substitutions along the linker with full codon coverage. By screening this library under different potassium concentrations, we have identified mutations in the linker and S6 that abolish WT complementation on potassium-depleted media (Loss of Function). This approach has allowed high-throughput screening of all possible amino acid identities along the linker that are incompatible with channel function. Here we present preliminary assessments of the expression profile, stability and functional properties of these mutants.