Central Ion Channel Research Articles

Alternative splicing of the Anopheles gambiae nicotinic acetylcholine receptor, Agamαβ9, generates both alpha and beta subunits

Nicotinic acetylcholine receptors (nAChRs) are the members of the cys-loop ligand-gated ion channel superfamily and are formed by five subunits arranged around a central ion channel. Each subunit is encoded by a separate gene and is classified as either alpha or non-alpha depending on the presence or absence, respectively, of two adjacent cysteine residues which are important for acetylcholine binding. Here, we report for the first time a single nAChR gene encoding both alpha and non-alpha subunits. Specifically, alternative splicing of the Anopheles gambiae nAChR subunit, previously called Agamalpha9 and renamed here Agamalphabeta9, generates two variants, one possessing the two cysteines (denoted Agamalphabeta9(alpha)) and the other lacking the cysteine doublet (Agamalphabeta9(beta)). Attempts to heterologously express functional nAChRs consisting of the Agamalphabeta9 splice variants in Xenopus laevis oocytes were unsuccessful. Our findings further characterise a potential target to control the malaria mosquito as well as provide insights into the diversification of nAChRs.

Biophysical And Pharmacological Profiling Of Multiple NaV Subtypes On QPatch HT

The voltage dependent sodium channel is responsible for the upstroke and directed propagation of action potentials in nerve and muscle cells, and is therefore a central ion channel in excitable tissues. The implication of voltage gated sodium channels in pain mediation, and diseases such as epilepsy and cardiac arrythmia has made them very important targets for drug discovery. Nine functional mammalian isoforms have been discovered so far with different functional and pharmacological properties. In our study, eight subtypes of the voltage gated sodium channel were tested in parallel on the automated patch clamp system QPatch HT. The new clone screening feature developed for QPatch 16 and QPatch HT allows running up to eight different cell lines (clones or subtypes) at the same time, thus ensuring that the exact same conditions (temperature, Ringer's, pH etc.) are applied for each of the cell lines tested. Nav1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 and 1.8 were tested together in a range of experiments on QPatch HT. All but Nav1.2 and Nav1.8 were co-expressed with the β1 subunit. Three types of experiments were designed to explore 1) TTX sensitivity, 2) IV-relationship for activation and inactivation, and 3) recovery from inactivation, for the entire panel of Nav channel subtypes in a single experiment. It was shown that QPatch experiments using the cell clone screening feature together with the QPatch Assay Software data analysis package, enables the experimenter to obtain IC50 values for TTX, IV-relationships and time constants for recovery from inactivation which are very similar to manual patch clamp data, for all Nav subtypes, thus successfully distinguishing one subtype from another.

Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors

Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors

Structural and Functional Changes Induced in the Nicotinic Acetylcholine Receptor by Membrane Phospholipids

Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernández et al., 1993; Fernández-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of cation translocation and Fourier transform-infrared (FT-IR) spectroscopy studies have been used to illustrate the lipid dependence of both AChR function and AChR secondary structure, respectively.

Ligands of neuronal nicotinic acetylcholine receptor (nAChR): inferences from the Hansch and 3-D quantitative structure-activity relationship (QSAR) Models.

