Desensitized State Research Articles

Enhancement of α9α10 Nicotinic Acetylcholine Receptor Desensitization by Nimodipine

Entry of calcium ions through nicotinic acetylcholine receptors hyperpolarizes outer hair cells (OHCs) by activating colocalized SK potassium channels. This process, termed efferent inhibition, plays a central role in the protective reflex by suppressing the amplification of incoming sound. OHC nicotinic receptors are composed of α9 and α10 subunits that are activated by acetylcholine (ACh) release from efferent neurons originating within the medial olivocochlear nucleus. Fast application of ACh to GH4C1 cells transiently transfected with plasmids encoding mouse α9 and α10 subunits, produced an inward current that displayed a concentration-dependent amplitude, with an EC50 of 41 ± 5.4 μM and Hill slope (n H) of 0.89 ± 0.1 (n=5). This current was antagonized by nicotine, displaying a KB of 9.0 ± 1.0 μM (n=8), consistent with the ACh-mediated current arising from activation of heteromeric α9α10 receptor channels. Inward ACh currents in the presence of the dihydropyridine antagonist nimodipine (10 μM) were reduced in amplitude and displayed faster decay kinetics. Exponential decay rates (τ) increased from 647.5 ± 66.0 ms (n=6) in control, to 160.6 ± 25.8 ms (n=6) in the presence of nimodipine (10 μM) (p 0.05), suggesting that the increased decay rate of ACh currents did not result from a decrease in agonist binding affinity. using a double-pulse protocol, ACh (300 μM)-mediated responses recovered from desensitization with an exponential time-course (τ = 3.81 ± 0.67 s, n=3). Recovery was slowed in the presence of nimodipine (τ = 8.88 ± 2.54 s; n=3), indicating that nimodipine stabilizes the desensitization state of the α9α10 receptor and suggests that these receptors can be modulated to fine-tune efferent inhibition.

ELIC Channel Conformational Changes Detected by 19F NMR

Conformations of the pentameric ligand-gated ion channel (pLGIC) from Erwinia chysanthemi (ELIC)change at different functional states. Agonist binding to ELIC elicits channel opening for seconds or minutes before the channel goes to a prolonged desensitization state, in which the channel is closed in the presence of agonists. Our functional studies suggest that the general anesthetic propofol modulates ELIC negatively and inhibits ELIC channel current. Details of ELIC conformational changes upon agonist or anesthetic binding, however, have not been elucidated. In this study, we mutated two pore-ling residues (F247C and S229C) and a residue in the TM2-3 loop (L256C), labeled them with 2,2,2-trifluoroethanethiol (TET), and performed a series of 19F NMR experiments on the samples. Several results are noteworthy. First, residues L256C and S229C have distinctly different 19F NMR resonance peaks: the former appears as an overlap of two broad peaks, but the latter has four peaks with varied line widths, suggesting co-existence of different conformations. Second, upon adding the agonist propylamine to the samples, the 19F NMR spectra of L256C show no significant changes, but the 19F NMR spectra of S229C or F247C/S229C change profoundly, including the occurrence of a fifth peak. The fifth peak amplitude increases along with the increase of agonist concentrations (3, 10 to 18 mM) and a gradual decrease of the broad peak components. The data suggest a conformational shift in the pore and an increase in desensitization conformation due to long exposure of agonists. Third, the anesthetic propofol further increases the population of desensitization conformation in the S229C or F247C/S229C ELIC by ∼4%. Together, we have demonstrated a powerful approach for investigating conformational changes of pGLICs under different stimulations or modulations. Supported by NIH (R01GM066358, R01GM056257, R37GM049202, and T32GM075770).

Effect of Membrane Hydrophobic Thickness on the Uncoupling of Binding/Gating in the Nicotinic Acetylcholine Receptor

The effects of allosteric modulators on the nicotinic acetylcholine receptor (nAChR) are usually interpreted in terms of a model involving pre-existing resting, open, and desensitized conformations. The nAChR also adopts a lipid-dependent non-activatable conformation, referred to as the “uncoupled” state. Neither the global secondary structure nor the thermal denaturation of the nAChR is greatly affected by uncoupling, although uncoupling leads to an increase in peptide hydrogen exchange suggesting that previously buried peptide hydrogens become exposed to aqueous solvent. We propose that uncoupling results from a weakening of the physical interactions at the interface between the agonist binding and transmembrane pore domains because of lipid-dependent alterations of M4/Cys-loop interactions. As a preliminary test of this model, we reconstituted the nAChR into phosphatidylcholine (PC) membranes with varying hydrophobic thicknesses. While the nAChR in each PC membrane adopts an uncoupled conformation, long acyl chain lipids promote very slow agonist-induced conformational transitions to the desensitized state. Hydrophobic thickness likely influences the ability of the nAChR to undergo agonist-induced conformational transitions by promoting α-helix/α-helix interactions, which appears to lower the activation energy barriers between uncoupled and coupled conformations.

Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held

Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle (N(Glu)) and its diffusion coefficient (D(Glu)) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of N(Glu) and D(Glu) with an optimum of 7000 and 0.3 μm(2) ms(-1) reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals.

Impact of Recovery from Desensitization on Acid-sensing Ion Channel-1a (ASIC1a) Current and Response to High Frequency Stimulation

Consecutive proton stimulation reduces ASIC1a peak currents leading to silencing of channels. Kinetic analysis using a fast perfusion system shows that human ASIC1a has two desensitized states with markedly different stabilities. High frequency trains of short stimuli prevent desensitization. The results predict steady ASIC1a responses to high frequency release of protons as in synaptic transmission. ASIC1a is a neuronal sodium channel activated by external H(+) ions. To date, all the characterization of ASIC1a has been conducted applying long H(+) stimuli lasting several seconds. Such experimental protocols weaken and even silence ASIC1a currents to repetitive stimulation. In this work, we examined ASIC1a currents by methods that use rapid application and removal of H(+). We found that brief H(+) stimuli, <100 ms, even if applied at high frequency, prevent desensitization thereby generate full and steady peak currents of human ASIC1a. Kinetic analysis of recovery from desensitization of hASIC1a revealed two desensitized states: short- and long-lasting with time constants of τ(Ds) ≤0.5 and τ(Dl) = 229 s, while in chicken ASIC1a the two desensitized states have similar values τ(D) 4.5 s. It is the large difference in stability of the two desensitized states that makes hASIC1a desensitization more pronounced and complex than in cASIC1a. Furthermore, recovery from desensitization was unrelated to cytosolic variations in pH, ATP, PIP(2), or redox state but was dependent on the hydrophobicity of key residues in the first transmembrane segment (TM1). In conclusion, brief H(+)-stimuli maintain steady the magnitude of peak currents thereby the ASIC1a channel is well poised to partake in high frequency signals in the brain.

Effect of Ifenprodil on GluN1/GluN2B N-Methyl-D-aspartate Receptor Gating

Ifenprodil is an allosteric inhibitor of GluN1/GluN2B N-methyl-D-aspartate receptors. Despite its widespread use as a prototype for drug development and a subtype-selective tool for physiologic experiments, its precise effect on GluN1/GluN2B gating is yet to be fully understood. Interestingly, recent crystallographic evidence identified that ifenprodil, unlike zinc, binds at the interface of the GluN1/GluN2B amino terminal domain dimer by an induced-fit mechanism. To delineate the effect of this unique binding on GluN1/GluN2B receptor gating, we recorded steady-state currents from cell-attached and outside-out patches. At pH 7.9 in cell-attached patches, ifenprodil increased the occupancy of the long-lived shut conformations, thereby reducing the open probability of the receptor with no change in the mean open time. In addition, ifenprodil selectively affected the area of shut time constants, but not the time constants themselves. Kinetic analyses suggested that ifenprodil prevents the transition of the receptor to an open state and increases its dwell time in an intrinsically occurring closed conformation or desensitized state. We found distinct differences in the action of ifenprodil at GluN1/GluN2B in comparison with previous studies on the effect of zinc on GluN1/GluN2A gating, which may arise due to their unique binding sites. Our data also uncover the potential pH-dependent action of ifenprodil on gating. At a low pH (pH 7.4), but not pH 7.9, ifenprodil reduces the mean open time of GluN1/GluN2B receptors, which may be responsible for its usefulness as a context-dependent inhibitor in conditions like ischemia and stroke, when the pH of the extracellular milieu becomes acidic.

