RESUMEN
Ligand binding usually moves the target protein from an ensemble of inactive states to a well-defined active conformation. Matthies et al. flip this scheme around, finding that, for the magnesium channel CorA, loss of ligand binding induces an ensemble of conformations that turn the channel on.
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Proteínas Bacterianas/ultraestructura , Proteínas de Transporte de Catión/ultraestructura , Magnesio/metabolismo , Thermotoga maritima/químicaRESUMEN
Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel.
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Proteínas Bacterianas/química , Gammaproteobacteria/metabolismo , Canales de Sodio Activados por Voltaje/química , Secuencia de Aminoácidos , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Desplegamiento Proteico , Alineación de SecuenciaRESUMEN
Voltage-gated ion channels (VGICs) comprise multiple structural units, the assembly of which is required for function1,2. Structural understanding of how VGIC subunits assemble and whether chaperone proteins are required is lacking. High-voltage-activated calcium channels (CaVs)3,4 are paradigmatic multisubunit VGICs whose function and trafficking are powerfully shaped by interactions between pore-forming CaV1 or CaV2 CaVα1 (ref. 3), and the auxiliary CaVß5 and CaVα2δ subunits6,7. Here we present cryo-electron microscopy structures of human brain and cardiac CaV1.2 bound with CaVß3 to a chaperone-the endoplasmic reticulum membrane protein complex (EMC)8,9-and of the assembled CaV1.2-CaVß3-CaVα2δ-1 channel. These structures provide a view of an EMC-client complex and define EMC sites-the transmembrane (TM) and cytoplasmic (Cyto) docks; interaction between these sites and the client channel causes partial extraction of a pore subunit and splays open the CaVα2δ-interaction site. The structures identify the CaVα2δ-binding site for gabapentinoid anti-pain and anti-anxiety drugs6, show that EMC and CaVα2δ interactions with the channel are mutually exclusive, and indicate that EMC-to-CaVα2δ hand-off involves a divalent ion-dependent step and CaV1.2 element ordering. Disruption of the EMC-CaV complex compromises CaV function, suggesting that the EMC functions as a channel holdase that facilitates channel assembly. Together, the structures reveal a CaV assembly intermediate and EMC client-binding sites that could have wide-ranging implications for the biogenesis of VGICs and other membrane proteins.
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Canales de Calcio Tipo L , Retículo Endoplásmico , Proteínas de la Membrana , Humanos , Sitios de Unión , Encéfalo , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/ultraestructura , Microscopía por Crioelectrón , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Gabapentina/farmacología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Miocardio/químicaRESUMEN
American bullfrog (Rana castesbeiana) saxiphilin (RcSxph) is a high-affinity "toxin sponge" protein thought to prevent intoxication by saxitoxin (STX), a lethal bis-guanidinium neurotoxin that causes paralytic shellfish poisoning (PSP) by blocking voltage-gated sodium channels (NaVs). How specific RcSxph interactions contribute to STX binding has not been defined and whether other organisms have similar proteins is unclear. Here, we use mutagenesis, ligand binding, and structural studies to define the energetic basis of Sxph:STX recognition. The resultant STX "recognition code" enabled engineering of RcSxph to improve its ability to rescue NaVs from STX and facilitated discovery of 10 new frog and toad Sxphs. Definition of the STX binding code and Sxph family expansion among diverse anurans separated by â¼140 My of evolution provides a molecular basis for understanding the roles of toxin sponge proteins in toxin resistance and for developing novel proteins to sense or neutralize STX and related PSP toxins.
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Neurotoxinas , Saxitoxina , Animales , Saxitoxina/genética , Ligandos , Guanidina , Proteínas Portadoras/metabolismo , Rana catesbeianaRESUMEN
Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) channels of the TREK subfamily generate 'leak' currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit. In contrast to other potassium channels, K2P channels use a selectivity filter 'C-type' gate as the principal gating site. Despite recent advances, poor pharmacological profiles of K2P channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators-an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402-define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation-π interaction that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate 'leak mode' and provide direct evidence for K2P selectivity filter gating.