Neuronal acetylcholine ion channel receptors (nAChRs), that exist in several subtypes resulting from a different organisation of various subunits around the central ion channel, are involved in a variety of functions and disorders of the central nervous system. There is evidence to implicate a deficit of nAChRs in the symptomatology of severe neurologic pathologies, such as Alzheimer s and Parkinson s diseases. Reliable three-dimensional structures of nAChRs are not available yet, and this hampers adopting structure-based approaches in designing new ligands. Also pharmacophore models are not reliable enough to be used in ligand-based approaches to drug design and little structure-activity work has been reported so far. This paper deals with structure-activity relationships of a wide series of nicotinic ligands. It provides results from a study of the quantitative structure activity relationships (QSARs) based on literature data of about 270 nicotinic agonists, belonging to various chemical classes. The QSAR study was carried out by using either a classical Hansch approach or a Comparative Molecular Field Analysis (CoMFA). Within each congeneric series, Hansch-type equations revealed detrimental steric effects as the factors mainly modulating the receptor affinity, whereas CoMFA allowed us to merge progressively models obtained for each class of congeners into a more general one that showed good cross-validation statistics. The CoMFA coefficient isocontour maps illustrated, at the 3-D level, the most relevant interactions responsible for a high receptor affinity, whereas the robustness of the global three-dimensional QSAR/CoMFA (n = 206, q(2) = 0.749, r(2) = 0.847, s= 0.600) model was supported by the high value of the prediction statistics (r(2)pred = 0.961) and confirmed by the satisfactory predictions of the affinity data of an external set of 18 recently published ligands with chemical structures even quite diverse from those included in the training set.

Le récepteur périphérique de lˈacétylcholine : physiologie, physiopathologie appliquée aux maladies neuromusculaires et à la curarisation

The nicotinic acetylcholine receptor of the motor endplate is composed of five subunits (2α, 1β, 1δ, 1ϵ and 1γ) arranged around a central ion channel. The binding of one ACh molecule to each of the two α subunits opens the channel and depolarizes the motor endplate. Molecules of ACh are released from the nerve terminals as quanta that give rise to miniature endplate potentials (MEPPS). Summation of MEPPS produces a larger endplate potential that spreads to the surrounding sarcolemma and initiates muscle contraction. There are several types of neuromuscular blocks defined by their site, pre- or postsynaptic, and by their mechanism: deficiency or excess of depolarization. The basic abnormality in autoimmune myasthenia is a decrease in the number of AChR, resulting in endplate potentials of diminished amplitude. Most congenital myasthenic syndromes are postsynaptic and caused by mutations in AChR subunit genes that result in deficiency or altered kinetics of the AChR. Two classes of curare are used in anesthesiology, either depolarizing (agonists of the AChR) or non-depolarizing (competitive antagonists of the AChR). Preexisting pathology of neuromuscular transmission and the use of drugs that may affect the state of the AChR must be considered when planning curarisation for anesthesia or reanimation.

Structural Dynamics and Cation Interactions of DNA Quadruplex Molecules Containing Mixed Guanine/Cytosine Quartets Revealed by Large-Scale MD Simulations

Large-scale molecular dynamics (MD) simulations have been utilized to study G-DNA quadruplex molecules containing mixed GCGC and all-guanine GGGG quartet layers. Incorporation of mixed GCGC quartets into G-DNA stems substantially enhances their sequence variability. The mixed quadruplexes form rigid assemblies that require integral monovalent cations for their stabilization. The interaction of cations with the all-guanine quartets is the leading contribution for the stability of the four-stranded assemblies, while the mixed quartets are rather tolerated within the structure. The simulations predict that two cations are preferred to stabilize a four-layer quadruplex stem composed of two GCGC and two all-guanine quartets. The distribution of cations in the structure is influenced by the position of the GCGC quartets within the quadruplex, the presence and arrangement of thymidine loops connecting the guanine/cytosine stretches forming the stems, and the cation type present (Na(+) or K(+)). The simulations identify multiple nanosecond-scale stable arrangements of the thymidine loops present in the molecules investigated. In these thymidine loops, several structured pockets are identified capable of temporarily coordinating cations. However, no stable association of cations to a loop has been observed. The simulations reveal several paths through the thymidine loop regions that can be followed by the cations when exchanging between the central ion channel in the quadruplex stem and the surrounding solvent. We have carried out 20 independent simulations while the length of simulations reaches a total of 90 ns, rendering this study one of the most extensive MD investigations carried out on nucleic acids so far. The trajectories provide a largely converged characterization of the structural dynamics of these four-stranded G-DNA molecules.

Photoaffinity labeling the torpedo nicotinic acetylcholine receptor with [(3)H]tetracaine, a nondesensitizing noncompetitive antagonist.

