Understand spiciness: mechanism of TRPV1 channel activation by capsaicin.

Capsaicin in chili peppers bestows the sensation of spiciness. Since the discovery of its receptor, transient receptor potential vanilloid 1 (TRPV1) ion channel, how capsaicin activates this channel has been under extensive investigation using a variety of experimental techniques including mutagenesis, patch-clamp recording, crystallography, cryo-electron microscopy, computational docking and molecular dynamic simulation. A framework of how capsaicin binds and activates TRPV1 has started to merge: capsaicin binds to a pocket formed by the channel's transmembrane segments, where it takes a "tail-up, head-down" configuration. Binding is mediated by both hydrogen bonds and van der Waals interactions. Upon binding, capsaicin stabilizes the open state of TRPV1 by "pull-and-contact" with the S4-S5 linker. Understanding the ligand-host interaction will greatly facilitate pharmaceutical efforts to develop novel analgesics targeting TRPV1

Crosslinking constraints and computational models as complementary tools in modeling the extracellular domain of the glycine receptor

The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel superfamily, is the major inhibitory neurotransmitter-gated receptor in the spinal cord and brainstem. In these receptors, the extracellular domain binds agonists, antagonists and various other modulatory ligands that act allosterically to modulate receptor function. The structures of homologous receptors and binding proteins provide templates for modeling of the ligand-binding domain of GlyR, but limitations in sequence homology and structure resolution impact on modeling studies. The determination of distance constraints via chemical crosslinking studies coupled with mass spectrometry can provide additional structural information to aid in model refinement, however it is critical to be able to distinguish between intra- and inter-subunit constraints. In this report we model the structure of GlyBP, a structural and functional homolog of the extracellular domain of human homomeric α1 GlyR. We then show that intra- and intersubunit Lys-Lys crosslinks in trypsinized samples of purified monomeric and oligomeric protein bands from SDS-polyacrylamide gels may be identified and differentiated by MALDI-TOF MS studies of limited resolution. Thus, broadly available MS platforms are capable of providing distance constraints that may be utilized in characterizing large complexes that may be less amenable to NMR and crystallographic studies. Systematic studies of state-dependent chemical crosslinking and mass spectrometric identification of crosslinked sites has the potential to complement computational modeling efforts by providing constraints that can validate and refine allosteric models. © 2014 Liu et al

A comparison of glycine-and ivermectin-mediated conformational changes in the glycine receptor ligand-binding domain

Glycine receptor chloride channels are Cys-loop receptor proteins that isomerize between a low affinity closed state and a high affinity ion-conducting state. There is currently much interest in understanding the mechanisms that link affinity changes with conductance changes. This essentially involves an agonist binding in the glycine receptor ligand-binding site initiating local conformational changes that propagate in a wave towards the channel gate. However, it has proved difficult to convincingly distinguish those agonist-induced domain movements that are critical for triggering activation from those that are simply local deformations to accommodate ligands in the site. We employed voltage-clamp fluorometry to compare conformational changes in the ligand-binding site in response to activation by glycine, which binds locally, and ivermectin, which binds in the transmembrane domain. We reasoned that ivermectin-mediated activation should initiate a conformational wave that propagates from the pore-lining domain towards the ligand-binding domain, eliciting conformational changes in those extracellular domains that are allosterically linked to the gate. We found that ivermectin indeed elicited conformational changes in ligand-binding domain loops C, D and F. This implies that conformational changes in these domains are important for activation. This result also provides a mechanism to explain how ivermectin potentiates glycine-induced channel activation

Opposing modulation of Cx26 gap junctions and hemichannels by CO2

Cx26 hemichannels open in response to moderate elevations of CO2 (PCO2 55 mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated PCO2 of 55 mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2. Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 5.6 nS, 95% confidence interval, 3.2 to 11.9 nS) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification, but using 30 mM propionate we found that acidification in the absence of a change in PCO2 caused a median reduction in the gap junction conductance of 5.3 nS (2.8 to 8.3 nS). This effect of propionate was unaffected by the K125R mutation (median reduction 7.7 nS, 4.1 to 11.0 nS). pH-dependent and CO2-dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the Keratitis Ichthyosis Deafness syndrome (N14K, A40V and A88V) also abolished the CO2-dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound, is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction

Estudio de la interacción lípido-proteína sobre la función y la organización del receptor de acetilcolina nicotínico en membranas y en sistemas modelos simples

