Sites and Functional Consequence of Alkylphenol Anesthetic Binding to Kv1.2 Channels

Abstract

Inhalational general anesthetics, such as sevoflurane and isoflurane, modulate a subset of brain Kv1 potassium channels. However, the Kv1.2 channel is resistant to propofol, a commonly used intravenous alkylphenol anesthetic. We hypothesize that propofol binds to a presumed pocket involving the channel's S4-S5 linker, but functional transduction is poor and, therefore, propofol efficacy is low. To test this hypothesis, we used a photoactive propofol analog (meta-aziPropofol = AziPm) to directly probe binding and electrophysiological and mutational analyses in Xenopus oocytes to probe function. We find that AziPm photolabels L321 in the S4-S5 linker of both the wild-type Kv1.2 and a mutant Kv1.2 (G329 T) with a novel gating phenotype. Furthermore, whereas propofol does not significantly modulate Kv1.2 WT but robustly potentiates Kv1.2 G329T, AziPm inhibits Kv1.2 WT and also potentiates Kv1.2 G329T. Kv1.2 modulation by AziPm was abolished by two mutations that decreased hydrophobicity at L321 (L321A and L321F), confirming the specific significance of the S4-S5 linker in the mechanism of general anesthetic modulation. Since AziPm binds to Kv1.2 G329T and shares the propofol ability to potentiate this mutant, the parent propofol likely also binds to the Kv1.2 channel. However, binding and alkylphenol-induced transduction are seemingly sensitive to the conformation of the S4-S5 linker site (altered by G329T) and subtle differences in the chemical structures of propofol and AziPm. Overall, the results are consistent with a mechanism of general anesthetic modulation that depends on the complementarity of necessary ligand binding and permissive ion channel conformations that dictate modulation and efficacy.

Binding of General Anesthetics to Ion Channels

“…S3). It is worth mentioning that these findings recapitulate recent photolabeling experiments demonstrating that photoactive analogs of sevoflurane do interact at the S4S5 linker and at the S6Phelix interface of the open conductive Kv1.2 channel (Bu et al, 2017;Woll et al, 2017). In detail, Leu317 and Thr384 were found to be protected from photoactive analogs, with the former being more protected though.…”

“…Among all other aspects that might impact channelanesthetic interactions in general, we are specifically interested in determining if sevoflurane binds the wellcharacterized openconductive (O) and restingclosed (C) structures of Kv1.2 (Long et al, 2005;Stock et al, 2013) in a conformationdependent manner to impact protein equilibrium. Very recently, we went through an innovative structurebased study (Stock et al, 2017) of concentrationdependent binding of small ligands to multiple saturable sites in proteins to show that sevoflurane binds the openpore structure of Kv1.2 at the S4S5 linker and the S6Phelix interface a result largely supported by independent photolabeling experiments (Bu et al, 2017;Woll et al, 2017). Here, we aim at extending these previous calculations to investigate sevoflurane interactions with the entire TMdomain of the channel and more importantly, to resolve any conformational dependence for its binding process to channel structures.…”

5-Aminolevulinic Acid Dehydratase Gene Dosage Affects Programmed Cell Death and Immunity

“…After denaturing in urea and gradient dialysis, refolded GhLMMD protein was obtained and then used for enzyme activity assay, SDS-PAGE analysis, and western blotting. HRPconjugated anti-His antibody (Proteintech) was used in western blotting as described (Bu et al, 2017).…”

Sites Contributing to TRPA1 Activation by the Anesthetic Propofol Identified by Photoaffinity Labeling

“…2 D). S6 and S5 form the channel gate within 6TM-channels; therefore it is not surprising that ligand binding near S6 influences the dynamics and activity of 6TM-channels (20,21). Interestingly, these residues do not share identity within the propofol-insensitive Drosophila TRPA1 (9,12), again suggesting that these residues may contribute to functionally relevant propofol-binding site(s) (Fig.…”