Tetracaine (N,N-dimethylaminoethyl-4-butylaminobenzoate) and related N,N-dialkylaminoethyl substituted benzoic acid esters have been used to characterize the high-affinity binding site for aromatic amine noncompetitive antagonists in the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Tetracaine binds at equilibrium to a single site with a K(eq) value of 0.5 microM in the absence of agonist or presence of alpha-bungarotoxin and with a K(eq) value of 30 microM in the presence of agonist (i.e., for nAChR in the desensitized state). Preferential binding to nAChR in the absence of agonist is also seen for N,N-DEAE and N,N-diethylaminopropyl esters, both binding with 10-fold higher affinity in the absence of agonist than in the presence, and for the 4-ethoxybenzoic acid ester of N, N-diethylaminoethanol, but not for the 4-amino benzoate ester (procaine). Irradiation at 302 nm of nAChR-rich membranes equilibrated with [(3)H]tetracaine resulted in covalent incorporation with similar efficiency into nAChR alpha, beta, gamma, and delta subunits. The pharmacological specificity of nAChR subunit photolabeling as well as its dependence on [(3)H]tetracaine concentration establish that the observed photolabeling is at the high-affinity [(3)H]tetracaine-binding site. Within alpha subunit, >/=95% of specific photolabeling was contained within a 20-kilodalton proteolytic fragment beginning at Ser(173) that contains the M1 to M3 hydrophobic segments. With all four subunits contributing to [(3)H]tetracaine site, the site in the closed channel state of the nAChR is most likely within the central ion channel domain.

Nanosecond Molecular Dynamics Simulations of Parallel and Antiparallel Guanine Quadruplex DNA Molecules

Molecular dynamics (MD) simulations are presented of four-stranded G-DNA molecules formed by the sequences d(G4) and d(G4T4G4). Starting coordinates are based on high-resolution X-ray structures or NMR data. Simulations of the all-parallel d(G4) quadruplex with sodium cations in the central ion channel yield exceptionally stable trajectories on the nanosecond scale. Simulations without cations in the channel show destabilization of the G-DNA structure, underscoring the central role of these ions for the structural integrity of the molecule. Further simulations reveal that the cation-stabilized d(G)4 stem can adopt an alternative very stable conformation involving a guanine base triad. Simulations of d(G4T4G4) quadruplexes indicate a similar rigidity and stability of the antiparallel guanine stem as for the parallel d(G4) conformer. The simulations further demonstrate significant geometrical plasticity of the thymine residues arranged in four-nucleotide loops, including loop geometries capable of coordinat...

Asymmetric Contribution of the Conserved Disulfide Loop to Subunit Oligomerization and Assembly of the Nicotinic Acetylcholine Receptor

The acetylcholine receptor (AChR) at the motor synapse is a pentamer of homologous subunits with the composition alpha2betagammadelta. Owing to the circular arrangement of subunits that forms a central ion channel, each subunit interface contains contributions from opposite faces of each subunit, designated + and -. Common to all subunits of the AChR and members of its superfamily is a disulfide loop formed between cysteines 128 and 142 of the major extracellular domain. To gain insight into the structural contribution of the disulfide loop and its possible location, we mutated the invariant proline at position 136 to glycine (P136G) and examined subunit assembly. When introduced into any AChR subunit, P136G disrupted assembly by affecting the - face of the subunit, suggesting equivalent positioning of the loop in each subunit and localization to the - face. Also, the contribution of the loop in the overall assembly process differed for each subunit. In the beta and gamma subunits, P136G prevented assembly of higher order heteroligomers, whereas in the alpha and delta subunits, P136G prevented transport of assembled pentamers to the cell surface. The results demonstrate asymmetry in the contribution of the disulfide loop to formation of subunit interfaces, and that the loop in each subunit contributes at different stages of assembly.

Quaternary structure of the native GABAA receptor determined by electron microscopic image analysis.