En esta Tesis doctoral se profundizó en el estudio de la interacción lípido-receptor de acetilcolina nicotínico (AChR) en dos aspectos: por un lado, el mecanismo de inhibición de ácidos grasos libres (AGLs), antagonistas no competitivos del AChR, y por el otro, la ubicación del AChR en dominios líquido-ordenados (Lo) condicionada por dos características particulares de la membrana. Con la finalidad de dilucidar el mecanismo de antagonismo de los AGLs sobre el AChR, se utilizaron AGLs con un doble enlace único en diferentes posiciones de una cadena acílica de 18 átomos de carbono. Estudios funcionales realizados con la técnica de patch-clamp han mostrado que solo el cis-6-18:1 y el cis-9-18:1 reducen la duración del estado de canal abierto del receptor, sugiriendo, por lo tanto, un mecanismo de bloqueo alostérico del canal. Mediante espectroscopía de fluorescencia se comprobó que todos los AGLs se ubican en la interfase lípido-AChR, quedando el cis-6-18:1 restringido a los sitios denominados sitios anulares, mientras que el resto de los AGLs ocupa también sitios no-anulares. Por otro lado, estudios de polarización de fluorescencia mostraron que el AGLs cis-9-18:1 es el que ocasiona el mayor desorden en la membrana. Se comprobó i) que todos los cis-AGLs generan cambios conformacionales del AChR a nivel transmembrana, ii) que los cis-9-18:1, cis-11-18:1 y cis-13-18:1 perturban al AChR en su estado de reposo e iii) que los cis-6-18:1 y cis-9-18:1 son los que causan una mayor perturbación del estado desensibilizado. De esta manera, la posición e isomería del ángulo de torsión de los AGLs insaturados serían un factor clave en el bloqueo del AChR, sugiriendo entonces que los AGLs con un único doble enlace y ubicados superficialmente en la membrana inhiben en forma directa la función del AChR, posiblemente al perturbar una secuencia aminoacídica transmembrana involucrada en los cambios alostéricos necesarios para la apertura del canal iónico. Se postula que en la membrana plasmática el AChR se encuentra en dominios lipídicos denominados ?balsas? (?rafts?). Sin embargo, el AChR no muestra preferencia por dominios Lo en sistemas modelo compuestos por esfingomielina (SM), colesterol (Col) y POPC (1:1:1), pero sí lo hace un segmento transmembrana (γM4) que exhibe mayor contacto con los lípidos. Es decir, su distribución no dependería exclusivamente de sus propiedades intrínsecas sino también de señales extrínsecas a la proteína. En este trabajo de Tesis se estudió la posible partición diferencial del AChR en los dominios Lo en dos sistemas modelo diferentes en función de: a) la presencia de diferentes especies puras de SM en la membrana, y b) la existencia de asimetría transbicapa en el sistema modelo, mediante el agregado de SM de cerebro (bSM) en la hemicapa externa. Tanto la existencia de asimetría como la presencia de 16:0-SM o 18:0-SM, en comparación con las bSM y 24:1-SM, producen una partición preferencial del AChR en los dominios Lo. De este modo, la localización del AChR en estos dominios depende no solo de sus propiedades sino también de las características propias de la membrana en la que se encuentra. Entender la interacción lípido-AChR es de gran importancia para determinar tratamientos que puedan mejorar o inhibir la función del AChR y tratar enfermedades que lo involucren.In this Ph. D. thesis the understanding of the lipid-niconitic acetylcholine receptor (AChR) interaction was furthered in two aspects, namely the inhibition mechanism of free fatty acids (FFA), non-competitive AChR antagonists, and AChR location in liquid-ordered (Lo) domains conditioned by two membrane characteristics. To elucidate FFA’s antagonism mechanism, FFA with only one double bond in different positions of an 18-carbon acyl chain were tested on AChR. Patch-clamp functional studies showed that only cis-6-18:1 and cis-9-18:1 reduce the duration of the AChR open state, thus suggesting an allosteric blocking mechanism. Fluorescence spectroscopy measurements demonstrated that all FFA locate in the AChR-lipid interfase, with cis-6-18:1 restricted to anular sites, while the rest of the FFA tested also ocupy non-anular sites. Fluorescence polarization studies showed that cis-9-18:1 causes the highest membrane disorder of all FFA tested. It was determined that i) all cis-FFA generate AChR conformational changes at a transmembrane level, ii) only cis-9-18:1, cis-11-18:1 and cis-13-18:1 disturb AChR resting state and iii) cis-6-18:1 and cis-9-18:1 are the ones that cause the highest disturbance of the desensitized state. Thus, the position and isomerism of the torsion angle of unsaturated FFAs are probably a key factor in terms of AChR blockage, possibly by perturbing a transmembrane aminoacidic sequence involved in the allosteric changes necessary for ion channel gating. In the plasma membrane, AChR is postulated to be located in lipid domains known as rafts. However, AChR shows no preference for Lo domains in model systems – composed of sphingomyelin (SM), cholesterol (Chol) and POPC (1:1:1) –, but a transmembrane segment (γM4) that in closest contact with lipids does have preference for them. This means that AChR distribution seems not to exclusively depend on its intrinsic properties but on signals external to the protein. In this Ph. D. thesis a posible differential AChR partitioning in Lo domains was studied in two model systems as a function of a) the presence of different pure SM species in the membrane and b) the existence of transbilayer asymmetry in the model system, by the addition of brain SM (bSM) in the external hemilayer. Both asymmetry and the presence of either 16:0-SM or 18:0-SM, in comparison with bSM or 24:1-SM, lead to an AChR preferential partitioning in Lo domains. AChR location in these domains depends not only on its properties but also on the characteristics of the membrane in which the ion channel is immersed. Understanding lipid-AChR interaction is of great importance to determine treatments that can either improve or inhibit AChR function and, this, in turn, will help determining the treatment of diseases in which AChR is involved.Fil: Perillo, Vanesa Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Autor

Decrypting the Sequence of Structural Events during the Gating Transition of Pentameric Ligand-Gated Ion Channels Based on an Interpolated Elastic Network Model

Despite many experimental and computational studies of the gating transition of pentameric ligand-gated ion channels (pLGICs), the structural basis of how ligand binding couples to channel gating remains unknown. By using a newly developed interpolated elastic network model (iENM), we have attempted to compute a likely transition pathway from the closed- to the open-channel conformation of pLGICs as captured by the crystal structures of two prokaryotic pLGICs. The iENM pathway predicts a sequence of structural events that begins at the ligand-binding loops and is followed by the displacements of two key loops (loop 2 and loop 7) at the interface between the extracellular and transmembrane domain, the tilting/bending of the pore-lining M2 helix, and subsequent movements of M4, M3 and M1 helices in the transmembrane domain. The predicted order of structural events is in broad agreement with the W-value analysis of a subunit of nicotinic acetylcholine receptor mutants, which supports a conserved core mechanism for ligand-gated channel opening in pLGICs. Further perturbation analysis has supported the critical role of certain intra-subunit and inter-subunit interactions in dictating the above sequence of events