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.
Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6') and A244(13'). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.
Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.
Quaternary distance restraints are essential to define the three-dimensional structures of protein assemblies. These distances often fall within a range of 10-18 Å, which challenges the high and low measurement limits of conventional nuclear magnetic resonance (NMR) and double electron-electron resonance electron spin resonance spectroscopies. Here, we report the use of 19F paramagnetic relaxation enhancement (PRE) NMR in combination with 19F/paramagnetic labeling to equivalent sites in different subunits of a protein complex in micelles to determine intersubunit distances. The feasibility of this strategy was evaluated on a pentameric ligand-gated ion channel, for which we found excellent agreement of the 19F PRE NMR results with previous structural information. The study suggests that 19F PRE NMR is a viable tool in extracting distance restraints to define quaternary structures.
Cannabinoids exert therapeutic effects on several diseases such as chronic pain and startle disease by targeting glycine receptors (GlyRs). Our previous studies have shown that cannabinoids target a serine residue at position 296 in the third transmembrane helix of the α1/α3 GlyR. This site is located on the outside of the ion channel protein at the lipid interface where the cholesterol concentrates. However, whether membrane cholesterol regulates cannabinoid-GlyR interaction remains unknown. Here, we show that GlyRs are closely associated with cholesterol/caveolin-rich domains at subcellular levels. Membrane cholesterol reduction significantly inhibits cannabinoid potentiation of glycine-activated currents in cultured spinal neurons and in HEK 293T cells expressing α1/α3 GlyRs. Such inhibition is fully rescued by cholesterol replenishment in a concentration-dependent manner. Molecular docking calculations further reveal that cholesterol regulates cannabinoid enhancement of GlyR function through both direct and indirect mechanisms. Taken together, these findings suggest that cholesterol is critical for the cannabinoid-GlyR interaction in the cell membrane.
The human glycine receptors (hGlyRs) are chloride-selective ion channels that mediate inhibitory neurotransmission in the brain stem and spinal cord. They are also targets for compounds of potential use in analgesic therapies. Here, we develop a strategy to discover analgesic drugs via structure-based virtual screening based on the recently published NMR structure of the hGlyR-α1 transmembrane domain (PDB ID: 2M6I) and the critical role of residue S296 in hGlyR-α1 potentiation by Δ9-tetrahydrocannabinol (THC). We screened 1549 FDA-approved drugs in the DrugBank database on an ensemble of 180 hGlyR-α1 structures generated from molecular dynamics simulations of the NMR structure of the hGlyR-α1 transmembrane domain in different lipid environments. Thirteen hit compounds from the screening were selected for functional validation in Xenopus laevis oocytes expressing hGlyR-α1. Only one compound showed no potentiation effects; seven potentiated hGlyR-α1 at a level greater than THC at 1 μM. Our virtual screening protocol is generally applicable to drug targets with lipid-facing binding sites.
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