RESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.
Asunto(s)
Epilepsia , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Activación del Canal Iónico , Mutación , Canales de Potasio , Propofol , Humanos , Sitios de Unión , Microscopía por Crioelectrón , Electrofisiología , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Epilepsia/metabolismo , Células HEK293 , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/antagonistas & inhibidores , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/ultraestructura , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Metionina/genética , Metionina/metabolismo , Modelos Moleculares , Movimiento/efectos de los fármacos , Fenilalanina/genética , Fenilalanina/metabolismo , Polimorfismo Genético , Canales de Potasio/química , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de Potasio/ultraestructura , Propofol/farmacología , Propofol/químicaRESUMEN
Quaternary ammonium blockers were previously shown to bind in the pore to block both open and closed conformations of large-conductance calcium-activated potassium (BK and MthK) channels. Because blocker entry was assumed through the intracellular entryway (bundle crossing), closed-pore access suggested that the gate was not at the bundle crossing. Structures of closed MthK, a Methanobacterium thermoautotrophicum homolog of BK channels, revealed a tightly constricted intracellular gate, leading us to investigate the membrane-facing fenestrations as alternative pathways for blocker access directly from the membrane. Atomistic free energy simulations showed that intracellular blockers indeed access the pore through the fenestrations, and a mutant channel with narrower fenestrations displayed no closed-state TPeA block at concentrations that blocked the wild-type channel. Apo BK channels display similar fenestrations, suggesting that blockers may use them as access paths into closed channels. Thus, membrane fenestrations represent a non-canonical pathway for selective targeting of specific channel conformations, opening novel ways to selectively drug BK channels.
Asunto(s)
Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Potasio/metabolismo , Conformación MolecularRESUMEN
Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive drug targets1-5. More than 210 structures from more than 20 different TRP channels have been determined, and all are tetramers4. Despite this wealth of structures, many aspects concerning TRPV channels remain poorly understood, including the pore-dilation phenomenon, whereby prolonged activation leads to increased conductance, permeability to large ions and loss of rectification6,7. Here, we used high-speed atomic force microscopy (HS-AFM) to analyse membrane-embedded TRPV3 at the single-molecule level and discovered a pentameric state. HS-AFM dynamic imaging revealed transience and reversibility of the pentamer in dynamic equilibrium with the canonical tetramer through membrane diffusive protomer exchange. The pentamer population increased upon diphenylboronic anhydride (DPBA) addition, an agonist that has been shown to induce TRPV3 pore dilation. On the basis of these findings, we designed a protein production and data analysis pipeline that resulted in a cryogenic-electron microscopy structure of the TRPV3 pentamer, showing an enlarged pore compared to the tetramer. The slow kinetics to enter and exit the pentameric state, the increased pentamer formation upon DPBA addition and the enlarged pore indicate that the pentamer represents the structural correlate of pore dilation. We thus show membrane diffusive protomer exchange as an additional mechanism for structural changes and conformational variability. Overall, we provide structural evidence for a non-canonical pentameric TRP-channel assembly, laying the foundation for new directions in TRP channel research.
Asunto(s)
Multimerización de Proteína , Canales Catiónicos TRPV , Anhídridos/química , Anhídridos/farmacología , Análisis de Datos , Difusión , Subunidades de Proteína/química , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/metabolismo , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/efectos de los fármacos , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/ultraestructura , Microscopía de Fuerza Atómica , Terapia Molecular Dirigida , Microscopía por Crioelectrón , Estructura Cuaternaria de Proteína/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacosRESUMEN
Cyclic nucleotide-gated ion channels are crucial in many physiological processes such as vision and pacemaking in the heart. SthK is a prokaryotic homolog with high sequence and structure similarities to hyperpolarization-activated and cyclic nucleotide-modulated and cyclic nucleotide-gated channels, especially at the level of the cyclic nucleotide binding domains (CNBDs). Functional measurements showed that cyclic adenosine monophosphate (cAMP) is a channel activator while cyclic guanosine monophosphate (cGMP) barely leads to pore opening. Here, using atomic force microscopy single-molecule force spectroscopy and force probe molecular dynamics simulations, we unravel quantitatively and at the atomic level how CNBDs discriminate between cyclic nucleotides. We find that cAMP binds to the SthK CNBD slightly stronger than cGMP and accesses a deep-bound state that a cGMP-bound CNBD cannot reach. We propose that the deep binding of cAMP is the discriminatory state that is essential for cAMP-dependent channel activation.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos , Nucleótidos Cíclicos , Canales Catiónicos Regulados por Nucleótidos Cíclicos/química , Activación del Canal Iónico/fisiología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismoRESUMEN
Tandem pore domain (K2P) potassium channels modulate resting membrane potentials and shape cellular excitability. For the mechanosensitive subfamily of K2Ps, the composition of phospholipids within the bilayer strongly influences channel activity. To examine the molecular details of K2P lipid modulation, we solved cryo-EM structures of the TREK1 K2P channel bound to either the anionic lipid phosphatidic acid (PA) or the zwitterionic lipid phosphatidylethanolamine (PE). At the extracellular face of TREK1, a PA lipid inserts its hydrocarbon tail into a pocket behind the selectivity filter, causing a structural rearrangement that recapitulates mutations and pharmacology known to activate TREK1. At the cytoplasmic face, PA and PE lipids compete to modulate the conformation of the TREK1 TM4 gating helix. Our findings demonstrate two distinct pathways by which anionic lipids enhance TREK1 activity and provide a framework for a model that integrates lipid gating with the effects of other mechanosensitive K2P modulators.
