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
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
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
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
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
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
Allosteric couplings underlie many cellular signaling processes and provide an exciting avenue for development of new diagnostics and therapeutics. A general method for identifying important residues in allosteric mechanisms would be very useful, but remains elusive due to the complexity of long-range phenomena. Here, we introduce an NMR method to identify residues involved in allosteric coupling between two ligand-binding sites in a protein, which we call chemical shift detection of allostery participants (CAP). Networks of functional groups responding to each ligand are defined through correlated NMR perturbations. In this process, we also identify allostery participants, groups that respond to both binding events and likely play a role in the coupling between the binding sites. Such residues exhibit multiple functional states with distinct NMR chemical shifts, depending on binding status at both binding sites. Such a strategy was applied to the prototypical ion channel KcsA. We had previously shown that the potassium affinity at the extracellular selectivity filter is strongly dependent on proton binding at the intracellular pH sensor. Here, we analyzed proton and potassium binding networks and identified groups that depend on both proton and potassium binding (allostery participants). These groups are viewed as candidates for transmitting information between functional units. The vital role of one such identified amino acid was validated through site-specific mutagenesis, electrophysiology functional studies, and NMR-detected thermodynamic analysis of allosteric coupling. This strategy for identifying allostery participants is likely to have applications for many other systems.
Asunto(s)
Regulación Alostérica , Modelos Moleculares , Proteínas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Espectroscopía de Resonancia Magnética , Mutación , Canales de Potasio/química , Canales de Potasio/metabolismo , Conformación Proteica , Proteínas/genética , Relación Estructura-ActividadRESUMEN
Cyclic nucleotide-modulated ion channels play crucial roles in signal transduction in eukaryotes. The molecular mechanism by which ligand binding leads to channel opening remains poorly understood, due in part to the lack of a robust method for preparing sufficient amounts of purified, stable protein required for structural and biochemical characterization. To overcome this limitation, we designed a stable, highly expressed chimeric ion channel consisting of the transmembrane domains of the well characterized potassium channel KcsA and the cyclic nucleotide-binding domains of the prokaryotic cyclic nucleotide-modulated channel MloK1. This chimera demonstrates KcsA-like pH-sensitive activity which is modulated by cAMP, reminiscent of the dual modulation in hyperpolarization-activated and cyclic nucleotide-gated channels that display voltage-dependent activity that is also modulated by cAMP. Using this chimeric construct, we were able to measure for the first time the binding thermodynamics of cAMP to an intact cyclic nucleotide-modulated ion channel using isothermal titration calorimetry. The energetics of ligand binding to channels reconstituted in lipid bilayers are substantially different from those observed in detergent micelles, suggesting that the conformation of the chimera's transmembrane domain is sensitive to its (lipid or lipid-mimetic) environment and that ligand binding induces conformational changes in the transmembrane domain. Nevertheless, because cAMP on its own does not activate these chimeric channels, cAMP binding likely has a smaller energetic contribution to gating than proton binding suggesting that there is only a small difference in cAMP binding energy between the open and closed states of the channel.
Asunto(s)
Proteínas Bacterianas/metabolismo , AMP Cíclico/metabolismo , Activación del Canal Iónico , Lípidos de la Membrana/metabolismo , Mesorhizobium/metabolismo , Canales de Potasio/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , AMP Cíclico/química , AMP Cíclico/genética , Concentración de Iones de Hidrógeno , Lípidos de la Membrana/química , Lípidos de la Membrana/genética , Mesorhizobium/química , Mesorhizobium/genética , Canales de Potasio/química , Canales de Potasio/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genéticaRESUMEN
To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl(+)) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H(+)). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C18:1 to C22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H(+) gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H(+) affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels' lipid bilayer environment or other regulatory processes.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Canales de Potasio/metabolismo , Espectrometría de Fluorescencia/métodos , Activación del Canal Iónico , Membrana Dobles de Lípidos/química , Fosfolípidos/metabolismoRESUMEN
