Insight into the modulation of Shaw2 Kv channels by general anesthetics: Structural and functional studies of S4–S5 linker and S6 C-terminal peptides in micelles by NMR

Abstract

The modulation of the Drosophila Shaw2 Kv channel by 1-alkanols and inhaled anesthetics is correlated with the involvement of the S4–S5 linker and C-terminus of S6, and consistent with stabilization of the channel’s closed state. Structural analysis of peptides from S4–S5 (L45) and S6 (S6c), by nuclear magnetic resonance and circular dichroism spectroscopy supports that an α-helical conformation was adopted by L45, while S6c was only in an unstable/dynamic partially folded α-helix in dodecylphosphocholine micelles. Solvent accessibility and paramagnetic probing of L45 revealed that L45 lies parallel to the surface of micelles with charged and polar residues pointing towards the solution while hydrophobic residues are buried inside the micelles. Chemical shift perturbation introduced by 1-butanol on residues Gln320, Thr321, Phe322 and Arg323 of L45, as well as Thr423 and Gln424 of S6c indicates possible anesthetic binding sites on these two important components in the channel activation apparatus. Diffusion measurements confirmed the association of L45, S6c and 1-butanol with micelles which suggests the capability of 1-butanol to influence a possible interaction of L45 and S6c in the micelle environment.

Propofol inhibits prokaryotic voltage-gated Na+ channels by promoting activation-coupled inactivation

“…S8) and given their shared functional modulation by propofol. This hypothesis is also strongly supported by previous work investigating the action of volatile general anesthetics on structurally related ion channels, such as NaChBac, Kv, and TRP channels (Woll et al, 2017a(Woll et al, , 2017bBarber et al, 2012Barber et al, , 2014Zhang et al, 2013;Liang et al, 2015;Kinde et al, 2016;Ton et al, 2017;Bu et al, 2018). Toward linking our findings to mechanisms of general anesthetic action in eukaryotic Navs, this binding pocket near the S4-S5 linker is especially intriguing in light of the recent cryo-EM structure of Nav1.4 from electric eel Electrophorus electricus (EeNav1.4) in complex with the β1 subunit (Yan et al, 2017).…”

Alkanols inhibit voltage-gated K+ channels via a distinct gating modifying mechanism that prevents gate opening

“…6 ). Whereas the binding determinants for 4-AP, including those for guanidine compounds that possibly work in a similar manner 30 , reside within S6 c 31 32 , alkanols are suggested to distort the coupling between the S4-S5 linker and S6 c 33 . The observation that alkanols rescued partly the kinetics of the Shaker -IR-P475A mutant, favors the idea that alkanols alter the conformation of the S4–S5 linker and/or S6 c without disrupting their communication completely.…”

“…Whereas their activating property is ascribed to their ability to shift the voltage dependence of channel opening towards more hyperpolarized potentials and to facilitate the late subunit-concerted transition of channel opening 39 40 , their molecular mechanism to induce channel inhibition is still debated 41 . Whereas alkanols most likely target the S4-S5 linker of K v channels 33 , PUFAs supposedly exert their effect through the VSD 39 40 , although a role for the S4-S5 linker has been suggested 42 .…”

Mechanistic Insights into the Modulation of Voltage-Gated Ion Channels by Inhalational Anesthetics

“…Biophysical Journal 109(10) 2003-2011 fully removes and confers inhibition by n-alcohols, respectively (36,47). Moreover, there is a positive correlation between the apparent anesthetic binding energy and the a-helicity of the S4-S5 linker (37,47,48); and a single mutation in the critical PVPV motif of the K-Shaw2 S6 tail (PVPA) converts the inhibition by n-alcohols or halothane into potentiation (39,49). This potentiation is greatly dependent on the context provided by the S4-S5 linker both in K-Shaw2 and other related chimeric Kv channels (47).…”