Coupled Control of Desensitization and Gating by the Ligand Binding Domain of Glutamate Receptors

The kinetics of ligand gated ion channels are tuned to permit diverse roles in cellular signaling. To follow high-frequency excitatory synaptic input, postsynaptic AMPA-type glutamate receptors must recover rapidly from desensitization. Chimeras between AMPA and the related kainate receptors demonstrate that the ligand binding domains alone control the lifetime of the desensitized state. Mutation of nonconserved amino acids in the lower lobe (domain 2) of the ligand binding domain conferred slow recovery from desensitization on AMPA receptors, and fast recovery on kainate receptors. Single-channel recordings and a correlation between the rate of deactivation and the rate of recovery across panels of mutant receptors revealed that domain 2 also controls ion channel gating. Our results demonstrate that the same mechanism that ensures fast recovery also sharpens the response of AMPA channels to synaptically released glutamate.

Potential State-selective Hydrogen Bond Formation Can Modulate Activation and Desensitization of the α7 Nicotinic Acetylcholine Receptor

A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on human α7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles), or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand-binding domain residue Gln-57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E, and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Gln-57 and replace it with hydrogen bond donating, hydrogen bond accepting, or nonhydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or -pyrroles than for their 3-substituted isomers, whereas the nonhydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596 agonist co-application data revealed that for wild-type α7 nAChR, the 3-furan desensitized state was relatively stabilized compared with that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.

Bupropion Binds to Two Sites in the Torpedo Nicotinic Acetylcholine Receptor Transmembrane Domain: A Photoaffinity Labeling Study with the Bupropion Analogue [125I]-SADU-3-72

Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(N-tert-butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone (SADU-3-72). Based on inhibition of [(125)I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC(50) = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC(50) = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [(125)I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [(125)I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K, and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu(265), βLeu(257)) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr(213) in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.

The Gating Mechanism of a P2X4 Receptor: Normal Mode Analysis and Molecular Dynamics Simulations

P2X4 receptors are trimeric ATP-gated non-selective cation channels which play crucial roles in various physiological processes. It remains unclear how ATP binding triggers channel opening. Here, we propose a gating mechanism of a P2X4 receptor based on normal mode analysis and molecular dynamics (MD) simulations. Starting with the first available P2X4 crystal structure in the resting state, a normal mode that couples the motions of three β strands (β1, β13 and β14) at the trimeric interface of the ligand binding domain (LBD) and the motion of the pore-forming helix (TM2) of the transmembrane domain (TMD) was identified. The resulting widening of the fenestrations above the TMD and the opening of the TMD pore are in close agreement with observed signatures of channel activation. Four charged residues implicated in ATP binding are located in β1, β13 and β14. P2X4 activation was further investigated by MD simulations in explicit water. ATP was placed near the putative binding site in two opposite orientations, with the adenine either promixal or distal to the TMD. In simulations with proximal adenine, the adenine ring inserted between β1 and β13 and the phosphate group moved downward. At the same time β1 and β14 approached each other to close in on the ATP, allowing close interactions with the four charged residues. The motions of these β strands are similar to those in the normal mode putatively representing channel activation. In simulations with distal adenine, the ATP hindered the closure between β1 and β14, perhaps representing a desensitized state. Our computational studies produced the first complete model, supported by electrophysiological data, for how ATP binding leads to P2X4 channel activation. The detailed gating mechanism will be essential for the rational drug design.

Oral desensitization for milk allergy in children

The purpose of this review is to research current evidence on cow's milk oral immunotherapy for the treatment of cow's milk allergy (CMA). The specific, active treatment for IgE-mediated food allergy included CMA, which is currently being investigated in human trials. Allergen-specific approaches include oral, sublingual and epicutaneous immunotherapy. Reports on oral immunotherapy (OIT) for the treatment of milk allergy have been more extensive and carried out mostly with native proteins. The aim of OIT with cow's milk is the achievement of desensitization or tolerance by patients suffering from CMA. Desensitization state can be achieved by approximately 36-92% of the children treated with specific immunotherapy; the rate of permanent tolerance is unknown. Longer duration of desensitization may result in permanent tolerance. The possibility of adverse events or reactions during OIT is quite frequent. Side-effects have been reported by patients in all published studies. OIT as an active treatment for CMA represents an emerging reality. Before this treatment can be used in clinical practice, additional studies are needed. Currently, many issues remain unanswered: severity and type of food allergy responsive to specific immunotherapy, degree of protection, 'shared schedules' of desensitization(s) in research settings and well established risk-to-benefit ratio. However the field of specific, active treatment of food allergy is poised for clinically important advances.