Asunto(s)
Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio de Dominio Poro en Tándem/genética , Fosfolípidos , Potenciales de la Membrana , Potasio/metabolismoRESUMEN
Carefully orchestrated opening and closing of ion channels control the diffusion of ions across cell membranes, generating the electrical signals required for fast transmission of information throughout the nervous system. Inactivation is a parsimonious means for channels to restrict ion conduction without the need to remove the activating stimulus. Voltage-gated channel inactivation plays crucial physiological roles, such as controlling action potential duration and firing frequency in neurons. The ball-and-chain moniker applies to a type of inactivation proposed first for sodium channels and later shown to be a universal mechanism. Still, structural evidence for this mechanism remained elusive until recently. We review the ball-and-chain inactivation research starting from its introduction as a crucial component of sodium conductance during electrical signaling in the classical Hodgkin and Huxley studies, through the discovery of its simple intuitive mechanism in potassium channels during the molecular cloning era, to the eventual elucidation of a potassium channel structure in a ball-and-chain inactivated state.
Asunto(s)
Canales de Potasio , Transducción de Señal , Canales de Potasio/química , Membrana CelularRESUMEN
Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker channel homolog, and HCN2, whose activity contributes to pacemaking in the heart and brain. Using SthK allowed the reconstitution of purified channels in controlled lipid compositions for functional and structural assays that are not available for the eukaryotic channels. We identified anionic lipids bound tightly to SthK and their exact binding locations and determined that they potentiate channel activity. Cryo-EM structures in the most potentiating lipids revealed an open state and identified a nonannular lipid bound with its headgroup near an intersubunit salt bridge that clamps the intracellular channel gate shut. Breaking this conserved salt bridge abolished lipid modulation in SthK and eukaryotic HCN2 channels, indicating that anionic membrane lipids facilitate channel opening by destabilizing these interactions. Our findings underline the importance of state-dependent protein-lipid interactions.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Lípidos de la Membrana , AnionesRESUMEN
Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos , Nucleótidos Cíclicos , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Activación del Canal Iónico , AMP Cíclico/metabolismo , LigandosRESUMEN
Conformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the ß-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Electrofisiología/métodos , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Canales Iónicos/metabolismo , Porinas/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Membrana Dobles de Lípidos/química , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Porinas/genética , Porinas/metabolismo , Conformación Proteica , Conformación Proteica en Lámina beta , Proteínas Recombinantes , Análisis Espectral , Relación Estructura-ActividadRESUMEN
Recent developments in cryogenic electron microscopy (cryo-EM) led to an exponential increase in high-resolution structures of membrane proteins, and in particular ion channels. However, structures alone can only provide limited information about the workings of these proteins. In order to understand ion channel function and regulation in molecular detail, the obtained structural data need to be correlated to functional states of the same protein. Here, we describe several techniques that can be employed to study ion channel structure and function in vitro and under defined, similar conditions. Lipid nanodiscs provide a native-like environment for membrane proteins and have become a valuable tool in membrane protein structural biology and biophysics. Combined with liposome-based flux assays for the kinetic analysis of ion channel activity as well as electrophysiological recordings, researchers now have access to an array of experimental techniques allowing for detailed structure-function correlations using purified components. Two examples are presented where we put emphasis on the lipid environment and time-resolved techniques together with mutations and protein engineering to interpret structural data obtained from single particle cryo-EM on cyclic nucleotide-gated or Ca2+-gated K+ channels. Furthermore, we provide short protocols for all the assays used in our work so that others can adapt these techniques to their experimental needs. Comprehensive structure-function correlations are essential in order to pharmacologically target channelopathies.
