Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology.

The main objective of the present study was to find detergents that can maintain the functionality and stability of the Torpedo californica nicotinic acetylcholine receptor (Tc-nAChR). We examined the functionality, stability, and purity analysis of affinity-purified Tc-nAChR solubilized in detergents from the Cyclofos (CF) family [cyclofoscholine 4 (CF-4), cyclofoscholine 6 (CF-6), and cyclofloscholine 7 (CF-7)]. The functionality of the CF-Tc-nAChR-detergent complex (DC) was evaluated using the Two Electrode Voltage Clamp (TEVC) method. To assess stability, we used the florescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) methodology. We also performed a lipidomic analysis using Ultra-Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS) to evaluate the lipid composition of the CF-Tc-nAChR-DCs. The CF-4-Tc-nAChR-DC displayed a robust macroscopic current (- 200 ± 60 nA); however, the CF-6-Tc-nAChR-DC and CF-7-Tc-nAChR-DC displayed significant reductions in the macroscopic currents. The CF-6-Tc-nAChR and CF-4-Tc-nAChR displayed higher fractional florescence recovery. Addition of cholesterol produced a mild enhancement of the mobile fraction on the CF-6-Tc-nAChR. The lipidomic analysis revealed that the CF-7-Tc-nAChR-DC displayed substantial delipidation, consistent with the lack of stability and functional response of this complex. Although the CF-6-nAChR-DC complex retained the largest amount of lipids, it showed a loss of six lipid species [SM(d16:1/18:0); PC(18:2/14:1); PC(14:0/18:1); PC(16:0/18:1); PC(20:5/20:4), and PC(20:4/20:5)] that are present in the CF-4-nAChR-DC. Overall, the CF-4-nAChR displayed robust functionality, significant stability, and the best purity among the three CF detergents; therefore, CF-4 is a suitable candidate to prepare Tc-nAChR crystals for structural studies.

Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.

In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyte plasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30 day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30 day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex display a significant delay in the ACh-induce channel activation. In summary, these results suggest that the physical properties of the lipid analog detergents (headgroup and acyl chain length) are the most effective in maintaining both the stability and functionality of the nAChR in the detergent solubilized complex.

Internal dose assessment of (-)-18F-flubatine, comparing animal model datasets of mice and piglets with first-in-human results.

UNLABELLED: (-)-(18)F-flubatine is a promising tracer for neuroimaging of nicotinic acetylcholine receptors (nAChRs), subtype α4ß2, using PET. Radiation doses after intravenous administration of the tracer in mice and piglets were assessed to determine the organ doses (ODs) and the effective dose (ED) to humans. The results were compared with subsequent clinical investigations in human volunteers. METHODS: Twenty-seven female CD1 mice (weight ± SD, 28.2 ± 2.1 g) received intravenous injection of 0.75 ± 0.33 MBq of (-)-(18)F-flubatine. Up to 240 min after injection, 3 animals per time point were sacrificed and the organs harvested, weighed, and counted in a γ counter to determine mass and activity, respectively. Furthermore, whole-body PET scans of 5 female piglets (age ± SD, 44 ± 3 d; weight ± SD, 13.7 ± 1.7 kg) and 3 humans (2 men and 1 woman; age ± SD, 59.6 ± 3.9 y; weight ± SD, 74.3 ± 3.1 kg) were obtained up to 236 min (piglets) and 355 min (humans) after injection of 186.6 ± 7.4 and 353.7 ± 10.2 MBq of (-)-(18)F-flubatine, respectively, using a PET/CT scanner. The CT was used for delineation of the organs. Exponential curves were fitted to the time-activity-data, and time and mass scales were adapted to the human anatomy. The ODs were calculated using OLINDA/EXM (version 1.0); EDs were calculated with the tissue-weighting factors of ICRP103. RESULTS: After the injection of (-)-(18)F-flubatine, there were no adverse or clinically detectable pharmacologic effects in any of the subjects. The highest activities after injection were found in the kidneys, urinary bladder, and liver. The urinary bladder receives the highest OD in all investigated species, followed by the kidneys and the liver for animals and humans, respectively. On the basis of mouse, piglet, and human kinetic data, the projected human ED of (-)-(18)F-flubatine was estimated to be 12.5 µSv/MBq in mice, 14.7 ± 0.7 µSv/MBq in piglets, and 23.4 ± 0.4 µSv/MBq in humans. CONCLUSION: As has been demonstrated for other PET radiotracers, preclinical (i.e., animal-derived) dosimetry underestimates the ED to humans, in the current case of (-)-(18)F-flubatine by 34%-44%.

Assessment of homology templates and an anesthetic binding site within the γ-aminobutyric acid receptor.

