Non-conductive Conformation Research Articles

Importance of molecular dynamics equilibrium protocol on protein-lipid interaction near channel pore

Multiscale molecular dynamics simulations using Martini coarse-grained (CG) and all-atom (AA) force fields are commonly used in membrane protein studies. In particular, reverse mapping an equilibrated CG model to an AA model offers an efficient way for preparing large membrane protein systems with complex protein shapes and lipid compositions. Here, we report that this hybrid CG-equilibrium-AA-production protocol may artificially increase lipid density and decrease hydration in ion channel pores walled with transmembrane gaps. To understand the origin of this conundrum, we conducted replicas of CG, AA, and CG reverse-mapped AA simulations of the pore domain of the mechanosensitive Piezo1 channel in a nonconducting conformation. Lipid/water density analysis and free energy calculations reveal that the lack of initial pore hydration allows excessive lipids to enter the upper pore lumen through gaps between pore helices during CG simulation. Due to the mismatch between CG and AA lipid kinetics, these pore lipids remain trapped in the subsequent AA simulations, despite unfavorable binding free energy. We tested several CG equilibrium protocols and found that a protocol restraining the whole lipid produces pore hydration consistent with AA results, thus eliminating this artifact for further studies of lipid gating and protein-lipid interactions.

Computer simulations of Piezo1 channel regulation by membrane and small molecule

PIEZO proteins are homotrimeric cation-selective mechanosensitive ion channels that transduce various forms of mechanical forces into intracellular biochemical signals. Using multi-scale molecular dynamics simulations, we have generated an open state of the PIEOZ1 channel from the cryo-EM structure of a nonconducting conformation that reproduces unitary ionic conductance and selectivity in wild-type and mutants channels (Jiang, et al. Commun. Biol. 2021). This open state further allowed us to validate a putative allosteric agonist binding site of Yoda1 we previously identified using MD simulation and electrophysiology (Botello-Smith, et al.

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel.

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.

The Selectivity Filter Is Involved in the U-Type Inactivation Process of Kv2.1 and Kv3.1 Channels

Voltage-gated potassium (Kv) channels display several types of inactivation processes, including N-, C-, and U-types. C-type inactivation is attributed to a nonconductive conformation of the selectivity filter (SF). It has been proposed that the activation gate and the channel’s SF are allosterically coupled because the conformational changes of the former affect the structure of the latter and vice versa. The second threonine of the SF signature sequence (e.g., TTVGYG) has been proven to be essential for this allosteric coupling. To further study the role of the SF in U-type inactivation, we substituted the second threonine of the TTVGYG sequence by an alanine in the hKv2.1 and hKv3.1 channels, which are known to display U-type inactivation. Both hKv2.1-T377A and hKv3.1-T400A yielded channels that were resistant to inactivation, and as a result, they displayed noninactivating currents upon channel opening; i.e., hKv2.1-T377A and hKv3.1-T400A remained fully conductive upon prolonged moderate depolarizations, whereas in wild-type hKv2.1 and hKv3.1, the current amplitude typically reduces because of U-type inactivation. Interestingly, increasing the extracellular K+ concentration increased the macroscopic current amplitude of both hKv2.1-T377A and hKv3.1-T400A, which is similar to the response of the homologous T to A mutation in Shaker and hKv1.5 channels that display C-type inactivation. Our data support an important role for the second threonine of the SF signature sequence in the U-type inactivation gating of hKv2.1 and hKv3.1.

Structural Basis of TRPV3 Activation and Inactivation

The TRPV3 ion channel is critically involved in skin physiology and pathophysiology. Here we present cryo-EM structures of TRPV3 in lipid nanodiscs in distinct functional states representing the gating cycle. The ligand-free, closed conformation reveals a dual gating mechanism involving both the extracellular selectivity-filter gate and the intracellular helix bundle-crossing gate, and lipids are identified throughout the channel assembly. Both gates expand upon channel activation, accompanied by substantial structural rearrangements in the cytoplasmic domains. Sustained application of an agonist inactivates TRPV3 and results in a distinct non-conducting conformation, which is dramatically different from the apo, closed state. The pore-lining helix S6 maintains a π-helical segment upon activation but becomes entirely α-helical during inactivation. These results are in sharp contrast to recently reported TRPV3 structures determined in detergent micelles and in amphiphilic polymers that are devoid of lipids. Taken together, multiple distinct TRPV3 structures in a lipid membrane environment provide unique insights into physiological activation and inactivation.