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Canales de Potasio de Dominio Poro en Tándem/agonistas , Canales de Potasio de Dominio Poro en Tándem/química , Animales , Ácido Araquidónico/química , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacología , Sitios de Unión/efectos de los fármacos , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Lípidos , Ratones , Modelos Moleculares , Pichia , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Conformación Proteica/efectos de los fármacos , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Tiofenos/química , Tiofenos/metabolismo , Tiofenos/farmacología , Xenopus laevisRESUMEN
Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca2+. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.
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Anoctamina-1/química , Anoctamina-1/ultraestructura , Calcio/química , Calcio/farmacología , Microscopía por Crioelectrón , Activación del Canal Iónico/efectos de los fármacos , Animales , Aniones/química , Aniones/metabolismo , Anoctamina-1/metabolismo , Calcio/metabolismo , Glucósidos/química , Células HEK293 , Humanos , Transporte Iónico/efectos de los fármacos , Ratones , Modelos Moleculares , Nanoestructuras/química , Nanoestructuras/ultraestructura , Conformación Proteica/efectos de los fármacosRESUMEN
K2P (KCNK) potassium channels form "background" or "leak" currents that have critical roles in cell excitability control in the brain, cardiovascular system, and somatosensory neurons. Similar to many ion channel families, studies of K2Ps have been limited by poor pharmacology. Of six K2P subfamilies, the thermo- and mechanosensitive TREK subfamily comprising K2P2.1 (TREK-1), K2P4.1 (TRAAK), and K2P10.1 (TREK-2) are the first to have structures determined for each subfamily member. These structural studies have revealed key architectural features that underlie K2P function and have uncovered sites residing at every level of the channel structure with respect to the membrane where small molecules or lipids can control channel function. This polysite pharmacology within a relatively small (~70 kDa) ion channel comprises four structurally defined modulator binding sites that occur above (Keystone inhibitor site), at the level of (K2P modulator pocket), and below (Fenestration and Modulatory lipid sites) the C-type selectivity filter gate that is at the heart of K2P function. Uncovering this rich structural landscape provides the framework for understanding and developing subtype-selective modulators to probe K2P function that may provide leads for drugs for anesthesia, pain, arrhythmia, ischemia, and migraine.
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Neuronas , Sitios de Unión , HumanosRESUMEN
Ion channels turn diverse types of inputs, ranging from neurotransmitters to physical forces, into electrical signals. Channel responses to ligands generally rely on binding to discrete sensor domains that are coupled to the portion of the channel responsible for ion permeation. By contrast, sensing physical cues such as voltage, pressure, and temperature arises from more varied mechanisms. Voltage is commonly sensed by a local, domain-based strategy, whereas the predominant paradigm for pressure sensing employs a global response in channel structure to membrane tension changes. Temperature sensing has been the most challenging response to understand and whether discrete sensor domains exist for pressure and temperature has been the subject of much investigation and debate. Recent exciting advances have uncovered discrete sensor modules for pressure and temperature in force-sensitive and thermal-sensitive ion channels, respectively. In particular, characterization of bacterial voltage-gated sodium channel (BacNaV) thermal responses has identified a coiled-coil thermosensor that controls channel function through a temperature-dependent unfolding event. This coiled-coil thermosensor blueprint recurs in other temperature sensitive ion channels and thermosensitive proteins. Together with the identification of ion channel pressure sensing domains, these examples demonstrate that "local" domain-based solutions for sensing force and temperature exist and highlight the diversity of both global and local strategies that channels use to sense physical inputs. The modular nature of these newly discovered physical signal sensors provides opportunities to engineer novel pressure-sensitive and thermosensitive proteins and raises new questions about how such modular sensors may have evolved and empowered ion channel pores with new sensibilities.