BACKGROUND: Anesthetics mediate portions of their activity via modulation of the γ-aminobutyric acid receptor (GABAaR). Although its molecular structure remains unknown, significant progress has been made toward understanding its interactions with anesthetics via molecular modeling. METHODS: The structure of the torpedo acetylcholine receptor (nAChRα), the structures of the α4 and ß2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH-sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50s. RESULTS: Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between α and ß subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50. CONCLUSION: Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed a correlation of ligand docking scores with experimentally measured GABAaR potentiation.

Single-channel current through nicotinic receptor produced by closure of binding site C-loop.

We investigated the initial coupling of agonist binding to channel gating of the nicotinic acetylcholine receptor using targeted molecular-dynamics (TMD) simulation. After TMD simulation to accelerate closure of the C-loops at the agonist binding sites, the region of the pore that passes through the cell membrane expands. To determine whether the structural changes in the pore result in ion conduction, we used a coarse-grained ion conduction simulator, Biology Boltzmann transport Monte Carlo, and applied it to two structural frames taken before and after TMD simulation. The structural model before TMD simulation represents the channel in the proposed "resting" state, whereas the model after TMD simulation represents the channel in the proposed "active" state. Under external voltage biases, the channel in the "active" state was permeable to cations. Our simulated ion conductance approaches that obtained experimentally and recapitulates several functional properties characteristic of the nicotinic acetylcholine receptor. Thus, closure of the C-loop triggers a structural change in the channel sufficient to account for the open channel current. This approach of applying Biology Boltzmann transport Monte Carlo simulation can be used to further investigate the binding to gating transduction mechanism and the structural bases for ion selection and translocation.

Modulation of proton-induced current fluctuations in the human nicotinic acetylcholine receptor channel.

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that switches upon activation from a closed state to a full conducting state. We found that the mutation delta S268K, located at 12' position of the second transmembrane domain of the delta subunit of the human nAChR generates a long-lived intermediate conducting state, from which openings to a wild-type like conductance level occur on a submillisecond time scale. Aiming to understand the interplay between structural changes near the 12' position and channel gating, we investigated the influence of various parameters: different ligands (acetylcholine, choline and epibatidine), ligand concentrations, transmembrane voltages and both fetal and adult nAChRs. Since sojourns in the high conductance state are not fully resolved in time, spectral noise analysis was used as a complement to dwell time analysis to determine the gating rate constants. Open channel current fluctuations are described by a two-state Markov model. The characteristic time of the process is markedly influenced by the ligand and the receptor type, whereas the frequency of openings to the high conductance state increases with membrane hyperpolarization. Conductance changes are discussed with regard to reversible transfer reaction of single protons at the lysine 12' side chain.

An energy-efficient gating mechanism in the acetylcholine receptor channel suggested by molecular and Brownian dynamics.

Acetylcholine receptors mediate electrical signaling between nerve and muscle by opening and closing a transmembrane ion conductive pore. Molecular and Brownian dynamics simulations are used to shed light on the location and mechanism of the channel gate. Four separate 5 ns molecular dynamics simulations are carried out on the imaged structure of the channel, a hypothetical open structure with a slightly wider pore and a mutant structure in which a central ring of hydrophobic residues is replaced by polar groups. Water is found to partially evacuate the pore during molecular simulations of the imaged structure, whereas ions face a large energy barrier and do not conduct through the channel in Brownian dynamics simulations. The pore appears to be in a closed configuration despite containing an unobstructed pathway across the membrane as a series of hydrophobic residues in the center of the channel provide an unfavorable home to water and ions. When the channel is widened slightly, water floods into the channel and ions conduct at a rate comparable to the currents measured experimentally in open channels. The pore remains permeable to ions provided the extracellular end of the pore-lining helix is restrained near the putative open configuration to mimic the presence of the ligand binding domain. Replacing some of the hydrophobic residues with polar ones decreases the barrier for ion permeation but does not result in significant currents. The channel is posited to utilize an energy efficient gating mechanism in which only minor conformational changes of the hydrophobic region of the pore are required to create macroscopic changes in conductance.

Nicotinic cholinergic receptors in the rat cerebellum: multiple heteromeric subtypes.

Nicotinic receptors (nAChRs) in the cerebellum have been implicated in the pathology of autism spectrum disorders (Lee et al., 2002; Martin-Ruiz et al., 2004). The subtypes of nAChRs in the cerebellum are not known in any detail, except that, in addition to the homomeric alpha7 subtype, there appears to be one or more heteromeric subtypes consisting of combinations of alpha and beta subunits. To begin to better understand the potential roles of these heteromeric nAChRs in cerebellar circuitry and their potential as targets for nicotinic drugs, we investigated their subunit composition. Using subunit-selective antibodies in sequential immunoprecipitation assays, we detected six structurally distinct heteromeric nAChR populations in the rat cerebellum. Among these were several subtypes that have not been encountered previously, including alpha3alpha4beta2 and alpha3alpha4beta4 nAChRs. This diversity suggests that nAChRs play multiple roles in cerebellar physiology.