Accessibility of Cations to the Selectivity Filter of KcsA in the Inactivated State: An Equilibrium Binding Study.

Cation binding under equilibrium conditions has been used as a tool to explore the accessibility of permeant and nonpermeant cations to the selectivity filter in three different inactivated models of the potassium channel KcsA. The results show that the stack of ion binding sites (S1 to S4) in the inactivated filter models remain accessible to cations as they are in the resting channel state. The inactivated state of the selectivity filter is therefore “resting-like” under such equilibrium conditions. Nonetheless, quantitative differences in the apparent KD’s of the binding processes reveal that the affinity for the binding of permeant cations to the inactivated channel models, mainly K+, decreases considerably with respect to the resting channel. This is likely to cause a loss of K+ from the inactivated filter and consequently, to promote nonconductive conformations. The most affected site by the affinity loss seems to be S4, which is interesting because S4 is the first site to accommodate K+ coming from the channel vestibule when K+ exits the cell. Moreover, binding of the nonpermeant species, Na+, is not substantially affected by inactivation, meaning that the inactivated channels are also less selective for permeant versus nonpermeant cations under equilibrium conditions.

Electrostatics, proton sensor, and networks governing the gating transition in GLIC, a proton-gated pentameric ion channel

The pentameric ligand-gated ion channel (pLGIC) from Gloeobacter violaceus (GLIC) has provided insightful structure-function views on the permeation process and the allosteric regulation of the pLGICs family. However, GLIC is activated by pH instead of a neurotransmitter and a clear picture for the gating transition driven by protons is still lacking. We used an electrostatics-based (finite difference Poisson-Boltzmann/Debye-Hückel) method to predict the acidities of all aspartic and glutamic residues in GLIC, both in its active and closed-channel states. Those residues with a predicted pKa close to the experimental pH50 were individually replaced by alanine and the resulting variant receptors were titrated by ATR/FTIR spectroscopy. E35, located in front of loop F far away from the orthosteric site, appears as the key proton sensor with a measured individual pKa at 5.8. In the GLIC open conformation, E35 is connected through a water-mediated hydrogen-bond network first to the highly conserved electrostatic triad R192-D122-D32 and then to Y197-Y119-K248, both located at the extracellular domain-transmembrane domain interface. The second triad controls a cluster of hydrophobic side chains from the M2-M3 loop that is remodeled during the gating transition. We solved 12 crystal structures of GLIC mutants, 6 of them being trapped in an agonist-bound but nonconductive conformation. Combined with previous data, this reveals two branches of a continuous network originating from E35 that reach, independently, the middle transmembrane region of two adjacent subunits. We conclude that GLIC's gating proceeds by making use of loop F, already known as an allosteric site in other pLGICs, instead of the classic orthosteric site.

Gating mechanism of the extracellular entry to the lipid pathway in a TMEM16 scramblase

Members of the TMEM16/ANO family of membrane proteins are Ca2+-activated phospholipid scramblases and/or Cl− channels. A membrane-exposed hydrophilic groove in these proteins serves as a shared translocation pathway for ions and lipids. However, the mechanism by which lipids gain access to and permeate through the groove remains poorly understood. Here, we combine quantitative scrambling assays and molecular dynamic simulations to identify the key steps regulating lipid movement through the groove. Lipid scrambling is limited by two constrictions defined by evolutionarily conserved charged and polar residues, one extracellular and the other near the membrane mid-point. The region between these constrictions is inaccessible to lipids and water molecules, suggesting that the groove is in a non-conductive conformation. A sequence of lipid-triggered reorganizations of interactions between these residues and the permeating lipids propagates from the extracellular entryway to the central constriction, allowing the groove to open and coordinate the headgroups of transiting lipids.