Transmembrane AMPA receptor regulatory protein regulation of competitive antagonism: a problem of interpretation

Synaptic AMPA receptors are greatly influenced by a family of transmembrane AMPA receptor regulatory proteins (TARPs) which control trafficking, channel gating and pharmacology. The prototypical TARP, stargazin (or γ2), shifts the blocking ability of several AMPAR-selective compounds including the commonly used quinoxalinedione antagonists, CNQX and NBQX. Stargazin's effect on CNQX is particularly intriguing as it not only apparently lowers the potency of block, as with NBQX, but also renders it a partial agonist. Given this, agonist behaviour by CNQX has been speculated to account for its weaker blocking effect on AMPAR-TARP complexes. Here we show that this is not the case. The apparent effect of stargazin on CNQX antagonism can be almost entirely explained by an increase in the apparent affinity for l-glutamate (l-Glu), a full agonist and neurotransmitter at AMPAR synapses. Partial agonism at best plays a minor role but not through channel gating per se but rather because CNQX elicits AMPAR desensitization. Our study reveals that CNQX is best thought of as a non-competitive antagonist at glutamatergic synapses due to the predominance of non-equilibrium conditions. Consequently, CNQX primarily reports the proportion of AMPARs available for activation but may also impose additional block by receptor desensitization.

Investigation of the Molecular Mechanism of the α7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulator PNU-120596 Provides Evidence for Two Distinct Desensitized States

Although α7 nicotinic acetylcholine receptors are considered potentially important therapeutic targets, the development of selective agonists has been stymied by the α7 receptor's intrinsically low probability of opening (P(open)) and the concern that an agonist-based therapeutic approach would disrupt endogenous cholinergic function. Development of α7 positive allosteric modulators (PAMs) holds promise of avoiding both issues. N-(5-Chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea (PNU-120596) is one of the most effective α7 PAMs, with a mechanism associated, at least in part, with the destabilization of desensitized states. We studied the mechanism of PNU-120596 potentiation of α7 receptors expressed in Xenopus laevis oocytes and outside-out patches from BOSC 23 cells. We identify two forms of α7 desensitization: one is destabilized by PNU-120596 (D(s)), and the other is induced by strong episodes of activation and is stable in the presence of the PAM (D(i)). Our characterization of prolonged bursts of single-channel currents that occur with PNU-120596 provide a remarkable contrast to the behavior of the channels in the absence of the PAM. Individual channels that avoid the D(i) state show a 100,000-fold increase in P(open) compared with receptors in the nonpotentiated state. In the presence of PNU-120596, balance between D(s) and D(i) is dynamically regulated by both agonist and PAM binding, with maximal ion channel activity at intermediate levels of binding to both classes of sites. In the presence of high agonist concentrations, competitive antagonists may have the effect of shifting the balance in favor of D(s) and increasing ion channel currents.

Functional analysis of the hippocampus using opto-fMRI

Channelrhodopsins (ChRs) are light-gated cation channels. After blue-light excitation, the protein undergoes a photocycle with different intermediates. Here, we have recorded transient absorbance changes of ChR2 from Chlamydomonas reinhardtii in the visible and infrared regions with nanosecond time resolution, the latter being accomplished using tunable quantum cascade lasers. Because proton transfer reactions play a key role in channel gating, we determined vibrational as well as kinetic isotope effects (VIEs and KIEs) of carboxylic groups of various key aspartic and glutamic acid residues by monitoring their C=O stretching vibrations in H2O and in D2O. D156 exhibits a substantial KIE (>2) in its deprotonation and reprotonation, which substantiates its role as the internal proton donor to the retinal Schiff base. The unusual VIE of D156, upshifted from 1736 cm−1 to 1738 cm−1 in D2O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8 cm−1 in D2O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9 cm−1 and a KIE of ∼2 for the de- and the reprotonation reactions during the lifetime of the late desensitized state. The KIE of 1 determined in the time range from 20 ns to 5 ms is incompatible with early deprotonation of E90.

