Evidence of inter- and intra-subunit alcohol and anesthetic binding cavities in the glycine receptor

textAlcohol is abundantly consumed by society and general anesthetics are used everyday in operating suites throughout the world, yet the sites and mechanisms of action for these drugs are not completely understood. Glycine receptors (GlyRs) are pentameric ion channels expressed throughout the brain and spinal cord and have become increasingly popular targets in the study of alcohol action. Each GlyR subunit is composed of four alpha helical transmembrane segments (TM1-4), and although amino acids involved with alcohol action have been previously identified in TM1-4, the orientation of each of these residues with respect to a putative alcohol/anesthetic binding cavity remains controversial. In order to better characterize this binding cavity within the GlyR, we conducted a series of experiments using cysteine mutagenesis and biochemical cross-linking. In Aim 1, the participation of TM1 with TM3 in a common alcohol/anesthetic binding cavity was further investigated. We used two-electrode voltage clamp electrophysiology in Xenopus oocytes to demonstrate the ability of A288 in TM3 to form cross-links with I229 in TM1, which reduced the ability of both alcohol and anesthetics to modulate GlyR function. Aim 2 investigated whether TM3 could also participate in a binding cavity with TM4. We have shown that residues in TM4 are able to form cross-links with A288 in TM3, and found that cross-linking between TM3 and those residues in TM4 also reduced the ability of alcohol and anesthetics to enhance GlyR function. Aim 3 determined whether these cross-links are formed between residues within the same subunit (intra-subunit) or between subunits (inter-subunit), and ultimately whether these residues participate in a common alcohol/anesthetic binding cavity within or between GlyR subunits. GlyR protein, which measures about 50 kDa, was extracted from oocytes injected with the cysteine mutants, and immunoblotting was used with a GlyR-specific antibody to subsequently help quantify band ratios between cross-linked and uncross-linked conditions. We found an increase in the 100:50 kDa band ratio for the TM1-3 mutant only, but not TM3-4 mutant or the wild-type, which suggests TM1-3 may participate in an alcohol binding cavity between GlyR subunits while TM3-4 may contribute to a binding cavity within a subunit.Pharmaceutical Science

Computational Protein Design and Molecular Dynamics Simulations: A Study of Membrane Proteins, Small Peptides and Molecular Systems

Molecular design and modeling can provide stringent assessment of our understanding of the structure and function of proteins. Due to the subtleness of the interactions that largely stabilize proteins, computational methods have been particularly valuable in establishing practical, formal and physically grounded protocols to study the structure and function of these biomolecules. Especifically, computational protein design seeks to identify sequences that fold into a desired structure and have specific structural and functional properties using computational methodologies. Among current techniques, an entropy-based formalism that efficiently determines the number and composition of sequences satisfying a predefined set of constraints seems particularly promising and powerful. Complementary to this methodology are the well-established molecular dynamics simulation techniques that have been extensively used to study structure, function and dynamics of biologically relevant systems. Herein different studies of systems using computational techniques to address particular molecular problems are described. Efforts to redesign membrane proteins to generate water-soluble variants were applied to a widely studied pentameric ligand-gated ion channel, the nicotinic acetylchoilne receptor (nAChR). NMR structures and binding studies demostrated the robustness and applicability of the computational design approach. Toward the creation of water-soluble variants of a G protein–coupled receptor (GPCR), comparative modeling and docking calculations were used to investigate the structure of the human μ opioid receptor and presented in light of previous mutagenesis studies of structure and agonist-induced activation. Candidate peptides for possible therapeutic agents were computationally analyzed. Peptide design, loop modeling and MD simulations were applied to investigate the stromal cell-derived factor-1&a; (SDF-1&a;). SDF-1&a; displays promising therapeutic benefits to treat blood-supply related heart disease and elicit growth of microvasculature. Simplified analogs of SDF-1&a; exhibit enhanced therapeutic properties in cell-based assays. MD simulations provide insights about the molecular features of this enhancement. One simplified peptide offers a potentially clinically translatable neovasculogenic therapy. Lastly, MD simulations were utilized to analyze a molecule with hindered internal rotors, a tribenzylamine hemicryptophane. The molecule was characterized by different experimental and computational techniques. The structural and dynamic features of the hemicryptophane molecule make it an attractive starting point for controlling internal rotation of aromatic rings within molecular systems

Bacterial Voltage-Gated Sodium Channels (BacNaVs) from the Soil, Sea, and Salt Lakes Enlighten Molecular Mechanisms of Electrical Signaling and Pharmacology in the Brain and Heart

AbstractVoltage-gated sodium channels (NaVs) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60years, functional studies of NaVs have occupied a central place in physiological and biophysical investigation of the molecular basis of excitability. Recently, structural studies of members of a large family of bacterial voltage-gated sodium channels (BacNaVs) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic NaVs have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNaVs as templates for drug development efforts

Halogen bonds involved in binding of halogenated ligands by protein kinases.