Theoretical studies of the M2 transmembrane segment of the glycine receptor: models of the open pore structure and current-voltage characteristics.

The pentameric glycine receptor (GlyR), a member of the nicotinicoid superfamily of ligand-gated ion channels, is an inhibitory Cl(-) channel that is gated by glycine. Using recently published NMR data of the second transmembrane segment (M2) of the human alpha1 GlyR, structural models of pentameric assemblies embedded in a lipid bilayer were constructed using a combination of experimentally determined constraints coupled with all-atom energy minimization. Based on this structure of the pentameric M2 "pore", Brownian dynamics simulations of ion permeation through this putative conducting open state of the channel were carried out. Simulated I-V curves were in good agreement with published experimental current-voltage curves and the anion/cation permeability ratio, suggesting that our open-state model may be representative of the conducting channel of the full-length receptor. These studies also predicted regions of chloride occupancy and suggested residues critical to anion permeation. Calculations of the conductance of the cation-selective mutant A251E channel are also consistent with experimental data. In addition, both rotation and untilting of the pore helices of our model were found to be broadly consistent with closing of the channel, albeit at distinct regions that may reflect alternate gates of the receptor.

Kinked-helices model of the nicotinic acetylcholine receptor ion channel and its complexes with blockers: simulation by the Monte Carlo minimization method.

A model of the nicotinic acetylcholine receptor ion channel was elaborated based on the data from electron microscopy, affinity labeling, cysteine scanning, mutagenesis studies, and channel blockade. A restrained Monte Carlo minimization method was used for the calculations. Five identical M2 segments (the sequence EKMTLSISVL10LALTVFLLVI20V) were arranged in five-helix bundles with various geometrical profiles of the pore. For each bundle, energy profiles for chlorpromazine, QX-222, pentamethonium, and other blocking drugs pulled through the pore were calculated. An optimal model obtained allows all of the blockers free access to the pore, but retards them at the rings of residues known to contribute to the corresponding binding sites. In this model, M2 helices are necessarily kinked. They come into contact with each other at the cytoplasmic end but diverge at the synaptic end, where N-termini of M1 segments may contribute to the pore. The kinks disengage alpha-helical H-bonds between Ala12 and Ser8. The uncoupled lone electron pairs of Ser8 carbonyl oxygens protrude into the pore, forming a hydrophilic ring that may be important for the permeation of cations. A split network of H-bonds provides a flexibility to the chains Val9-Ala12, the numerous conformations of which form only two or three intrasegment H-bonds. The cross-ectional dimensions of the interface between the flexible chains vary essentially at the level of Leu11. We suggest that conformational transitions in the chains Val9-Ala12 are responsible for the channel gating, whereas rotations of more stable alpha-helical parts of M2 segments may be necessary to transfer the channel in the desensitized state.

Inversion of Markov processes to determine rate constants from single-channel data.

The determination of rate constants from single-channel data can be very difficult, in part because the single-channel lifetime distributions commonly analyzed by experimenters often have a complicated mathematical relation to the channel gating mechanism. The standard treatment of channel gating as a Markov process leads to the prediction that lifetime distributions are exponential functions. As the number of states of a channel gating scheme increases, the number of exponential terms in the lifetime distribution increases, and the weights and decay constants of the lifetime distributions become progressively more complicated functions of the underlying rate constants. In the present study a mathematical strategy for inverting these functions is introduced in order to determine rate constants from single-channel lifetime distributions. This inversion is easy for channel gating schemes with two or fewer states of a given conductance, so the present study focuses on schemes with more states. The procedure is to derive explicit equations relating the parameters of the lifetime distribution to the rate constants of the scheme. Such equations can be derived using the equality between symmetric functions of eigenvalues of a matrix and sums over principle minors, as well as expressions for the moments, derivatives, and weights of a lifetime distribution. The rate constants are then obtained as roots to this system of equations. For a gating scheme with three sequential closed states and a single gateway state, exact analytical expressions were found for each rate constant in terms of the parameters of the three-exponential closed-time distribution. For several other gating schemes, systems of equations were found that could be solved numerically to obtain the rate constants. Lifetime distributions were shown to specify a unique set of real rate constants in sequential gating schemes with up to five closed or five open states. For kinetic schemes with multiple gating pathways, the analysis of simulated data revealed multiple solutions. These multiple solutions could be distinguished by examining two-dimensional probability density functions. The utility of the methods introduced here are demonstrated by analyzing published data on nicotinic acetylcholine receptors, GABA(A) receptors, and NMDA receptors.