A single amino acid residue controls Ca2+signaling by an octopamine receptor fromDrosophila melanogaster

Rhythmic activity of cells and cellular networks plays an important role in physiology. In the nervous system oscillations of electrical activity and/or second messenger concentrations are important to synchronize neuronal activity. At the molecular level, rhythmic activity can be initiated by different routes. We have recently shown that an octopamine-activated G-protein-coupled receptor (GPCR; DmOctα1Rb, CG3856) from Drosophila initiates Ca(2+) oscillations. Here, we have unraveled the molecular basis of cellular Ca(2+) signaling controlled by the DmOctα1Rb receptor using a combination of pharmacological intervention, site-directed mutagenesis, and functional cellular Ca(2+) imaging on heterologously expressed receptors. Phosphorylation of a single amino acid residue in the third intracellular loop of the GPCR by PKC is necessary and sufficient to desensitize the receptor. From its desensitized state, DmOctα1Rb is resensitized by dephosphorylation, and a new Ca(2+) signal occurs on octopamine stimulation. Our findings show that transient changes of the receptor's surface profile have a strong effect on its physiological signaling properties. We expect that the detailed knowledge of DmOctα1Rb-dependent signal transduction fosters the identification of specific drugs that can be used for GPCR-mediated pest control, since octopamine serves important physiological and behavioral functions in arthropods.

Structure of the M2 Transmembrane Segment of GLIC, a Prokaryotic Cys Loop Receptor Homologue from Gloeobacter violaceus, Probed by Substituted Cysteine Accessibility

GLIC is a homopentameric proton-gated, prokaryotic homologue of the Cys loop receptor family of neurotransmitter-gated ion channels. Recently, crystal structures of GLIC hypothesized to represent an open channel state were published. To explore the channel structure in functional GLIC channels, we tested the ability of p-chloromercuribenzenesulfonate to react with 30 individual cysteine substitution mutants in and flanking the M2 channel-lining segment in the closed state (pH 7.5) and in a submaximally activated state (pH 5.0). Nine mutants did not tolerate cysteine substitution and were not functional. From positions 10' to 27', p-chloromercuribenzenesulfonate significantly modified the currents at pH 7.5 and 5.0 in all mutants except H234C (11'), I235C (12'), V241C (18'), T243C (20'), L245C (22'), and Y250C (27'), which were not functional, except for 12'. Currents for P246C (23') and K247C (24') were only significantly altered at pH 5.0. The reaction rates were all >1000 m(-1) s(-1). The reactive residues were more accessible in the activated than in the resting state. We infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is more loosely packed from 10' to the extracellular end than the x-ray structures suggest. We infer that the charge selectivity filter is in the cytoplasmic half of the channel. We also show that below pH 5.0, GLIC desensitizes on a time scale of minutes and infer that the crystal structures may represent a desensitized state.

Structural Differences in the Nicotinic Acetylcholine Receptor (NACHR) ion Channel Between Open and Desensitized States Revealed by Time-Resolved Photolabeling with [ 3H]Chlorpromazine (CPZ)

Extensive photoaffinity labeling studies and mutational analyses of Torpedo/muscle-type nAChR indicate only subtle differences in the structure of the ion channel between closed, open and desensitized states, with amino acids on the same face of each M2 helix lining the ion channel in each state (cytoplasmic to synaptic: M2-2, M2-6, M2-9, M2-13, M2-16/17, & M2-20). To provide further information about the differences in channel structure between the open and desensitized states, we have used time-resolved photolabeling to determine where the channel blocker [3H]CPZ binds in the open channel state after 30 msec exposure to [3H]CPZ and agonist, compared to its binding site(s) in nAChRs in the desensitized state exposed to [3H]CPZ for 30 msec or at equilibrium. In the desensitized state, at equilibrium [3H]CPZ binds to and photolabels a pocket near the cytoplasmic end of the channel (bottom, M2-2/M2-6/M2-9) and a pocket near the extracellular end of the channel (top, M2-16/17/M2-20), while in the closed, resting state it photolabels only the site at the bottom of the channel [Chiara,DC, et.al. Biochemistry 2009 48:10066-77]. For nAChRs pre-equilibrated with agonist (desensitized state), after 30 msec [3H]CPZ photolabels only the site at the top of the channel (M2-16/17/20), while for nAChRs in the open state (30 msec agonist and CPZ), the pockets at both the bottom (M2-2/M2-6/M2-9) and top (M2-16/17/M2-20) were photolabeled. From these results we conclude that: 1) a structural alteration at the top of the ion channel occurs between closed and open states, whereas this region is similar in structure in the open and desensitized states; and 2) in the equilibrium desensitized state, there is an access barrier for CPZ at or near M2-13 (Supported by GM58448).