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Sensación Térmica , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Humanos , Transducción de Señal , Canales de Sodio/química , Canales de Sodio/metabolismo , Canales de Potencial de Receptor Transitorio/químicaRESUMEN
K(2P)2.1 (TREK-1) is a polymodal two-pore domain leak potassium channel that responds to external pH, GPCR-mediated phosphorylation signals, and temperature through the action of distinct sensors within the channel. How the various intracellular and extracellular sensory elements control channel function remains unresolved. Here, we show that the K(2P)2.1 (TREK-1) intracellular C-terminal tail (Ct), a major sensory element of the channel, perceives metabolic and thermal commands and relays them to the extracellular C-type gate through transmembrane helix M4 and pore helix 1. By decoupling Ct from the pore-forming core, we further demonstrate that Ct is the primary heat-sensing element of the channel, whereas, in contrast, the pore domain lacks robust temperature sensitivity. Together, our findings outline a mechanism for signal transduction within K(2P)2.1 (TREK-1) in which there is a clear crosstalk between the C-type gate and intracellular Ct domain. In addition, our findings support the general notion of the existence of modular temperature-sensing domains in temperature-sensitive ion channels. This marked distinction between gating and sensory elements suggests a general design principle that may underlie the function of a variety of temperature-sensitive channels.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Secuencia de Aminoácidos , Animales , Fenómenos Electrofisiológicos , Femenino , Activación del Canal Iónico/genética , Activación del Canal Iónico/fisiología , Metabolismo/fisiología , Ratones , Modelos Biológicos , Modelos Moleculares , Oocitos/química , Oocitos/metabolismo , Estimulación Física , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Dominios y Motivos de Interacción de Proteínas/genética , Temperatura , XenopusRESUMEN
Transmembrane proteins with unknown function 16 (TMEM16A) is a calcium-activated chloride channel (CaCC) important for neuronal, exocrine, and smooth muscle functions. TMEM16A belongs to a family of integral membrane proteins that includes another CaCC, TMEM16B, responsible for controlling action potential waveform and synaptic efficacy, and a small-conductance calcium-activated nonselective cation channel, TMEM16F, linked to Scott syndrome. We find that these channels in the TMEM16 family share a homodimeric architecture facilitated by their cytoplasmic N termini. This dimerization domain is important for channel assembly in eukaryotic cells, and the in vitro association of peptides containing the dimerization domain is consistent with a homotypic protein-protein interaction. Amino acid substitutions in the dimerization domain affect functional TMEM16A-CaCC channel expression, as expected from its critical role in channel subunit assembly.
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Canales de Cloruro/química , Modelos Moleculares , Familia de Multigenes/genética , Proteínas de Neoplasias/química , Anoctamina-1 , Western Blotting , Canales de Cloruro/genética , Dimerización , Células HEK293 , Humanos , Inmunoprecipitación , Proteínas de Neoplasias/genética , Estructura Terciaria de Proteína/genéticaRESUMEN
Members of the K(2P) potassium channel family regulate neuronal excitability and are implicated in pain, anaesthetic responses, thermosensation, neuroprotection, and mood. Unlike other potassium channels, K(2P)s are gated by remarkably diverse stimuli that include chemical, thermal, and mechanical modalities. It has remained unclear whether the various gating inputs act through separate or common channel elements. Here, we show that protons, heat, and pressure affect activity of the prototypical, polymodal K(2P), K(2P)2.1 (KCNK2/TREK-1), at a common molecular gate that comprises elements of the pore-forming segments and the N-terminal end of the M4 transmembrane segment. We further demonstrate that the M4 gating element is conserved among K(2P)s and is employed regardless of whether the gating stimuli are inhibitory or activating. Our results define a unique gating mechanism shared by K(2P) family members and suggest that their diverse sensory properties are achieved by coupling different molecular sensors to a conserved core gating apparatus.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio de Dominio Poro en Tándem/fisiología , Secuencia de Aminoácidos , Animales , Calor , Ratones , Datos de Secuencia Molecular , Canales de Potasio de Dominio Poro en Tándem/genética , Presión , ProtonesRESUMEN
Interactions between voltage-gated calcium channels (Ca(V)s) and calmodulin (CaM) modulate Ca(V) function. In this study, we report the structure of a Ca(2+)/CaM Ca(V)1.2 C-terminal tail complex that contains two PreIQ helices bridged by two Ca(2+)/CaMs and two Ca(2+)/CaM-IQ domain complexes. Sedimentation equilibrium experiments establish that the complex has a 2:1 Ca(2+)/CaM:C-terminal tail stoichiometry and does not form higher order assemblies. Moreover, subunit-counting experiments demonstrate that in live cell membranes Ca(V)1.2s are monomers. Thus, contrary to previous proposals, the crystallographic dimer lacks physiological relevance. Isothermal titration calorimetry and biochemical experiments show that the two Ca(2+)/CaMs in the complex have different properties. Ca(2+)/CaM bound to the PreIQ C-region is labile, whereas Ca(2+)/CaM bound to the IQ domain is not. Furthermore, neither of lobes of apo-CaM interacts strongly with the PreIQ domain. Electrophysiological studies indicate that the PreIQ C-region has a role in calcium-dependent facilitation. Together, the data show that two Ca(2+)/CaMs can bind the Ca(V)1.2 tail simultaneously and indicate a functional role for Ca(2+)/CaM at the C-region site.
