Transient receptor potential vanilloid (TRPV) cation channels are polymodal sensors involved in a variety of physiological processes. TRPV2, a member of the TRPV family, is regulated by temperature, by ligands, such as probenecid and cannabinoids, and by lipids. TRPV2 has been implicated in many biological functions, including somatosensation, osmosensation and innate immunity. Here we present the atomic model of rabbit TRPV2 in its putative desensitized state, as determined by cryo-EM at a nominal resolution of ~4 Å. In the TRPV2 structure, the transmembrane segment 6 (S6), which is involved in gate opening, adopts a conformation different from the one observed in TRPV1. Structural comparisons of TRPV1 and TRPV2 indicate that a rotation of the ankyrin-repeat domain is coupled to pore opening via the TRP domain, and this pore opening can be modulated by rearrangements in the secondary structure of S6.

Transient receptor potential melastatin (TRPM) cation channels are poly-modal sensors that are involved in a variety of physiological processes. Within the TRPM family, member 8 (TRPM8) is the primary cold- and menthol-sensor in humans. We determined the cryo-electron microscopy structure of the full-length TRPM8 from the collared flycatcher at an overall resolution of ~4.1 Å. Our TRPM8 structure reveals a three-layered architecture. The amino-terminal domain with a fold distinct among known TRP structures, together with the carboxyl-terminal region, forms a large two-layered cytosolic ring that extensively interacts with the transmembrane channel layer. The structure suggests that the menthol binding site is located within the voltage-sensor-like domain and thus provides a structural glimpse of the design principle of the molecular transducer for cold and menthol sensation.

Voltage-dependent ion channels gate open in response to changes in cell membrane voltage. This form of gating permits the propagation of action potentials. We present two structures of the voltage-dependent K ؉ channel KvAP, in complex with monoclonal Fv fragments (3.9 Å) and without antibody fragments (8 Å). We also studied KvAP with disulfide cross-bridges in lipid membranes. Analyzing these data in the context of the crystal structure of Kv1.2 and EPR data on KvAP we reach the following conclusions: (i) KvAP is similar in structure to Kv1.2 with a very modest difference in the orientation of its voltage sensor; (ii) mAb fragments are not the source of non-native conformations of KvAP in crystal structures; (iii) because KvAP contains separate loosely adherent domains, a lipid membrane is required to maintain their correct relative orientations, and (iv) the model of KvAP is consistent with the proposal of voltage sensing through the movement of an argininecontaining helix-turn-helix element at the protein-lipid interface.membrane protein ͉ protein-lipid interface ͉ voltage-gated ion channel ͉ voltage sensor V oltage-dependent ion channels ''sense'' voltage differences across the cell membrane and open or close in response to its value (1). These channels contain a centrally located pore surrounded by four voltage sensors. Voltage-dependent K ϩ (Kv) channels are tetramers with four identical subunits, each with six transmembrane segments (S1-S6): S5 and S6 form the central pore at the interface between the subunits, and S1-S4 form the voltage sensors (2, 3). The voltage sensors have four to seven positively charged amino acids (usually arginine) on S4, known as gating charges, and fewer negatively charged amino acids (aspartate or glutamate) distributed on S1, S2, and S3. The voltage sensors undergo a conformational change when the pore gates open, coupling movement of the S4 gating charges within the membrane electric field to channel open probability.The first structure of a Kv channel, termed KvAP, from the archeabacterium Aeropyrum Pernix (4), was determined by crystallizing the channel as a complex with a monoclonal Fab fragment attached to its voltage sensors [Protein Data Bank (PDB) ID code 1ORQ] (5). In that structure the voltage sensors are in a non-native conformation, displaced toward the intracellular side of the transmembrane pore. Together with a second structure of the voltage sensor alone, termed the isolated voltage sensor (PDB ID code 1ORS), the following ideas about voltagedependent gating were proposed (5, 6): that the voltage sensor is a very mobile structure, presumably because it must carry charged amino acids through the membrane electric field; that the charge bearing S4 forms a helix-turn-helix with the Cterminal half of S3 (S3b), and that the helix-turn-helix moves at the protein-lipid interface a large distance. Experiments using avidin capture of biotin linked to the voltage sensor indicated that four of the S4 arginine amino acids translate 15-20 Å across the membrane. These struc...

