Structural Analysis of S4-S5 Linker Architecture in Depolarization and Hyperpolarization activated K+ Channels

Abstract

MVP, the hyperpolarization-activated potassium channel from the archaeon M. jannaschii, provides a unique opportunity for investigating electromechanical coupling, or how the voltage-sensing domain (VSD) couples to the pore domain, in voltage-gated potassium (Kv) channels. In contrast to depolarization-activated Kv channels, the pore domain of MVP opens when the S4 helix moves inwards at hyperpolarizing potentials, and closes when the S4 helix moves outwards at depolarizing potentials. Because of this inverse relationship between the pore domain and VSD, MVP is in a closed conformation in the absence of a membrane potential whereas depolarization-activated channels, such as KvAP, are in an open conformation. We have taken advantage of this property to compare the local structure and dynamics of the S4-S5 linker of these two channels using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Structural and biochemical studies of depolarization-activated channels suggest that the movement of the S4 is transmitted to the pore domain via the S4-S5 linker. The S4-S5 linker is proposed to rest against the S6 helix as a rigid helical lever to hold the pore closed at hyperpolarizing potentials. However, directed mutagenesis in the S4-S5 linker region in MVP testing properties such as length, helicity, and sequence dependence have suggested that MVP may not be using the S4-S5 linker as a helical lever to couple the VSD and pore domain. using EPR, we present a comparison of the local structure and dynamics of this region in MVP and KvAP from recombinant protein samples reconstituted in liposomes. We expect these results will inform the mechanism for electromechanical coupling in MVP and add to the overall understanding of electromechanical coupling in Kv channels.