Asunto(s)
Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/química , Calmodulina/química , Membrana Celular/metabolismo , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , XenopusRESUMEN
Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (Na(V)Sp1) PD forms a stand-alone, ion selective pore (Na(V)Sp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na(V)Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na(V)Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca(V)Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na(V)s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na(V)Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na(V)Ae1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.
Asunto(s)
Canales de Sodio/química , Canales de Sodio/metabolismo , Alcanivoraceae/genética , Alcanivoraceae/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fenómenos Biofísicos , Ectothiorhodospiraceae/genética , Ectothiorhodospiraceae/metabolismo , Fenómenos Electrofisiológicos , Datos de Secuencia Molecular , Mutación , Filogenia , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Rhodobacteraceae/genética , Rhodobacteraceae/metabolismo , Homología de Secuencia de Aminoácido , Canales de Sodio/genéticaRESUMEN
The halothane-inhibited K2P leak potassium channel K2P13.1 (THIK-1)1-3 is found in diverse cells1,4 including neurons1,5 and microglia6-8 where it affects surveillance6, synaptic pruning7, phagocytosis7, and inflammasome-mediated interleukin-1ß release6,8,9. As with many K2Ps1,5,10-14 and other voltage-gated ion channel (VGIC) superfamily members3,15,16, polyunsaturated fatty acid (PUFA) lipids modulate K2P13.1 (THIK-1)1,5,14,17 via a poorly understood mechanism. Here, we present cryo-electronmicroscopy (cryo-EM) structures of human K2P13.1 (THIK-1) and mutants in lipid nanodiscs and detergent. These reveal that, unlike other K2Ps13,18-24, K2P13.1 (THIK-1) has a two-chamber aqueous inner cavity obstructed by a M4 transmembrane helix tyrosine (Tyr273, the flow restrictor). This hydrophilic barrier can be opened by an activatory mutation, S136P25, at natural break in the M2 transmembrane helix and by intrinsic channel dynamics. The structures also reveal a buried lipid in the P1/M4 intersubunit interface at a location, the PUFA site, that coincides with the TREK subfamily K2P modulator pocket for small molecule agonists18,26,27. This overlap, together with the effects of mutation on K2P13.1 (THIK-1) PUFA responses, indicates that the PUFA site lipids are K2P13.1 (THIK-1) cofactors. Comparison with the PUFA-responsive VGIC Kv7.1 (KCNQ1)28-31 reveals a shared role for the equivalent pore domain intersubunit interface in lipid modulation, providing a framework for dissecting the effects of PUFAs on the VGIC superfamily. Our findings reveal the unique architecture underlying K2P13.1 (THIK-1) function, highlight the importance of the P1/M4 interface in control of K2Ps by both natural and synthetic agents, and should aid development of THIK subfamily modulators for diseases such as neuroinflammation6,32 and autism6.