Structural and Functional Studies Uncover Two Networks Stabilizing the Active Form of GLIC, a Bacterial Proton-Gated Pentameric Ion Channel

The proton-activated pentameric ligand-gated ion channel from Gloeobacter violaceus GLIC can be used as a model system to study the structural and functional properties of its eukaryotic counterparts, which mediate signaling transduction in animal neuron cells by controlling permeation of ions flux gated by neurotransmitter binding. Here we used the Finite Difference Poisson-Boltzmann/Debye-Hückel method to predict the pKas of all the Asp and Glu in GLIC both in the active and resting states. Those residues with a high deviation of predicted pKas in both forms were titrated by Fourier-transform infra-red spectroscopy after reconstitution in lipid bilayers. The results showed that E35 is the main key proton-sensor residue. Examination of the active form structure shows that E35 interacts with T158 from Loop F. Here we verified that breaking this interaction hinders the proton-elicited currents. We next probed the interfacial crevice immediately below E35, shaped by Loop F, Q193 (pre-M1) and the neighboring M2-M3 loop, where a previously unnoticed hydrogen bond network may help maintaining the open channel. We show that breaking this hydrophilic network originating from Q193 favors the ion channel in a nonconductive conformation in the crystal state. Replacing side-chains of residues just below Q193, such as Y197 and I201, with other hydrophobic side-chains blocks the receptor in a locally closed form. Two signal transduction networks are proposed, both originating from the key proton-sensing residue E35, loop F and the pre-M1 region: i) one hydrophilic network extends to the M2-M3 loop from the neighboring subunit and ii) one hydrophobic network interacts with the Cys-loop and the M2-M3 loop of the same subunit. Due to the strong conservation of these loop regions, this model could be generalized to the entire pLGIC family.

A Universal Molecular Mechanism for C-type Inactivation in Potassium Channels

C-type inactivation of K+ channels is an elusive molecular process that is thought to involve a subtle conformational change at the level of the selectivity filter. C-type inactivation is also of great physiological significance, as it affects the firing patterns of neurons in the central nervous system, and the repolarization of cardiac cells in the heart. In the present study, extended molecular dynamics simulation of the hERG channel with an open inner gate converged toward a stable constricted conformation for the selectivity filter. This conformation displays both similarities and differences with the known constricted conformation observed in X-ray structures of the KcsA channel. The constricted hERG filter involves a considerable reorientation of phenylalanine at position 627 along the selectivity filter to dock into the binding pocket occupied by the inactivating water molecules in the constricted KcsA structure. This observation supports the notion that constriction of the filter is the molecular basis of inactivation, but with alternative structural factors stabilizing the conformation. Additional MD simulations and free energy calculations carried out on the KcsA channel show that the filter can remain conductive as long as the intracellular gate is not fully open, but rapidly transitions to a constricted non-conductive conformation on a microsecond timescale once the inner gate is fully open. The short microsecond lifetime of the conductive filter with a fully open gate, cannot explain the relatively long periods of sustained ion conduction observed experimentally, suggesting that the conduction period must predominantly correspond to long-lived states of the channel with a partially open activation gate. In summary, C-type inactivation is linked to a conductive-to-constricted transition of the selectivity filter that is allosterically driven by the slow opening of the intracellular gate.

The Role of the Selectivity Filter in TRPV1 Channel Gating

The TRPV1 channel is a homotetrameric non-selective cation channel that functions in nociceptors as an integrator of external noxious stimuli and endogenous pro-inflammatory molecules. Although the binding sites for some TRPV1 modulators have been characterized, we still don’t understand how any of these stimuli influence ion conduction in this receptor. The identification and characterization of the regions that function as activation gates are therefore central to understanding the mechanisms of gating in the TRPV1 and other related channels. The structures of TRPV1 in open and closed states suggested that in addition to the intracellular gate formed by the pore-lining S6 helices, the selectivity filter could also function as an activation gate. The available structures of other TRP channels also support the existence of two gates, suggesting that it could be a conserved and unique feature of the TRP family. Here we set out to determine whether the selectivity filter of TRPV1 functions as a gate. We have substituted cysteines along the pore using a cysteineless channel background and assessed their accessibility to externally applied silver ions in both the open and the closed states in patch clamp recordings. First we found that external silver ions act as high-affinity, voltage-dependent, permeant blockers of the cysteineless TRPV1 channel, with a higher affinity for the closed state relative to the open state, suggesting that the filter does not function as a gate. We have provided further support for this hypothesis by showing that accessibility to external silver ions to a cysteine more intracellular than the filter is not affected by channel gating. It is possible that the filter adopts a non-conducting conformation under specific conditions yet to be identified, or that it constitutes a highly dynamic region of the receptor.

Chemical substitutions in the selectivity filter of potassium channels do not rule out constricted-like conformations for C-type inactivation

In many K+ channels, prolonged activating stimuli lead to a time-dependent reduction in ion conduction, a phenomenon known as C-type inactivation. X-ray structures of the KcsA channel suggest that this inactivated state corresponds to a "constricted" conformation of the selectivity filter. However, the functional significance of the constricted conformation has become a matter of debate. Functional and structural studies based on chemically modified semisynthetic KcsA channels along the selectivity filter led to the conclusion that the constricted conformation does not correspond to the C-type inactivated state. The main results supporting this view include the observation that C-type inactivation is not suppressed by a substitution of D-alanine at Gly77, even though this modification is believed to lock the selectivity filter into its conductive conformation, whereas it is suppressed following amide-to-ester backbone substitutions at Gly77 and Tyr78, even though these structure-conserving modifications are not believed to prevent the selectivity filter from adopting the constricted conformation. However, several untested assumptions about the structural and functional impact of these chemical modifications underlie these arguments. To make progress, molecular dynamics simulations based on atomic models of the KcsA channel were performed. The computational results support the notion that the constricted conformation of the selectivity filter corresponds to the functional C-type inactivated state of the KcsA. Importantly, MD simulations reveal that the semisynthetic KcsAD-ala77 channel can adopt an asymmetrical constricted-like nonconductive conformation and that the amide-to-ester backbone substitutions at Gly77 and Tyr78 perturb the hydrogen bonding involving the buried water molecules stabilizing the constricted conformation.

Mechanism of partial agonism in AMPA-type glutamate receptors

Neurotransmitters trigger synaptic currents by activating ligand-gated ion channel receptors. Whereas most neurotransmitters are efficacious agonists, molecules that activate receptors more weakly—partial agonists—also exist. Whether these partial agonists have weak activity because they stabilize less active forms, sustain active states for a lesser fraction of the time or both, remains an open question. Here we describe the crystal structure of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) ligand binding domain (LBD) tetramer in complex with the partial agonist 5-fluorowillardiine (FW). We validate this structure, and others of different geometry, using engineered intersubunit bridges. We establish an inverse relation between the efficacy of an agonist and its promiscuity to drive the LBD layer into different conformations. These results suggest that partial agonists of the AMPAR are weak activators of the receptor because they stabilize multiple non-conducting conformations, indicating that agonism is a function of both the space and time domains.

Structural Insights into the Atomistic Mechanisms of Action of Small Molecule Inhibitors Targeting the KCa3.1 Channel Pore.

The intermediate-conductance Ca2+-activated K+ channel (KCa3.1) constitutes an attractive pharmacological target for immunosuppression, fibroproliferative disorders, atherosclerosis, and stroke. However, there currently is no available crystal structure of this medically relevant channel that could be used for structure-assisted drug design. Using the Rosetta molecular modeling suite we generated a molecular model of the KCa3.1 pore and tested the model by first confirming previously mapped binding sites and visualizing the mechanism of TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole), senicapoc (2,2-bis-(4-fluorophenyl)-2-phenylacetamide), and NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one) inhibition at the atomistic level. All three compounds block ion conduction directly by fully or partially occupying the site that would normally be occupied by K+ before it enters the selectivity filter. We then challenged the model to predict the receptor sites and mechanisms of action of the dihydropyridine nifedipine and an isosteric 4-phenyl-pyran. Rosetta predicted receptor sites for nifedipine in the fenestration region and for the 4-phenyl-pyran in the pore lumen, which could both be confirmed by site-directed mutagenesis and electrophysiology. While nifedipine is thus not a pore blocker and might be stabilizing the channel in a nonconducting conformation or interfere with gating, the 4-phenyl-pyran was found to be a classical pore blocker that directly inhibits ion conduction similar to the triarylmethanes TRAM-34 and senicapoc. The Rosetta KCa3.1 pore model explains the mechanism of action of several KCa3.1 blockers at the molecular level and could be used for structure-assisted drug design.

The Selective Nav1.7 Inhibitor, PF-05089771, Interacts Equivalently with Fast and Slow Inactivated Nav1.7 Channels.