Structures of the prokaryotic K(+) channel, KcsA, highlight the role of the selectivity filter carbonyls from the GYG signature sequence in determining a highly selective pore, but channels displaying this sequence vary widely in their cation selectivity. Furthermore, variable selectivity can be found within the same channel during a process called C-type inactivation. We investigated the mechanism for changes in selectivity associated with inactivation in a model K(+) channel, KcsA. We found that E71A, a noninactivating KcsA mutant in which a hydrogen-bond behind the selectivity filter is disrupted, also displays decreased K(+) selectivity. In E71A channels, Na(+) permeates at higher rates as seen with and flux measurements and analysis of intracellular Na(+) block. Crystal structures of E71A reveal that the selectivity filter no longer assumes the "collapsed," presumed inactivated, conformation in low K(+), but a "flipped" conformation, that is also observed in high K(+), high Na(+), and even Na(+) only conditions. The data reveal the importance of the E71-D80 interaction in both favoring inactivation and maintaining high K(+) selectivity. We propose a molecular mechanism by which inactivation and K(+) selectivity are linked, a mechanism that may also be at work in other channels containing the canonical GYG signature sequence.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Activación del Canal Iónico , Canales de Potasio/química , Canales de Potasio/metabolismo , Conformación Proteica , Proteínas Bacterianas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Potasio/metabolismo , Canales de Potasio/genética , Radioisótopos de Rubidio/química , Radioisótopos de Rubidio/metabolismo , Radioisótopos de Sodio , Difracción de Rayos XRESUMEN
Potassium channels are involved in a tremendously diverse range of physiological applications requiring distinctly different functional properties. Not surprisingly, the amino acid sequences for these proteins are diverse as well, except for the region that has been ordained the "selectivity filter". The goal of this review is to examine our current understanding of the role of the selectivity filter and regions adjacent to it in specifying selectivity as well as its role in gating/inactivation and possible mechanisms by which these processes are coupled. Our working hypothesis is that an amino acid network behind the filter modulates selectivity in channels with the same signature sequence while at the same time affecting channel inactivation properties. This article is part of a Special Issue entitled: Membrane protein structure and function.
Asunto(s)
Canales de Potasio/química , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Canales de Potasio/genética , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled KcsA, a bacterial potassium channel that has high homology with mammalian channels, for solid-state NMR studies. The expression and purification is optimized for an average yield of â¼35-40mg/L of M9 media in a time-efficient way. The protein purity is confirmed by gel electrophoresis and the protein concentration is quantified by UV-vis absorption spectroscopy. Protocols to efficiently reconstitute KcsA into liposomes are also presented. The presence of liposomes is confirmed by cryo-electron microscopy images and the effect of magic angle spinning on liposome packing is shown. High-resolution solid-state NMR spectra of uniformly isotope labeled KcsA in these liposomes reveal that our protocol yields to a very homogenous KcsA sample with high signal to noise and several well-resolved residues in NMR spectra. Electrophysiology of our samples before and after solid-state NMR show that channel function and selectivity remain intact after the solid-state NMR.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Liposomas/química , Canales de Potasio/química , Canales de Potasio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Marcaje Isotópico , Liposomas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Canales de Potasio/genética , Canales de Potasio/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
PorB is the second most prevalent outer membrane protein in Neisseria meningitidis. PorB is required for neisserial pathogenesis and can elicit a Toll-like receptor mediated host immune response. Here, the x-ray crystal structure of PorB has been determined to 2.3 A resolution. Structural analysis and cocrystallization studies identify three putative solute translocation pathways through the channel pore: One pathway transports anions nonselectively, one transports cations nonselectively, and one facilitates the specific uptake of sugars. During infection, PorB likely binds host mitochondrial ATP, and cocrystallization with the ATP analog AMP-PNP suggests that binding of nucleotides regulates these translocation pathways both by partial occlusion of the pore and by restricting the motion of a putative voltage gating loop. PorB is located on the surface of N. meningitidis and can be recognized by receptors of the host innate immune system. Features of PorB suggest that Toll-like receptor mediated recognition outer membrane proteins may be initiated with a nonspecific electrostatic attraction.
Asunto(s)
Neisseria meningitidis/metabolismo , Porinas/metabolismo , Aniones , Proteínas de la Membrana Bacteriana Externa/química , Transporte Biológico , Carbohidratos/química , Cristalografía por Rayos X/métodos , Humanos , Inmunidad Innata , Liposomas/química , Modelos Moleculares , Conformación Molecular , Nucleótidos/química , Porinas/química , Receptores Toll-Like/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
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
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
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
The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Despite prior attempts to determine the mechanism responsible for pH gating, the proton sensor has remained elusive. We have constructed a KcsA channel mutant that remains open up to pH 9.0 by replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge. A series of individual and combined mutations were investigated by using single-channel recordings in lipid bilayers. We propose that these residues are the proton-binding sites and at neutral pH they form a complex network of inter- and intrasubunit salt bridges and hydrogen bonds near the bundle crossing that greatly stabilize the closed state. In our model, these residues change their ionization state at acidic pH, thereby disrupting this network, modifying the electrostatic landscape near the channel gate, and favoring channel opening.