RESUMEN
Anuran saxiphilins (Sxphs) are "toxin sponge" proteins thought to prevent the lethal effects of small-molecule neurotoxins through sequestration. Here, we present a protocol for the expression, purification, and characterization of Sxphs. We describe steps for using thermofluor, fluorescence polarization, and isothermal titration calorimetry assays that probe Sxph:saxitoxin interactions using a range of sample quantities. These assays are generalizable and can be used for other paralytic shellfish poisoning toxin-binding proteins. For complete details on the use and execution of this protocol, please refer to Chen et al. (2022).1.
Asunto(s)
Neurotoxinas , Saxitoxina , Saxitoxina/metabolismo , Calorimetría , Polarización de FluorescenciaRESUMEN
K2P potassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2P function remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (covalent activation of TREK family K+ channels to clamp membrane potential) that leverages the discovery of a K2P modulator pocket site that reacts with electrophile-bearing derivatives of a TREK subfamily small-molecule activator, ML335, to activate the channel irreversibly. We show that CATKLAMP can be used to probe fundamental aspects of K2P function, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a means to alter K2P channel activity that should facilitate molecular and systems level studies of K2P function and enable the search for new K2P modulators.
Asunto(s)
Canales de Potasio de Dominio Poro en Tándem , Humanos , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio de Dominio Poro en Tándem/genética , Animales , Células HEK293 , Ratones , Potenciales de la Membrana/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de los fármacos , RatasRESUMEN
Based on sequence similarity, the sp7 gene product, MscSP, of the sulfur-compound-decomposing Gram-negative marine bacterium Silicibacter pomeroyi belongs to the family of MscS-type mechanosensitive channels. To investigate MscSP channel properties, we measured its response to membrane tension using the patch-clamp technique on either a heterologous expression system using giant spheroplasts of MJF465 Escherichia coli strain (devoid of mechanosensitive channels MscL, MscS, and MscK), or on purified MscSP protein reconstituted in azolectin liposomes. These experiments showed typical pressure-dependent gating properties of a stretch-activated channel with a current/voltage plot indicating a rectifying behavior and weak preference for anions similar to the MscS channel of E. coli. However, the MscSP channel exhibited functional differences with respect to conductance and desensitization behavior, with the most striking difference between the two channels being the lack of inactivation in MscSP compared with MscS. This seems to result from the fact that although MscSP has a Gly in an equivalent position to MscS (G113), a position that is critical for inactivation, MscSP has a Glu residue instead of an Asn in a position that was recently shown to allosterically influence MscS inactivation, N117. To our knowledge, this study describes the first electrophysiological characterization of an MscS-like channel from a marine bacterium belonging to sulfur-degrading α-proteobacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mecanotransducción Celular , Proteínas de la Membrana/metabolismo , Rhodobacteraceae/citología , Rhodobacteraceae/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas de la Membrana/química , Modelos Moleculares , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Target identification is a core challenge in chemical genetics. Here we use chemical similarity to computationally predict the targets of 586 compounds that were active in a zebrafish behavioral assay. Among 20 predictions tested, 11 compounds had activities ranging from 1 nM to 10,000 nM on the predicted targets. The roles of two of these targets were tested in the original zebrafish phenotype. Prediction of targets from chemotype is rapid and may be generally applicable.
Asunto(s)
Simulación por Computador , Evaluación Preclínica de Medicamentos/métodos , Animales , Conducta Animal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fenotipo , Relación Estructura-Actividad , Pez CebraRESUMEN
Gabapentinoid drugs for pain and anxiety act on the CaVα2δ-1 and CaVα2δ-2 subunits of high-voltage-activated calcium channels (CaV1s and CaV2s). Here we present the cryo-EM structure of the gabapentin-bound brain and cardiac CaV1.2/CaVß3/CaVα2δ-1 channel. The data reveal a binding pocket in the CaVα2δ-1 dCache1 domain that completely encapsulates gabapentin and define CaVα2δ isoform sequence variations that explain the gabapentin binding selectivity of CaVα2δ-1 and CaVα2δ-2.