Asunto(s)
Canales Iónicos , Proteínas de la Membrana , Microscopía por Crioelectrón , Cinética , Lípidos , Proteínas de la Membrana/genéticaRESUMEN
Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a "microbody," in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of ß coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/farmacología , Fragmentos Fc de Inmunoglobulinas/metabolismo , Microcuerpos/metabolismo , SARS-CoV-2/efectos de los fármacos , Secuencia de Aminoácidos , Animales , COVID-19/prevención & control , COVID-19/virología , Modelos Animales de Enfermedad , Disulfuros/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones Transgénicos , Dominios Proteicos , Multimerización de Proteína , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Virión/metabolismo , Internalización del Virus/efectos de los fármacosRESUMEN
SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that the cis/trans conformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms: trans Pro300 SthK with high ligand binding affinity and fast activation, and cis Pro300 SthK with low affinity and slow activation. Following channel activation, the cis/trans equilibrium, catalyzed by prolyl isomerases, is shifted towards trans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/química , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Spirochaeta/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , AMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Ciclosporina/farmacología , Activación del Canal Iónico/fisiología , Isomerismo , Cinética , Modelos Moleculares , Isomerasa de Peptidilprolil/metabolismo , Prolina/metabolismoRESUMEN
K2P potassium channels are known to be modulated by volatile anesthetic (VA) drugs and play important roles in clinically relevant effects that accompany general anesthesia. Here, we utilize a photoaffinity analog of the VA isoflurane to identify a VA-binding site in the TREK1 K2P channel. The functional importance of the identified site was validated by mutagenesis and biochemical modification. Molecular dynamics simulations of TREK1 in the presence of VA found multiple neighboring residues on TREK1 TM2, TM3, and TM4 that contribute to anesthetic binding. The identified VA-binding region contains residues that play roles in the mechanisms by which heat, mechanical stretch, and pharmacological modulators alter TREK1 channel activity and overlaps with positions found to modulate TASK K2P channel VA sensitivity. Our findings define molecular contacts that mediate VA binding to TREK1 channels and suggest a mechanistic basis to explain how K2P channels are modulated by VAs.
Asunto(s)
Anestésicos por Inhalación/farmacología , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Anestésicos por Inhalación/metabolismo , Animales , Sitios de Unión , Humanos , Isoflurano/farmacología , Ratones , Simulación del Acoplamiento Molecular , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Xenopus laevis , Pez CebraRESUMEN
Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca2+-activated K+ channel from Methanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K+ concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF's four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K+ affinity (â¼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K+, similar to KcsA, but that even a single K+ binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K+ concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK's S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Dominios Proteicos/fisiología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Cristalografía por Rayos X , Canales de Potasio de Gran Conductancia Activados por el Calcio/aislamiento & purificación , Canales de Potasio de Gran Conductancia Activados por el Calcio/ultraestructura , Methanobacterium , Simulación de Dinámica Molecular , Potasio/metabolismoRESUMEN
We present an approach for preparing cryo-electron microscopy (cryo-EM) grids to study short-lived molecular states. Using piezoelectric dispensing, two independent streams of ~50-pl droplets of sample are deposited within 10 ms of each other onto the surface of a nanowire EM grid, and the mixing reaction stops when the grid is vitrified in liquid ethane ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.
Asunto(s)
Microscopía por Crioelectrón/métodos , Nanocables , Robótica , Manejo de Especímenes/métodos , Factores de TiempoRESUMEN
Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present1. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency1,2. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a 'ball-and-chain' mechanism3-7. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum-a purely calcium-gated and inactivating channel-in a lipid environment. In the absence of Ca2+, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca2+-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism.
Asunto(s)
Microscopía por Crioelectrón , Activación del Canal Iónico , Methanobacterium/química , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Canales de Potasio Calcio-Activados/ultraestructura , Calcio/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Canales de Potasio Calcio-Activados/química , Canales de Potasio Calcio-Activados/metabolismo , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , TermodinámicaRESUMEN
Integral membrane proteins have historically been challenging targets for biophysical research due to their low solubility in aqueous solution. Their importance for chemical and electrical signaling between cells, however, makes them fascinating targets for investigators interested in the regulation of cellular and physiological processes. Since membrane proteins shunt the barrier imposed by the cell membrane, they also serve as entry points for drugs, adding pharmaceutical research and development to the interests. In recent years, detailed understanding of membrane protein function has significantly increased due to high-resolution structural information obtained from single-particle cryo-EM, X-ray crystallography, and NMR. In order to further advance our mechanistic understanding on membrane proteins as well as foster drug development, it is crucial to generate more biophysical and functional data on these proteins under defined conditions. To that end, different techniques have been developed to stabilize integral membrane proteins in native-like environments that allow both structural and biophysical investigations-amphipols, lipid bicelles, and lipid nanodiscs. In this chapter, we provide detailed protocols for the reconstitution of membrane proteins according to these three techniques. We also outline some of the possible applications of each technique and discuss their advantages and possible caveats.
Asunto(s)
Biofisica/métodos , Membrana Dobles de Lípidos/química , Microdominios de Membrana , Proteínas de la Membrana/química , Renaturación de Proteína , Química Analítica , Detergentes/química , Detergentes/farmacología , Membrana Dobles de Lípidos/metabolismo , Liposomas/química , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Micelas , Modelos Moleculares , Nanoestructuras/química , Polímeros/química , Polímeros/farmacología , Propilaminas/química , Propilaminas/farmacología , Conformación Proteica , Pliegue de Proteína , Renaturación de Proteína/efectos de los fármacos , Estabilidad Proteica , SolubilidadRESUMEN
Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, a mutation that decreases ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites.