A Potential Route of Capsaicin to Its Binding Site in the TRPV1 Ion Channel.

Transient receptor potential (TRP) ion channels are important pharmacological targets because of their role in the perception of pain, and so, understanding their chemical regulation is essential for the development of analgesic drugs. Among the currently known TRP channel chemical agonists, capsaicin, the active compound of chili pepper, is probably the most exhaustively studied. The availability of the three-dimensional structure of the vanilloid receptor 1 (TRPV1) has fueled computational studies revealing the molecular details of capsaicin binding modes. Although this is a significant step, a comprehensible binding mechanism or pathway is invaluable for targeting TRP channels in modern pharmacology. In the present work, free-energy and enhanced sampling techniques have been used to explore a possible membrane-mediated pathway for capsaicin to enter the TRPV1 binding pocket where capsaicin accesses the protein starting at the extracellular milieu through the outer leaflet and into its binding site in the protein. The main states visited along this route have been characterized and include (i) a bound state in agreement with the binding mode "head-down, tail-up" and (ii) an alternative state corresponding to a "head-up, tail-down" binding mode. In agreement with previous reports, binding is mediated by both hydrogen bonds and van der Waals interactions, and residue Y511 is crucial for stabilizing the bound state and during the binding process. Together, these results provide a foundation to further understand TRPV channels, and they could be used to guide therapeutic design of selective inhibitors potentially leading to novel avenues for pharmacological applications targeting the TRPV1 channel.

Capturing the Molecular Mechanism of Anesthetic Action by Simulation Methods.

Significant computational efforts have been focused toward exposing the molecular mechanisms of anesthesia in recent years. In the past decade, this has been aided considerably by a momentous increase in the number of high-resolution structures of ion channels, which are putative targets for the anesthetic agents, as well as advancements in high-performance computing technologies. In this review, typical simulation methods to investigate the behavior of model membranes and membrane-protein systems are briefly reviewed, and related computational studies are surveyed. Both lipid- and protein-mediated mechanisms of anesthetic action are scrutinized, focusing on the behavior of ion channels in the latter case.

Exposure of the HIV-1 broadly neutralizing antibody 10E8 MPER epitope on the membrane surface by gp41 transmembrane domain scaffolds.

The 10E8 antibody achieves near-pan neutralization of HIV-1 by targeting the remarkably conserved gp41 membrane-proximal external region (MPER) and the connected transmembrane domain (TMD) of the HIV-1 envelope glycoprotein (Env). Thus, recreating the structure that generates 10E8-like antibodies is a major goal of the rational design of anti-HIV vaccines. Unfortunately, high-resolution information of this segment in the native Env is lacking, limiting our understanding of the behavior of the crucial 10E8 epitope residues. In this report, two sequences, namely, MPER-TMD1 (gp41 residues 671-700) and MPER-TMD2 (gp41 residues 671-709) were compared both experimentally and computationally, to assess the TMD as a potential membrane integral scaffold for the 10E8 epitope. These sequences were selected to represent a minimal (MPER-TMD1) or full-length (MPER-TMD2) TMD membrane anchor according to mutagenesis results reported by Yue et al. (2009) J. Virol. 83, 11,588. Immunochemical assays revealed that MPER-TMD1, but not MPER-TMD2, effectively exposed the MPER C-terminal stretch, harboring the 10E8 epitope on the surface of phospholipid bilayers containing a cholesterol concentration equivalent to that of the viral envelope. Molecular dynamics simulations, using the recently resolved TMD trimer structure combined with the MPER in a cholesterol-enriched model membrane confirmed these results and provided an atomistic mechanism of epitope exposure which revealed that TMD truncation at position A700 combined with N-terminal addition of lysine residues positively impacts epitope exposure. Overall, these results provide crucial insights into the design of effective MPER-TMD derived immunogens.

Stereospecific Interactions of Cholesterol in a Model Cell Membrane: Implications for the Membrane Dipole Potential.

Cholesterol is a major constituent of the plasma membrane in higher order eukaryotes. The effect of cholesterol on the structure and organisation of cell membranes has been studied extensively by both experimental and computational means. In recent years, a wealth of data has been accumulated illustrating how subtle differences in the structure of cholesterol equate to considerable changes in the physical properties of the membrane. The effect of cholesterol stereoisomers, in particular, has been established, identifying a direct link with the activity of specific membrane proteins. In this study, we perform extensive molecular dynamics simulations of phospholipid bilayers containing three isomers of cholesterol, the native form (nat-cholesterol), the enantiomer of the native form (ent-cholesterol), and an epimer of cholesterol that differs by the orientation of the polar hydroxyl group (epi-cholesterol). Based on these simulations, an atomic-level description of the stereospecific cholesterol-phospholipid interactions is provided, establishing a potential mechanism for the perturbation of membrane properties, specifically the membrane dipole potential.

Exploring the Dynamics of the TWIK-1 Channel.

Potassium channels in the two-pore domain family (K2P) have various structural attributes that differ from those of other K(+) channels, including a dimeric assembly constituted of nonidentical domains and an expansive extracellular cap. Crystallization of the prototypical K2P channel, TWIK-1, finally revealed the structure of these characteristics in atomic detail, allowing computational studies to be undertaken. In this study, we performed molecular-dynamics simulations for a cumulative time of ∼1 µs to discern the mechanism of ion transport throughout TWIK-1. We observed the free passage of ions beneath the extracellular cap and identified multiple high-occupancy sites in close proximity to charged residues on the protein surface. Despite the overall topological similarity of the x-ray structure of the selectivity filter to other K(+) channels, the structure diverges significantly in molecular-dynamics simulations as a consequence of nonconserved residues in both pore domains contributing to the selectivity filter (T118 and L228). The behavior of such residues has been linked to channel inactivation and the phenomenon of dynamic selectivity, where TWIK-1 displays robust Na(+) inward flux in response to subphysiological K(+) concentrations.

Lateral Fenestrations in K(+)-Channels Explored Using Molecular Dynamics Simulations.

Potassium channels are of paramount physiological and pathological importance and therefore constitute significant drug targets. One of the keys to rationalize the way drugs modulate ion channels is to understand the ability of such small molecules to access their respective binding sites, from which they can exert an activating or inhibitory effect. Many computational studies have probed the energetics of ion permeation, and the mechanisms of voltage gating, but little is known about the role of fenestrations as possible mediators of drug entry in potassium channels. To explore the existence, structure, and conformational dynamics of transmembrane fenestrations accessible by drugs in potassium channels, molecular dynamics simulation trajectories were analyzed from three potassium channels: the open state voltage-gated channel Kv1.2, the G protein-gated inward rectifying channel GIRK2 (Kir3.2), and the human two-pore domain TWIK-1 (K2P1.1). The main results of this work were the identification of the sequence identity of four main lateral fenestrations of similar length and with bottleneck radius in the range of 0.9-2.4 Å for this set of potassium channels. It was found that the fenestrations in Kv1.2 and Kir3.2 remain closed to the passage of molecules larger than water. In contrast, in the TWIK-1 channel, both open and closed fenestrations are sampled throughout the simulation, with bottleneck radius shown to correlate with the random entry of lipid membrane molecules into the aperture of the fenestrations. Druggability scoring function analysis of the fenestration regions suggests that Kv and Kir channels studied are not druggable in practice due to steric constraining of the fenestration bottleneck. A high (>50%) fenestration sequence identity was found in each potassium channel subfamily studied, Kv1, Kir3, and K2P1. Finally, the reported fenestration sequence of TWIK-1 compared favorably with another channel, K2P channel TREK-2, reported to possess open fenestrations, suggesting that K2P channels could be druggable via fenestrations, for which we reported atomistic detail of the fenestration region, including the flexible residues M260 and L264 that interact with POPC membrane in a concerted fashion with the aperture and closure of the fenestrations.

Effect of anionic lipids on ion permeation through the KcsA K+-channel.

K+-channels are responsible for the efficient and selective conduction of K+ ions across the plasma membrane. The bacterial K+-channel KcsA has historically been used to characterize various aspects of K+ conduction via computational means. The energetic barriers associated with ion translocation across the KcsA selectivity filter have been computed in various studies, leading to the proposal of two alternate mechanisms of conduction, involving or neglecting the presence of water molecules in between the permeating ions. Here, the potential of mean force of K+ permeation is evaluated for KcsA in lipid bilayers containing anionic lipids, which is known to increase the open probability of the channel. In addition, the effect of the protonation/deprotonation of residue E71, which directly interacts with the selectivity filter sequence, is assessed. Both conduction mechanisms are considered throughout. The results obtained provide novel insights into the molecular functioning of K+-channels including the inactivation process.

Insights into the Mechanisms of K+ Permeation in K+ Channels from Computer Simulations.

Ion permeation, selectivity, and the behavior of the K+ channel selectivity filter have been studied intensively in the previous two decades. The agreement among multiple approaches used to study ion flux in K+ channels suggests a consensus mechanism of ion permeation across the selectivity that has been put to the test in recent years with the proposal of an alternative way by which ions can cross the selectivity filter of K+ channels via direct Coulomb repulsion between contacting cations. Past experimental work by Zhou and MacKinnon (J. Mol. Biol. 2004, 338, 839) showed that mutation of the site S4 reduces the total occupancy of the selectivity filter to less than two ions on average by lowering the occupancy of the S2-S4 configuration without changing the S1-S3 configuration much, and this reduction of occupancy means that ion configurations different from the ones involved in the canonical mechanism are likely to be involved. At that time, calculations using complicated kinetic networks to relate occupancy to conduction did not provide deeper insight into the conduction mechanism. Here, to help solve this enigma, umbrella sampling simulations have been performed to evaluate the potential of mean force of two KcsA mutant channels where the S4 site is substituted. Our new results provide insights into the significance of threonine in this position, revealing the effect of substitution on the alternate mechanisms of conduction proposed, involving either water or vacant sites.

Cytomegalovirus Retinitis Diagnosis and Treatment in a Kidney Transplant Recipient.

Cytomegalovirus retinitis is a rare complication in kidney transplant patients with only one other reported incidence. A 70-year-old female with a previous history of kidney transplant began experiencing a decreased vision of her right eye and was diagnosed with CMV retinitis and started on ganciclovir treatment. After completing the treatment, the patient has had no signs of recurrence or any other complaints. There is only one other published case report of CMV retinitis in a kidney transplant patient, therefore appropriate treatment and predictability of recurrence are unknown.

Combining Structural Data with Computational Methodologies to Investigate Structure-Function Relationships in TRP Channels.

Since the emergence of high-resolution three-dimensional structures of membrane proteins, and the increasing availability of state-of-the-art algorithms and high-performance-computing facilities, classical molecular dynamics (MD) simulations have become a routine device to explore the molecular behavior of these proteins. The rise of cryo-electron microscopy (cryo-EM) as a credible experimental tool to resolve structures at an atomic level has revolutionized structural biology in recent years, culminating in the disclosure of the first high-resolution three-dimensional structure of a transient receptor potential (TRP) channel, the vanilloid receptor 1 (TRPV1). As a result, the number of research articles investigating the molecular behavior of TRP channels using macromolecular simulation techniques has proliferated. This review provides an overview of the current state of this field, including our understanding of TRP channel structure, the framework of classical MD simulations, and how to perform such simulations to investigate structure-function relationships in TRP channels.

Influence of Cholesterol and Its Stereoisomers on Members of the Serotonin Receptor Family.

Despite the ubiquity of cholesterol within the cell membrane, the mechanism by which it influences embedded proteins remains elusive. Numerous G-protein coupled receptors exhibit dramatic responses to membrane cholesterol with regard to the ligand-binding affinity and functional properties, including the 5-HT receptor family. Here, we use over 25 µs of unbiased atomistic molecular dynamics simulations to identify cholesterol interaction sites in the 5-HT1B and 5-HT2B receptors and evaluate their impact on receptor structure. Susceptibility to membrane cholesterol is shown to be subtype dependent and determined by the quality of interactions between the extracellular loops. Charged residues are essential for maintaining the arrangement of the extracellular surface in 5-HT2B; in the absence of such interactions, the extracellular surface of the 5-HT1B is malleable, populating a number of distinct conformations. Elevated cholesterol density near transmembrane helix 4 is considered to be conducive to the conformation of extracellular loop 2. Occupation of this site is also shown to be stereospecific, illustrated by differential behavior of nat-cholesterol isomers, ent- and epi-cholesterol. In simulations containing the endogenous agonist, serotonin, cholesterol binding at transmembrane helix 4 biases bound serotonin molecules toward an unexpected binding mode in the extended binding pocket. The results highlight the capability of membrane cholesterol to influence the mobility of the extracellular surface in the 5-HT1 receptor family and manipulate the architecture of the extracellular ligand-binding pocket.

Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites.

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites. Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.

Oxytocin Modulates Nociception as an Agonist of Pain-Sensing TRPV1.

Oxytocin is a hormone with various actions. Oxytocin-containing parvocellular neurons project to the brainstem and spinal cord. Oxytocin release from these neurons suppresses nociception of inflammatory pain, the molecular mechanism of which remains unclear. Here, we report that the noxious stimulus receptor TRPV1 is an ionotropic oxytocin receptor. Oxytocin elicits TRPV1 activity in native and heterologous expression systems, regardless of the presence of the classical oxytocin receptor. In TRPV1 knockout mice, DRG neurons exhibit reduced oxytocin sensitivity relative to controls, and oxytocin injections significantly attenuate capsaicin-induced nociception in in vivo experiments. Furthermore, oxytocin potentiates TRPV1 in planar lipid bilayers, supporting a direct agonistic action. Molecular modeling and simulation experiments provide insight into oxytocin-TRPV1 interactions, which resemble DkTx. Together, our findings suggest the existence of endogenous regulatory pathways that modulate nociception via direct action of oxytocin on TRPV1, implying its analgesic effect via channel desensitization.