Analysis of 664 known structures of protein kinase complexes with halogenated ligands revealed 424 short contacts between a halogen atom and a potential protein X-bond acceptor, the topology and geometry of which were analyzed according to the type of a halogen atom (X = Cl, Br, I) and a putative protein X-bond acceptor. Among 236 identified halogen bonds, the most represented ones are directed to backbone carbonyls of the hinge region and may replace the pattern of ATP-like hydrogen bonds. Some halogen-π interactions with either aromatic residues or peptide bonds, that accompany the interaction with the hinge region, may possibly enhance ligand selectivity. Interestingly, many of these halogen-π interactions are bifurcated. Geometrical preferences identify iodine as the strongest X-bond donor, less so bromine, while virtually no such preferences were observed for chlorine; and a backbone carbonyl as the strongest X-bond acceptor. The presence of a halogen atom in a ligand additionally affects the properties of proximal hydrogen bonds, which according to geometrical parameters get strengthened, when a nitrogen of a halogenated ligand acts as the hydrogen bond donor

Mechanisms of NMDA receptor inhibition by memantine and ketamine

NMDA receptors (NMDARs), a subfamily of ionotropic glutamate receptors, have unique biophysical properties including high permeability to Ca2+. Activation of NMDARs increases the concentration of intracellular Ca2+ that can activate a vast array of signaling pathways. NMDARs are necessary for many processes including synaptic plasticity, dendritic integration, and cell survival. Aberrant NMDAR activation is implicated in many central nervous system disorders including neurodegenerative disorders, neuronal loss following ischemia, and neuropsychiatric disorders. Hope that NMDARs may serve as useful therapeutic targets is bolstered by the clinical success of two NMDAR antagonists, memantine and ketamine. Memantine and ketamine act as open channel blockers of the NMDAR-associated ion channel, and exhibit similar IC50 values and kinetics. Memantine is approved for treatment of Alzheimer's disease and shows promise in treatments of Huntington's disease, and ischemia. Ketamine was initially approved for use as a general anesthetic, but has recently shown efficacy in treatment of depression and of pain. Notably, memantine is not effective in treatment of depression or pain. In addition, memantine is well tolerated, whereas ketamine induces psychotomimetic side effects. The basis for the divergent clinical profiles of memantine and ketamine is not clear. One recently-proposed hypothesis is that memantine and ketamine inhibit overlapping but distinct subpopulations of NMDARs. However, mechanisms underlying inhibition of distinct NMDAR subpopulations by memantine or by ketamine are not fully understood. We therefore examined and compared mechanisms of inhibition by memantine and by ketamine. We also describe a novel fast perfusion system optimized for brief synaptic-like glutamate applications to lifted cells. We found that: (1) inhibition by memantine and ketamine exhibit differential dependence on duration of receptor activation and on NMDAR subtype; (2) the dependence of memantine inhibition on duration of NMDAR activation results from stabilization of a Ca2+-dependent desensitized state; (3) the endogenous NMDAR open channel blocker Mg2+ slows the binding kinetics of both memantine and ketamine, and, unexpectedly, speeds recovery from memantine inhibition; (4) although inhibition by memantine was thought to be mediated by only the charged form of memantine, the uncharged form of memantine also binds to and inhibits NMDARs, and exhibits surprisingly slow unbinding kinetics

Light-Enabled Identification of the Neuronal Substrates of Alkylphenol Anesthetics

General anesthetics are a critical class of drugs in modern medicine; however, the precise mechanisms by which they cause unconsciousness and unwanted side effects are largely undefined. In order to understand pharmacologic mechanisms of anesthetic action, drug interactions with macromolecular substrates and the subsequent functional consequences must be characterized. Analogs of general anesthetics that function as photolabels have been developed to assist in the identification of molecular targets. One such photolabel, meta-azi-propofol (AziPm), is an analog of the clinically used alkylphenol anesthetic propofol. In this work, AziPm is employed in a variety of experiments that aim to identify molecular substrates of propofol. Two proteins identified as propofol targets are more thoroughly examined: (1) the sirtuin deacetylase SIRT2 and (2) the mitochondrial voltage-dependent anion channel (VDAC). The binding sites of propofol on these proteins, and the in vitro functional consequences of propofol binding, are determined. Also described are the molecular interactions of VDAC with a separate ligand, cholesterol, which shares a binding site with propofol. In addition to molecular studies, a novel in vivo photolabeling technique, called optoanesthesia, that utilizes AziPm is introduced, and the behavioral phenotype induced by optoanesthesia in Xenopus laevis tadpoles is characterized. Finally, optoanesthesia is demonstrated with other ligands, including a photoactive analog of an anthracene anesthetic, and mechanistic insight into the pharmacology of this anthracene is revealed