Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action.

Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K+ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating

A structurally precise mechanism links an epilepsy-associated KCNC2 potassium channel mutation to interneuron dysfunction

De novo heterozygous variants in KCNC2 encoding the voltage-gated potassium (K+) channel subunit Kv3.2 are a recently described cause of developmental and epileptic encephalopathy (DEE). A de novo variant in KCNC2 c.374G > A (p.Cys125Tyr) was identified via exome sequencing in a patient with DEE. Relative to wild-type Kv3.2, Kv3.2-p.Cys125Tyr induces K+ currents exhibiting a large hyperpolarizing shift in the voltage dependence of activation, accelerated activation, and delayed deactivation consistent with a relative stabilization of the open conformation, along with increased current density. Leveraging the cryogenic electron microscopy (cryo-EM) structure of Kv3.1, molecular dynamic simulations suggest that a strong π-π stacking interaction between the variant Tyr125 and Tyr156 in the α-6 helix of the T1 domain promotes a relative stabilization of the open conformation of the channel, which underlies the observed gain of function. A multicompartment computational model of a Kv3-expressing parvalbumin-positive cerebral cortex fast-spiking γ-aminobutyric acidergic (GABAergic) interneuron (PV-IN) demonstrates how the Kv3.2-Cys125Tyr variant impairs neuronal excitability and dysregulates inhibition in cerebral cortex circuits to explain the resulting epilepsy

Analysis of <i>G</i>-<i>V</i> relations from Kv1.2, ΔT1-Kv1.2, K-Shaw2, K-Shaw2 T330G and ΔT1-K-Shaw2.

<p>(A) Best-fit Boltzmann parameters (<i>V</i>_1/2, <i>z</i> and <i>G</i>_max) from individual paired measurements before (Ctr) and after exposure to 1 mM sevoflurane (Sevo). Each pair of symbols connected by a <i>solid</i> line represents an individual paired experiment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#sec002" target="_blank">Materials and Methods</a>). The <i>G</i>_max graphs depict raw values before normalization (in mS). The <i>P</i> value resulting from a paired Student-<i>t</i> test is shown above each graph, and the <i>red</i> marks indicate the mean values of the sample. (B)–(E) are as described for panel A. The number oocytes examined for each Kv channel was 6, 6, 4, 6 and 6, respectively.</p

Kv1.2 G329T recapitulates the magnified positive modulation of Kv1.2 FRAKT by sevoflurane.

<p>(A) Effects of 1 mM sevoflurane on mutant whole-oocyte Kv1.2 currents evoked by a voltage step to +60 mV from a holding voltage of -100 mV. <i>Black</i>, <i>red</i> and <i>grey</i> current traces correspond to control, anesthetic-exposed, and washout, respectively. The scale bars indicate 50 ms and 1 μA. (B) Concentration-response relations of various general anesthetics acting on wild type and mutant Kv1.2 currents. <i>Solid</i> lines are the best fits assuming the double Hill equation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#sec002" target="_blank">Materials and Methods</a>). N = 4–8 oocytes for each dose. Best-fit parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.t001" target="_blank">Table 1</a>.</p

The T330G mutation eliminates the voltage-dependent potentiation of the K-Shaw2 conductance by sevoflurane.

<p>(A) Families of whole-oocyte K-Shaw2 (N = 4) and K-Shaw2 T330G (N = 6) currents in the absence (<i>left</i>) and presence of 1 mM sevoflurane (<i>right</i>). Currents were evoked by step depolarizations from a holding voltage of -100 mV. The steps were delivered in increments of 10 mV from -90 to +100 mV. The scale bars indicate 100 ms and 1 μA. (B) <i>G</i>-<i>V</i> relations of K-Shaw2 (<i>black</i>) and K-Shaw2 T330G (<i>red</i>) in the absence (<i>open</i>) and presence of 1 mM sevoflurane (<i>filled</i>). <i>Solid</i> lines are the best-fits to the Boltzmann equation. Best-fit parameters are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.g002" target="_blank">Fig 2</a> and Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.s001" target="_blank">S1 File</a>. (C) The voltage dependence of the conductance ratio (<i>G</i>_Sevo/<i>G</i>₀) K-Shaw2 (<i>black</i>) and K-Shaw2 T330G (<i>red</i>).</p

Positive modulation of the Kv1.2 conductance by sevoflurane.

<p>(A) Families of whole-oocyte Kv1.2 currents before (<i>left</i>) and after exposure to 1 mM sevoflurane (<i>center</i>). Currents were evoked by step depolarizations from a holding voltage of -100 mV. The steps were delivered in increments of 10 mV from -50 to +50 mV. The overlay (<i>right</i>) directly compares selected currents in the absence (<i>black</i>) and presence (<i>red</i>) of sevoflurane (currents evoked by steps to the indicated voltages). (B) Normalized <i>G</i>-<i>V</i> relations of Kv1.2 in the absence (<i>black</i>) and presence of 1 mM sevoflurane (<i>red</i>) (N = 6). The solid lines are the best-fit Boltzmann functions. <i>G</i>_max is the control maximum conductance before exposure to sevoflurane (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#sec002" target="_blank">Materials and Methods</a>). The mean best-fit parameters are summarized in Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143363#pone.0143363.s001" target="_blank">S1 File</a>. (C) The voltage dependence of the Kv1.2 conductance ratio (<i>G</i>_Sevo/<i>G</i>₀). This ratio was calculated from paired measurements of the <i>G</i>-<i>V</i> relations before (<i>G</i>₀) and after exposure to sevolfurane (<i>G</i>_Sevo).</p