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.

Action potentials (AP) are basic functional units of electrical signaling in excitable cells. These electrical signals are involved in many biological processes, including muscle contraction, synaptic transmission and hormone release. In general, the plasma membrane is polarized, displaying a difference in electric potential (membrane potential) with a negative intracellular voltage with respect to the extracellular space. During an AP, the membrane potential is momentarily canceled (depolarized) or reverted (anti-polarized) by a inwardly-rectifying current typically mediated by sodium-selective voltage-gated channels (VGC); the membrane potential is returned back (repolarize) to its initial voltage (resting potential) by a outwardlyrectifying current mediated by potassium-selective VGC. The temporal and electrical characteristics of APs depend on which VGCs are present in the membrane. Understanding the role of VGCs in AP generation in their native cells constitutes a difficult task, commonly riddled with the use of pharmacological agents to isolate each specific conductance. Here, we have developed a model to study cellular excitability using Xenopus oocytes. Spontaneous and evoked APs were readily recorded from oocytes expressing Nav1.4, Drosophila Kv1.1 (Shaker), human Kv7.2 and Kv7.3. These APs were around 5-ms long. However, in the absence of Shaker, the AP lasted about 50 ms. These observations indicated that we were able to modify the temporal characteristic of APs by removing the fastactivating Shaker. To further validate this model, we used the Kv7.2-7.3 agonist diclofenac seeking to decrease excitability. The addition of diclofenac drove the resting potential to more negative voltages and raised the threshold for excitation, effectively decreasing excitability. These results constitute proof of concept showing that this type of models can be used as functional scaffolds for the evaluation of pharmacological agents and the assessment of the effect of mutations in VGCs on the generation of bioelectrical signals.