The olfactory system must recognize and discriminate amongst an enormous variety of chemicals in the environment. To contend with such diversity, insects have evolved a family of odorant-gated ion channels comprised of a highly conserved co-receptor (Orco) and a divergent odorant receptor (OR) that confers chemical specificity. Here, we present the single-particle cryo-electron microscopy structure of an Orco homomer from the parasitic fig wasp Apocrypta bakeri at 3.5 Å resolution, providing structural insight into this receptor family. Orco possesses a novel channel architecture, with four subunits symmetrically arranged around a central pore that diverges into four lateral conduits that open to the cytosol. The Orco tetramer has few inter-subunit interactions within the membrane and is bound together by a small cytoplasmic anchor domain. The minimal sequence conservation among ORs maps largely to the pore and anchor domain, shedding light on how the architecture of this receptor family accommodates its remarkable sequence diversity and facilitates the evolution of odour tuning.

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Voltage-sensor domains (VSDs) are specialized transmembrane segments that confer voltage sensitivity to many proteins such as ion channels and enzymes. The activities of these domains are highly dependent on both the chemical and physical properties of the surrounding membrane environment. To learn about VSD-lipid interactions, we used nuclear magnetic resonance (NMR) spectroscopy to determine the structure and phospholipid interface of the VSD from the voltagedependent K + channel KvAP. The solution structure of the KvAP VSD solubilized within phospholipid micelles is similar to a previously determined crystal structure solubilized by a nonionic detergent and complexed with an antibody fragment. Two differences observed include a previously unidentified short amphipathic α-helix that precedes the first transmembrane helix and a subtle rigid body repositioning of the S3-S4 voltage-sensor paddle. Using 15 N relaxation experiments, we show that most of the VSD, including the pronounced kink in S3 and the S3-S4 paddle, is relatively rigid on the ps-ns time scale. In contrast, the kink in S3 is mobile on the μs-ms time scale and may act as a hinge in the movement of the paddle during channel gating. We characterized the VSD-phospholipid micelle interactions using nuclear Overhauser effect spectroscopy and show that the micelle uniformly coats the KvAP VSD and approximates the chemical environment of a phospholipid bilayer. Using paramagnetically labeled phospholipids, we show that bilayer-forming lipids interact with the S3 and S4 helices more strongly than with S1 and S2.

Pulsed Electron-Electron Double Resonance (PELDOR) measurements are presented from the potassium ion channel KcsA both solubilized in detergent and reconstituted in lipids. Site-directed spin labeling using MTSL was performed with a R64C mutant of the protein. The orientations of the spin labels in the tetramer were determined by PELDOR experiments performed at two magnetic field strengths (0.3 T / X-band and 1.2 T / Q-band) and variable probe frequency. Quantitative simulation of the PELDOR data supports a strongly restricted nitroxide, oriented at an angle of 65 degrees relative to the central channel axis. In general, poorer quality PELDOR data was obtained from membrane-reconstituted preparations compared to soluble proteins or detergent-solubilized samples. One reason for this is the reduced transverse spin relaxation time T 2 of nitroxides due to crowding of tetramers within the membrane that occurs even at low protein to lipid ratios. This reduced T 2 can be overcome by reconstituting mixtures of unlabeled and labeled proteins, yielding high-quality PELDOR data. Identical PELDOR oscillation frequencies and their dependencies on the probe frequency were observed in the detergent and membrane-reconstituted preparations indicating that the position and orientation of the spin labels are the same in both environments.