RESUMO
Transient receptor potential (TRP) proteins are a large family of cation-selective channels, surpassed in variety only by voltage-gated potassium channels. Detailed molecular mechanisms governing how membrane voltage, ligand binding, or temperature can induce conformational changes promoting the open state in TRP channels are still a matter of debate. Aiming to unveil distinctive structural features common to the transmembrane domains within the TRP family, we performed phylogenetic reconstruction, sequence statistics, and structural analysis over a large set of TRP channel genes. Here, we report an exceptionally conserved set of residues. This fingerprint is composed of twelve residues localized at equivalent three-dimensional positions in TRP channels from the different subtypes. Moreover, these amino acids are arranged in three groups, connected by a set of aromatics located at the core of the transmembrane structure. We hypothesize that differences in the connectivity between these different groups of residues harbor the apparent differences in coupling strategies used by TRP subgroups.
Assuntos
Canais de Potencial de Receptor Transitório , Filogenia , Domínios Proteicos , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Polyamines such as spermidine and spermine are found in nearly all cells, at concentrations ranging up to 0.5 mM. These cations are endogenous regulators of cellular K+ efflux, binding tightly in the pores of inwardly rectifying K+ (Kir) channels in a voltage-dependent manner. Although the voltage dependence of Kir channel polyamine blockade is thought to arise at least partially from the energetically coupled movements of polyamine and K+ ions through the pore, the nature of physical interactions between these molecules is unclear. Here we analyze the polyamine-blocking mechanism in the model K+ channel MthK, using a combination of electrophysiology and computation. Spermidine (SPD3+) and spermine (SPM4+) each blocked current through MthK channels in a voltage-dependent manner, and blockade by these polyamines was described by a three-state kinetic scheme over a wide range of polyamine concentrations. In the context of the scheme, both SPD3+ and SPM4+ access a blocking site with similar effective gating valences (0.84 ± 0.03 e0 for SPD3+ and 0.99 ± 0.04 e0 for SPM4+), whereas SPM4+ binds in the blocked state with an â¼20-fold higher affinity than SPD3+ (Kd = 28.1 ± 3.1 µM for SPD3+ and 1.28 ± 0.20 µM for SPM4+), consistent with a free energy difference of 1.8 kcal/mol. Molecular simulations of the MthK pore in complex with either SPD3+ or SPM4+ are consistent with the leading amine interacting with the hydroxyl groups of T59, at the selectivity filter threshold, with access to this site governed by outward movement of K+ ions. These coupled movements can account for a large fraction of the voltage dependence of blockade. In contrast, differences in binding energetics between SPD3+ and SPM4+ may arise from distinct electrostatic interactions between the polyamines and carboxylate oxygens on the side chains of E92 and E96, located in the pore-lining helix.
Assuntos
Poliaminas , Canais de Potássio Corretores do Fluxo de Internalização , Fenômenos Eletrofisiológicos , Cinética , Potássio , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Espermidina , EsperminaRESUMO
The transient receptor potential channel vanilloid type 1 (TRPV1) is activated by a variety of endogenous and exogenous stimuli and is involved in nociception and body temperature regulation. Although the structure of TRPV1 has been experimentally determined in both the closed and open states, very little is known about its activation mechanism. In particular, the conformational changes that occur in the pore domain and result in ionic conduction have not yet been identified. Here we suggest a hypothetical molecular mechanism for TRPV1 activation, which involves rotation of a conserved asparagine in S6 from a position facing the S4-S5 linker toward the pore. This rotation is associated with hydration of the pore and dehydration of the four peripheral cavities located between each S6 and S4-S5 linker. In light of our hypothesis, we perform bioinformatics analyses of TRP and other evolutionary related ion channels, evaluate newly available structures, and reexamine previously reported water accessibility and mutagenesis experiments. These analyses provide several independent lines of evidence to support our hypothesis. Finally, we show that our proposed molecular mechanism is compatible with the prevailing theory that the selectivity filter acts as a secondary gate in TRPV1.
Assuntos
Canais de Cátion TRPV/metabolismo , Asparagina , Simulação de Dinâmica Molecular , Conformação Proteica , RotaçãoRESUMO
Propofol is widely used in the clinic for the induction and maintenance of general anesthesia. As with most general anesthetics, however, our understanding of its mechanism of action remains incomplete. Local and general anesthetics largely inhibit voltage-gated Na+ channels (Navs) by inducing an apparent stabilization of the inactivated state, associated in some instances with pore block. To determine the biophysical and molecular basis of propofol action in Navs, we investigated NaChBac and NavMs, two prokaryotic Navs with distinct voltage dependencies and gating kinetics, by whole-cell patch clamp electrophysiology in the absence and presence of propofol at clinically relevant concentrations (2-10 µM). In both Navs, propofol induced a hyperpolarizing shift of the pre-pulse inactivation curve without any significant effects on recovery from inactivation at strongly hyperpolarized voltages, demonstrating that propofol does not stabilize the inactivated state. Moreover, there was no evidence of fast or slow pore block by propofol in a non-inactivating NaChBac mutant (T220A). Propofol also induced hyperpolarizing shifts of the conductance-voltage relationships with negligible effects on the time constants of deactivation at hyperpolarized voltages, indicating that propofol does not stabilize the open state. Instead, propofol decreases the time constants of macroscopic activation and inactivation. Adopting a kinetic scheme of Nav gating that assumes preferential closed-state recovery from inactivation, a 1.7-fold acceleration of the rate constant of activation and a 1.4-fold acceleration of the rate constant of inactivation were sufficient to reproduce experimental observations with computer simulations. In addition, molecular dynamics simulations and molecular docking suggest that propofol binding involves interactions with gating machinery in the S4-S5 linker and external pore regions. Our findings show that propofol is primarily a positive gating modulator of prokaryotic Navs, which ultimately inhibits the channels by promoting activation-coupled inactivation.
Assuntos
Proteínas de Bactérias/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Propofol/farmacologia , Canais de Sódio/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células HEK293 , Humanos , Cinética , Mutação , Técnicas de Patch-Clamp , Canais de Sódio/genética , Canais de Sódio/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Voltage-gated sodium (NaV) channels are important targets of general anesthetics, including the intravenous anesthetic propofol. Electrophysiology studies on the prokaryotic NaV channel NaChBac have demonstrated that propofol promotes channel activation and accelerates activation-coupled inactivation, but the molecular mechanisms of these effects are unclear. Here, guided by computational docking and molecular dynamics simulations, we predict several propofol-binding sites in NaChBac. We then strategically place small fluorinated probes at these putative binding sites and experimentally quantify the interaction strengths with a fluorinated propofol analogue, 4-fluoropropofol. In vitro and in vivo measurements show that 4-fluoropropofol and propofol have similar effects on NaChBac function and nearly identical anesthetizing effects on tadpole mobility. Using quantitative analysis by 19F-NMR saturation transfer difference spectroscopy, we reveal strong intermolecular cross-relaxation rate constants between 4-fluoropropofol and four different regions of NaChBac, including the activation gate and selectivity filter in the pore, the voltage sensing domain, and the S4-S5 linker. Unlike volatile anesthetics, 4-fluoropropofol does not bind to the extracellular interface of the pore domain. Collectively, our results show that propofol inhibits NaChBac at multiple sites, likely with distinct modes of action. This study provides a molecular basis for understanding the net inhibitory action of propofol on NaV channels.