In the transmitter-gated ion channel class of receptors, the members of which are all believed to be heterooligomers, the number and arrangement of the subunits are only known with any certainty for the nicotinic acetylcholine receptor from Torpedo electric fish. That receptor has been shown to possess a pentameric rosette structure, with five homologous subunits (alpha 2, beta gamma delta) arranged to enclose the central ion channel. The data were obtained by electron image analysis of two-dimensional receptor arrays, which form as a consequence of that receptor's exceptionally high abundance in the Torpedo membranes and are therefore not attainable for other receptors. We have applied another direct approach to determine the quaternary structure of native ionotropic GABA receptors. We have purified those receptors from porcine brain cortex and analysed the rotational symmetry of isolated receptors visualized by electron microscopy. The results show the receptor to have a pentameric structure with a central water-filled pore, which can now be said to be characteristic of the entire superfamily.

Cholinergic receptors and anesthesia

Postsynaptic chemically controlled ion channels are a possible location for the action of anaesthetics. The nicotinic acetylcholine receptor (AcChoR) is a member of a superfamily of chemically controlled ion channels, which have a large number of structural similarities in common. This receptor provides an excellent model for studying the effects of anaesthetics on excitable membrane proteins since it is well characterised and available in large quantities. The AcChoR consists of five subunits that surround a central ion channel. Anaesthetics can inhibit the function of the AcChoR by several different mechanisms: 1. By directly blocking the ion channel, 2. by an allosteric inhibition of the ion channel, 3. by changing the properties of the cell membrane surrounding the receptor. In high concentrations, anaesthetics stabilize the receptor in a desensitized state. Investigation of the mode of action of anaesthetics on the AcChoR provides important information as to the mechanisms of anaesthesia.

Three-dimensional structure of the acetylcholine receptor by cryoelectron microscopy and helical image reconstruction.

Long tubular vesicles have been grown from isolated Torpedo postsynaptic membranes, in which the receptors are arranged helically on the vesicle surface. The structures of these tubes have been analyzed by cryoelectron microscopy of specimens embedded in thin films of ice, combined with helical image reconstruction. Complete data sets from tubes belonging to several helical families have been obtained to a resolution of 17 A in all directions. Confirming a preliminary study (Toyoshima, C., and N. Unwin. 1988. Nature (Lond.). 336:247-250), the central ion channel has an almost constant diameter throughout the molecule except for the portion extending through the hydrophobic part of the lipid bilayer, where the pore is too small to be resolved. However, the density on the pseudo fivefold axis running through the pore is consistently highest in the cytoplasmic half of the bilayer, suggesting the gate is located in that region. The path followed by each subunit has been identified throughout the length of the receptor. The two alpha subunits follow equivalent paths. All subunits have similar features which change in character at the same level relative to the membrane.

Orientation of cobra alpha-toxin on the nicotinic acetylcholine receptor. Fluorescence studies.

Four flourescein isothiocyanate (FITC) derivatives of Naja naja siamemsis 3 neurotoxin (alpha-toxin), labeled at the epsilon-amino groups of Lys-23, Lys-35, Lys-49, or Lys-69, and a tetramethylrhodamine isothiocyanate (TRITC) derivative, labeled at epsilon-amino group of Lys-23, were prepared and used to analyze the orientation of cobra alpha-toxin on the nicotinic acetylcholine receptor (AcChR) relative to both the plane of the membrane and the central ion channel. Fluorescence-quenching studies of the AcChR-bound FITC derivatives indicated significant solute accessibility to each site of labeling and suggested that none of the sites of FITC labeling is included in the binding surface of the alpha-toxin. Labeling of Lys-23 with TRITC did not affect the affinity of the alpha-toxin toward the AcChR and confirmed, contrary to some previous reports, a minimal role of Lys-23 in the binding surface of the alpha-toxin. Measurements of energy transfer between the lipid-membrane surface and the sites of labeling on receptor-bound alpha-toxin derivatives show that the relative distances of closest approach between the surface of the lipid membrane domain and the sites of labeling are in the order Lys-23 less than or equal to Lys-49 less than Lys-35 less than or equal to Lys-69. Energy transfer between AcChR tryptophans and the sites of labeling of bound derivatives was about 50% greater to Lys-49 than to Lys-23, Lys-35, or Lys-69, suggesting that Lys-49 is closer to receptor tryptophans and to the center of the extracellular domain of the receptor than Lys-23, Lys-35, or Lys-69. Combined with previous observations that the tip of the central loop of the alpha-toxin directly interacts with the AcChR, the above results suggest a model of the approximate orientation of the snake neurotoxins on the receptor. This model shows the tip of the central loop of the toxin directly interacting with the receptor surface and the major axis of the neurotoxin tilting from a perpendicular projection from the membrane. The surface of the alpha-toxin that includes Lys-23 projects away from the central ion channel and the surface that includes Lys-35 and Lys-69 faces the ion channel.