Venomous animals produce small protein toxins that inhibit ion channels with high affinity. In several well-studied cases the inhibitory proteins are water-soluble and bind at a channel's aqueous-exposed extracellular surface. Here we show that a voltage-sensor toxin (VSTX1) from the Chilean Rose Tarantula (Grammostola spatulata) reaches its target by partitioning into the lipid membrane. Lipid membrane partitioning serves two purposes: to localize the toxin in the membrane where the voltage sensor resides and to exploit the free energy of partitioning to achieve apparent high-affinity inhibition. VSTX1, small hydrophobic poisons and anaesthetic molecules reveal a common theme of voltage sensor inhibition through lipid membrane access. The apparent requirement for such access is consistent with the recent proposal that the sensor in voltage-dependent K+ channels is located at the membrane-protein interface.

In voltage-gated Na ؉ , K ؉ , and Ca 2؉ channels, four voltage-sensor domains operate on a central pore domain in response to membrane voltage. In contrast, the voltage-gated proton channel (Hv) contains only a voltage-sensor domain, lacking a separate pore domain. The subunit stoichiometry and organization of Hv has been unknown. Here, we show that human Hv1 forms a dimer in the membrane and define regions that are close to the dimer interface by using cysteine cross-linking. Two dimeric interfaces appear to exist in Hv1, one mediated by S1 and the adjacent extracellular loop, and the other mediated by a putative intracellular coiled-coil domain. It may be significant that Hv1 uses for its dimer interface a surface that corresponds to the interface between the voltage sensor and pore in Kv channels.membrane protein ͉ voltage-dependent ion channel ͉ voltage sensor V oltage-gated six-transmembrane cation channels (Na ϩ , K ϩ , and Ca 2ϩ ) contain voltage-sensor and pore domains (1). In this class of ion channels voltage-sensor and pore domains carry out voltage sensing and cation conduction, respectively. Four voltage-sensor domains surround a single, centrally located ion conduction pathway. Each voltage sensor is attached to the pore in a specific manner so that conformational changes within the voltage sensors are transmitted to the pore's gate (2). It was originally thought that voltage-sensor domains existed only in the context of voltage-gated cation channels. However, the cloning of voltage-gated proton (Hv) channels and voltagesensor phosphatase enzymes revealed that voltage-sensor domains also exist in other contexts, apparently as independent (of a cation pore) membrane proteins (3-5), corroborating the studies of MacKinnon and coworkers (2, 6, 7) and Lu and coworkers (8) that supported the idea that voltage sensors, even in the context of voltage-dependent cation channels, are rather loosely attached to the central pore.The long-sought molecular identity of Hv1 (9) showed that it contains only a voltage-sensor domain without a separate pore domain in the membrane (3, 4). This observation suggested that in contrast to canonical voltage-dependent cation channels, the voltage-sensor domain of Hv1 is responsible for both H ϩ conduction and voltage sensing. In this study we evaluate the subunit stoichiometry of the Hv1 channel in cell membranes. ResultsHv1 Is a Dimer in the Membrane. To probe the oligomeric state of Hv1, cell membranes isolated from tsA201 cells (HEK293 derivatives) transfected with human Hv1 cDNAs were subjected to the amino-group specific bifunctional cross-linker disuccinimidyl suberate (DSS) and visualized by Western blot analysis using antibodies directed against the Hv1 channel. Amino groupspecific cross-linkers have been successful in defining the oligomeric status of several membrane proteins (10-12). Recombinant Hv1 makes functional channels in HEK293 cells (3, 4). In Fig. 1a, human Hv1 migrates at Ϸ35 kDa in SDS/PAGE under reducing conditions, which is consistent with the molecula...

MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg2+ within the active site, and provide a structural basis of catalysis for this class of enzyme.

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Summary Voltage-gated Na+ (NaV) channels initiate neuronal action potentials. NaV channels are composed of a transmembrane domain responsible for voltage-dependent Na+ conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins, including fibroblast growth factor homologous factors (FHFs) and calmodulin (CaM). Most ion channel structural studies have focused on mechanisms of permeation and voltage-dependent gating but less is known about how intracellular domains modulate channel function. Here we report the crystal structure of the ternary complex of a human NaV CTD, an FHF, and Ca2+-free CaM at 2.2 Å. Combined with functional experiments based on structural insights, we present a platform for understanding the roles of these auxiliary proteins in NaV channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity of individual NaV CTD isoforms for distinctive FHFs.