Assigning Functional States to Structural Models of Nicotinic-Receptor Type ion Channels

Aromatic-aromatic interactions are a prominent feature of the crystal structure of ELIC a bacterial member of the nicotinic-receptor superfamily of ion channels where five pore-facing phenylalanines come together to form a structure akin to a narrow iris that occludes the transmembrane pore. To identify the functional state of the channel that this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine receptor, including the position that aligns with the native phenylalanine 246 of ELIC, and assessed the consequences of such mutations using electrophysiological and toxin-binding assays. From our experiments, we conclude that the interaction among the side chains of pore-facing phenylalanines leads to the formation of a non-conductive conformation that is unresponsive to the application of acetylcholine and is highly stable even in the absence of ligand. Moreover, electrophysiological recordings from a GLIC channel (another bacterial member of the superfamily) engineered to have a ring of phenylalanines at the corresponding pore-facing position suggest that this novel refractory state is distinct from the well-known desensitized state. It seems reasonable to propose, then, that it is in this peculiar non-conductive conformation that the ELIC channel was crystallized. It seems also reasonable to propose that, in the absence of rings of pore-facing aromatic side chains, such stable conformation may never be attained by the acetylcholine receptor. Incidentally, we also noticed that the response of the proton-gated, wild-type GLIC channel to a fast change in pH from 7.4 to 4.5 (on the extracellular side) is only transient, with the evoked current fading completely in a matter of seconds. This raises the possibility that the crystal structures of GLIC obtained at pH 4.0 and pH 4.6 correspond to the (well-known) desensitized state.

Catharanthine Alkaloids are Noncompetitive Inhibitors of Muscle-Type Nicotinic Acetylcholine Receptors

This is an attempt to characterize the binding sites and the mechanisms of action of several catharanthine alkaloids including ibogaine, vincristine, and vinblastine, on muscle-type nicotinic acetylcholine receptors (AChRs), by comparing their pharmacological properties with that for the well characterized high-affinity noncompetitive antagonist (NCA) phencyclidine (PCP). In this regard, structural and functional approaches were used including radioligand equilibrium and competition binding assays using [3H]ibogaine and the analog of PCP, [piperidyl-3, 4-3H(N)]-N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine ([3H]TCP), Ca2+ influx determinations, thermodynamic and kinetic measurements using column-immobilized Torpedo AChRs, and molecular docking and dynamics studies The results established that: (a) the alkaloidsinhibit (±)-epibatidine-induced Ca2+ influx in embryonic muscle AChRs with the following potencies (in μM): ibogaine (17 ± 3) > vinblastine (20 ± 5) > vincristine (25 ± 4), that are slightly higher than that for PCP (31 ± 2), (b) the alkaloids inhibit [3H]TCP binding, and ibogaine and PCP inhibit [3H]ibogaine binding, to the desensitized Torpedo AChR with higher affinity compared to the resting AChR, (c) ibogaine binds to the Torpedo AChR by an entropy-driven process, and (d) ibogaine interacts with a binding domain located between the serine (position 6’) and valine (position 13’) rings, by a network of van der Waals and polar interactions. Collectively our data indicate that catharanthine alkaloids block agonist-activated ion channels by interacting with a binding domain that is shared with PCP located between the serine and valine rings. This supports the view that the catharanthine moiety, which is shared by ibogaine and vinca alkaloids, is a minimum structural requirement for the interaction of these molecules with the ion channel. In addition, ibogaine and vinca alkaloids may induce and maintain the desensitized state by a mechanism where their dissociation rates are decreased.

Characterizing the Energetic States of the GluR2 Ligand Binding Domain Core-Dimer

Tetrameric ligand binding domains of the family of ionotropic glutamate receptors assemble as dimers-of-dimers. Crystallographic studies of several glutamate receptor subtype isolated core-dimers suggest a single stable dimeric conformation. A binding domain dimer has not been captured in other conformations without the aid of biochemical methods to disrupt a critical dimer interface. Molecular dynamics simulations and continuum electrostatics calculations reveal that the active glutamate bound form of the ligand-binding domain found in typical crystal structures is the preferred energetic state of the isolated core-dimer in the presence of agonist glutamate. A desensitized conformational state is a higher energy ligand-bound state of the core-dimer. The resting apo conformational state is comparatively the least energetically favored conformation and does not contain a single state but a set of energetically equivalent conformational core-dimer states. We hypothesize the energetic balance of an open versus closed transmembrane region must be included to characterize the absolute energetic states of the full receptor, which in the presence of the ligand is believed to be a desensitized state.