Arrangement of the acetylcholine receptor subunits in the resting and desensitized states, determined by cryoelectron microscopy of crystallized Torpedo postsynaptic membranes.

Two conformational states of the nicotinic acetylcholine receptor have been investigated by cryoelectron microscopy of flattened vesicular crystals grown from Torpedo marmorata postsynaptic membranes. One was obtained from the vesicles without acetylcholine present, and is presumed to correspond to the native, or resting state; the other was obtained from the vesicles after exposure to 100 microM to 5 mM carbamylcholine (an acetylcholine analogue) and is presumed to correspond to a desensitized state. Both conformations were determined in three-dimensions to a resolution of 18 A, sufficient to reveal the configurations of the five subunits around the central ion channel over most of their length. The subunits of either structure have a similar appearance, consistent with their amino acid homology. They are each aligned almost parallel to the axis of the receptor, conferring a high degree of pentagonal symmetry to the bilayer portion and a contiguous region on the synaptic side. Their external surfaces form a pronounced ridge in the bilayer portion, which broadens toward the synaptic end. Comparison of features in the two three-dimensional maps reveals that carbamylcholine induces a quaternary rearrangement, involving predominantly the delta-subunit. The densities corresponding to this subunit are tilted by approximately 10 degrees tangential to the axis of the receptor over a large fraction of its length, and become misaligned relative to the densities corresponding to the other four subunits. The gamma-subunit is also affected, being displaced slightly away from the axis of the receptor. The alpha- and beta-subunits may be affected on a more localized scale. The overall changes are most pronounced in the synaptic region, where the ligand-binding site is located, and in the cytoplasmic region, which may be closer to the gate of the channel. The physiological process of desensitization appears to be associated with a structural transition in which the subunits switch to a less symmetrical configuration.

Topological mapping of acetylcholine receptor: evidence for a model with five transmembrane segments and a cytoplasmic COOH-terminal peptide.

Antibodies were raised against two synthetic peptides whose sequences correspond respectively to the COOH-terminal end (residues 501-516) of the protein encoded by the gene for the delta chain and to a proposed cytoplasmic region (residues 350-358) of the beta chain of the acetylcholine receptor from Torpedo californica. Binding of the COOH-terminal antibody to the acetylcholine receptor in intact, receptor-rich vesicles was tested by radioimmunoassay and by precipitation with immobilized protein A. In both cases, binding was detected only after treatment of the vesicles with detergent, suggesting that the segment of the receptor that is recognized by this antibody is on the cytoplasmic side of the membrane. Electron microscopy of tissue from Torpedo electric organ labeled with colloidal gold-conjugated second antibodies established that both anti-receptor antibodies bind to the cytoplasmic surface of the postsynaptic membrane. These experiments give ultrastructural evidence that the COOH-terminal segment of the delta chain as well as residues 350-358 of the beta chain are on the cytoplasmic surface. They strongly support a model in which each of the receptor subunits crosses the membrane five times in which one transmembrane segment of each chain contributes to the formation of a central ion channel.