Voltage-gated sodium (Nav) channel inhibitors are used clinically as analgesics and local anesthetics. However, the absence of Nav channel isoform selectivity of current treatment options can result in adverse cardiac and central nervous system side effects, limiting their therapeutic utility. Human hereditary gain- or loss-of-pain disorders have demonstrated an essential role of Nav1.7 sodium channels in the sensation of pain, thus making this channel an attractive target for new pain therapies. We previously identified a novel, state-dependent human Nav1.7 selective inhibitor (PF-05089771, IC50 = 11 nM) that interacts with the voltage-sensor domain (VSD) of domain IV. We further characterized the state-dependent interaction of PF-05089771 by systematically varying the voltage, frequency, and duration of conditioning prepulses to provide access to closed, open, and fast- or slow-inactivated states. The current study demonstrates that PF-05089771 exhibits a slow onset of block that is depolarization and concentration dependent, with a similarly slow recovery from block. Furthermore, the onset of block by PF-05089771 develops with similar rates using protocols that bias channels into predominantly fast- or slow-inactivated states, suggesting that channel inhibition is less dependent on the availability of a particular inactivated state than the relative time that the channel is depolarized. Taken together, the inhibitory profile of PF-05089771 suggests that a conformational change in the domain IV VSD after depolarization is necessary and sufficient to reveal a high-affinity binding site with which PF-05089771 interacts, stabilizing the channel in a nonconducting conformation from which recovery is slow.

Gating, Regulation, and Structure in K2P K+ Channels: In Varietate Concordia?

K2P K(+) channels with two pore domains in tandem associate as dimers to produce so-called background conductances that are regulated by a variety of stimuli. Whereas gating in K2P channels has been poorly understood, recent developments have provided important clues regarding the gating mechanism for this family of proteins. Two modes of gating present in other K(+) channels have been considered. The first is the so-called activation gating that occurs by bundle crossing and the splaying apart of pore-lining helices commanding ion passage. The second mode involves a change in conformation at the selectivity filter (SF), which impedes ion flow at this narrow portion of the conduction pathway and accounts for extracellular pH modulation of several K2P channels. Although some evidence supports the existence of an activation gate in K2P channels, recent results suggest that perhaps all stimuli, even those sensed at a distant location in the protein, are also mediated by SF gating. Recently resolved crystal structures of K2P channels in conductive and nonconductive conformations revealed that the nonconductive state is reached by blockade by a lipid acyl chain that gains access to the channel cavity through intramembrane fenestrations. Here we discuss whether this novel type of gating, proposed so far only for membrane tension gating, might mediate gating in response to other stimuli or whether SF gating is the only type of opening/closing mechanism present in K2P channels.

Signal Transduction at the Domain Interface of Prokaryotic Pentameric Ligand-Gated Ion Channels

Pentameric ligand-gated ion channels are activated by the binding of agonists to a site distant from the ion conduction path. These membrane proteins consist of distinct ligand-binding and pore domains that interact via an extended interface. Here, we have investigated the role of residues at this interface for channel activation to define critical interactions that couple conformational changes between the two structural units. By characterizing point mutants of the prokaryotic channels ELIC and GLIC by electrophysiology, X-ray crystallography and isothermal titration calorimetry, we have identified conserved residues that, upon mutation, apparently prevent activation but not ligand binding. The positions of nonactivating mutants cluster at a loop within the extracellular domain connecting β-strands 6 and 7 and at a loop joining the pore-forming helix M2 with M3 where they contribute to a densely packed core of the protein. An ionic interaction in the extracellular domain between the turn connecting β-strands 1 and 2 and a residue at the end of β-strand 10 stabilizes a state of the receptor with high affinity for agonists, whereas contacts of this turn to a conserved proline residue in the M2-M3 loop appear to be less important than previously anticipated. When mapping residues with strong functional phenotype on different channel structures, mutual distances are closer in conducting than in nonconducting conformations, consistent with a potential role of contacts in the stabilization of the open state. Our study has revealed a pattern of interactions that are crucial for the relay of conformational changes from the extracellular domain to the pore region of prokaryotic pentameric ligand-gated ion channels. Due to the strong conservation of the interface, these results are relevant for the entire family.

