dissection-drosophila-melanogaster-indirect-flight-muscles-for

Dissection of <em>Drosophila melanogaster</em> Indirect Flight Muscles for Microscopy Approaches

The indirect flight muscles (IFMs) of Drosophila melanogaster are a powerful genetic model to explore foundational principles of myogenesis. The ...

University of Missouri Kansas City

High-resolution structures of voltage-gated sodium channels (Nav) were first obtained from a prokaryotic ortholog NavAb, which provided important mechanistic insights into Na selectivity and voltage gating. Unlike eukaryotic Navs, the NavAb channel is formed by four identical subunits, but its ion selectivity and pharmacological profiles are very similar to eukaryotic Navs. Recently, the structures of the NavAb voltage sensor at resting and activated states were obtained by cryo-EM, but its intermediate states and transition dynamics remain unclear. In the present work, we used liposome flux assays to show that purified NavAb proteins were functional to conduct both H and Na and were blocked by the local anesthetic lidocaine. Additionally, we examined the real-time conformational dynamics of the NavAb voltage sensor using single-molecule FRET. Our single-molecule FRET measurements on the tandem NavAb channel labeled with Cy3/5 FRET fluorophore pair revealed spontaneous transitions of the NavAb S4 segment among three conformational states, which fitted well with the kinetic model developed for the S4 segment of the human voltage-gated proton channel hHv1. Interestingly, even under strong activating voltage, the NavAb S4 segment seems to adopt a conformational distribution similar to that of the hHv1 S4 segment at a deep resting state. The conformational behaviors of the NavAb voltage sensor under different voltages need to be further examined to understand the mechanisms of voltage sensing and gating in the canonical voltage-gated ion channel superfamily.

Voltage sensor dynamics of a bacterial voltage-gated sodium channel NavAb reveal three conformational states.

Unlike other members of the voltage-gated ion channel superfamily, voltage-gated proton (Hv) channels are solely composed of voltage sensor domains without separate ion-conducting pores. Due to their unique dependence on both voltage and transmembrane pH gradients, Hv channels normally open to mediate proton efflux. Multiple cellular ligands were also found to regulate the function of Hv channels, including Zn, cholesterol, polyunsaturated arachidonic acid, and albumin. Our previous work showed that Zn and cholesterol inhibit the human voltage-gated proton channel (hHv1) by stabilizing its S4 segment at resting state conformations. Released from phospholipids by phospholipase A2 in cells upon infection or injury, arachidonic acid regulates the function of many ion channels, including hHv1. In the present work, we examined the effects of arachidonic acid on purified hHv1 channels using liposome flux assays and revealed underlying structural mechanisms using single-molecule FRET. Our data indicated that arachidonic acid strongly activates hHv1 channels by promoting transitions of the S4 segment toward opening or "preopening" conformations. Moreover, we found that arachidonic acid even activates hHv1 channels inhibited by Zn and cholesterol, providing a biophysical mechanism to activate hHv1 channels in nonexcitable cells upon infection or injury.

Article	Year
Voltage sensor dynamics of a bacterial voltage-gated sodium channel NavAb reveal three conformational states. The Journal of biological chemistry\| PubMed ID: 36736429	2023
Arachidonic acid reverses cholesterol and zinc inhibition of human voltage-gated proton channels. The Journal of biological chemistry\| PubMed ID: 37315791	2023

Jenna DeCata

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