Engineering Subunit and Nonsense Suppression Stoichiometry in Potassium Channels

Multi-subunit membrane proteins employ chemically diverse inter- and intramolecular contacts to regulate their function. Potassium channel slow inactivation relies on a hydrogen bond network around the selectivity filter which supports an internal timing mechanism to control the transition between conducting and non-conducting conformations. Previously we used targeted replacement of W434 with the isosteric non-canonical amino acid 2-amino-3-indol-1-yl-propionic acid (Ind) to highlight the importance of the indole H-bond of this highly conserved Trp residue. However, these experiments were unable to nuance the specific contributions of individual channel subunits to channel inactivation. We therefore designed an engineered protein approach whereby complementary intein sequences are incorporated into the N- or C- termini of Shaker monomers, respectively, flanked by previously identified endoplasmic reticulum (ER) retention signals. The data shows that functional channels composed of monomeric ER-intein subunits are restricted from the surface membrane. However, co-expression of complementary ER-intein monomers results in robust expression of functional K+ channels through intein ligation and excision of both the ER retention signals and intein sequences. This technique was used to express Shaker channels with two W434Ind monomers within a single tetramer, producing an intermediate inactivation rate, consistent with the breakage of multiple H-bonds required for full inactivation. In addition, the fidelity of the stoichiometric assembly was assayed by TEA block of channels with zero, two or four T449F mutations. The development of this approach allows for regulation of subunit stoichiometry in multimeric complexes and advances the use of nonsense suppression by demonstrating a solution to incorporate multiple non-canonical amino acids into a single peptide and multimeric complexes. Specifically, the reduction of overall protein expression that is normally compounded at the second suppression site is effectively abrogated because two transcripts are suppressed independently then ligated as proteins.

A Structural Model of the Human α7 Nicotinic Receptor in an Open Conformation.

Nicotinic acetylcholine receptors (nAchRs) are ligand-gated ion channels that regulate chemical transmission at the neuromuscular junction. Structural information is available at low resolution from open and closed forms of an eukaryotic receptor, and at high resolution from other members of the same structural family, two prokaryotic orthologs and an eukaryotic GluCl channel. Structures of human channels however are still lacking. Homology modeling and Molecular Dynamics simulations are valuable tools to predict structures of unknown proteins, however, for the case of human nAchRs, they have been unsuccessful in providing a stable open structure so far. This is due to different problems with the template structures: on one side the homology with prokaryotic species is too low, while on the other the open eukaryotic GluCl proved itself unstable in several MD studies and collapsed to a dehydrated, non-conductive conformation, even when bound to an agonist. Aim of this work is to obtain, by a mixing of state-of-the-art homology and simulation techniques, a plausible prediction of the structure (still unknown) of the open state of human α7 nAChR complexed with epibatidine, from which it is possible to start structural and functional test studies. To prevent channel closure we employ a restraint that keeps the transmembrane pore open, and obtain in this way a stable, hydrated conformation. To further validate this conformation, we run four long, unbiased simulations starting from configurations chosen at random along the restrained trajectory. The channel remains stable and hydrated over the whole runs. This allows to assess the stability of the putative open conformation over a cumulative time of 1 μs, 800 ns of which are of unbiased simulation. Mostly based on the analysis of pore hydration and size, we suggest that the obtained structure has reasonable chances to be (at least one of the possible) structures of the channel in the open conformation.

The analgesic-like properties of the alpha7 nAChR silent agonist NS6740 is associated with non-conducting conformations of the receptor

The α7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of neurological disorders including chronic pain and inflammatory diseases. Since α7 can function as a ligand-gated ion channel, drug development initially focused on ligands that were selective activators of the α7 ion channel. However, the best α7 drugs for chronic pain and inflammation indications may not be ion channel activators but rather “silent agonists”, which bind to the receptor but preferentially induce non-conducting states that modulate signal transduction in non-neuronal cells. One such compound is NS6740. We show that NS6740 selectively induces prolonged desensitization of α7 nAChRs. There are two forms of α7 desensitization that can be distinguished by their sensitivity to the positive allosteric modulators (PAMs). At high concentrations, NS6740 preferentially induces PAM-insensitive desensitization, which over the course of several minutes reverts to the sensitive form. NS6740 was tested in several pain models after in vivo administration in the mouse. Although it had no effects in acute thermal pain, NS6740 induced significant dose- and time-dependent antinociceptive activity in formalin- and acetic acid-induced nociceptive behaviors as well as in the chronic constrictive nerve injury (CCI) model for neuropathic pain. The antinociceptive activity of NS6740 in these models was α7-dependent. In addition, NS6740 administration reversed pain-induced aversion, an important affective component of pain. The time and concentration dependence of the effects were consistent with NS6740 induction of PAM-insensitive non-conducting states, suggesting that signal transduction required for analgesia is accomplished by α7 receptors in that conformation.