Human Glycine Receptor Research Articles

Specific recognition mechanism between RNA and the KH3 domain of Nova-2 protein.

The KH3 domain of Nova-2 protein can precisely recognize the sequence-specific target RNA of human glycine receptor α2. However, the recognition mechanism between the protein and its target RNA is still hotly debated. In this study, molecular dynamic simulations in explicit solvent were utilized to understand the recognition mechanism. The structural analysis and the Kolmogorov-Smirnov P-test statistics reveal that the KH3 domain might obey a conformational selection mechanism upon RNA binding. However, the induced fit mechanism could not be completely ruled out. Unfolding kinetics indicates that the folding of RNA and KH3 happens first and then the binding between RNA and KH3 follows. Principle component analysis shows that the invariant Gly-Lys-Gly-Gly loop moves toward to the RNA molecule but the C-terminal domain moves away from the RNA molecule upon binding. These specific dominant motions were hypothesized to stabilize the complex structure. The hydrophobic and hydrogen bonding interactions were found to be the driving forces for the specific recognition, in contrast to the dominant electrostatic interactions for nonspecific recognition.

A mouse line for inducible and reversible silencing of specific neurons

BackgroundGenetic methods for inducibly and reversibly inhibiting neuronal activity of specific neurons are critical for exploring the functions of neuronal circuits. The engineered human glycine receptor, called ivermectin (IVM)-gated silencing receptor (IVMR), has been shown to possess this ability in vitro.ResultsHere we generated a mouse line, in which the IVMR coding sequence was inserted into the ROSA26 locus downstream of a loxP-flanked STOP cassette. Specific Cre-mediated IVMR expression was revealed by mis-expression of Cre in the striatum and by crossing with several Cre lines. Behavioral alteration was observed in Rosa26-IVMR mice with unilateral striatal Cre expression after systemic administration of IVM, and it could be re-initiated when IVM was applied again. A dramatic reduction in neuron firing was recorded in IVM-treated free moving Rosa26-IVMR;Emx1-Cre mice, and neuronal excitability was reduced within minutes as shown by recording in brain slice.ConclusionThis Rosa26-IVMR mouse line provides a powerful tool for exploring selective circuit functions in freely behaving mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-014-0068-8) contains supplementary material, which is available to authorized users.

Agonist and antagonist binding in human glycine receptors.

The human glycine receptor (hGlyR) is an anion-permeable ligand-gated channel that is part of a larger superfamily of receptors called the Cys-loop family. hGlyRs are particularly amenable to single-channel recordings, thus making them a model experimental system for understanding the Cys-loop receptor family in general. Understanding the relationship between agonist binding and efficacy in Cys-loop receptors should improve our future prospects for making specific agonists or antagonists. However, at present, there is no high-resolution structure for the complete hGlyR, and thus, modeling is needed to provide a physical framework on which to interpret single-channel data. The structure of the glutamate-gated chloride channel from Caenorhabditis elegans shows a much higher level of sequence identity to human hGlyR than previous templates such as AChBP or the bacterial channels, GLIC and ELIC. Thus, we constructed a model of the hGlyR and validated it against previously reported mutagenesis data. We used molecular dynamics to refine the model and to explore binding of both an agonist (glycine) and an antagonist (strychnine). The model shows excellent agreement with previous data but also suggests some unique features: (i) a water molecule that forms part of the binding site and allows us to account for some previous results that were difficult to reconcile, (ii) an interaction of the glycine agonist with S129, and (iii) an effect from E211, both of which we confirmed with new site-directed mutagenesis and patch clamp recordings. Finally, examination of the simulations suggests that strychnine binding induces movement to a conformational state distinct from the glycine-bound or apo state, not only within the ligand-binding domain but also in the transmembrane domain.

Opposing Effects of the Anesthetic Propofol at Pentameric Ligand-Gated Ion Channels Mediated by a Common Site

Propofol is an intravenous general anesthetic that alters neuronal excitability by modulating agonist responses of pentameric ligand-gated ion channels (pLGICs). Evidence suggests that propofol enhancement of anion-selective pLGICs is mediated by a binding site between adjacent subunits, whereas propofol inhibition of cation-selective pLGICs occurs via a binding site contained within helices M1-M4 of individual subunits. We considered this idea by testing propofol modulation of homomeric human glycine receptors (GlyRs) and nematode glutamate-gated chloride channels (GluCls) recombinantly expressed in Xenopus laevis oocytes with electrophysiology. The Haemonchus contortus AVR-14B GluCl was inhibited by propofol with an IC50 value of 252 ± 48 μM, providing the first example of propofol inhibition of an anion-selective pLGIC. Remarkably, inhibition was converted to enhancement by a single I18'S substitution in the channel-forming M2 helix (EC50 = 979 ± 88 μM). When a previously identified site between adjacent subunits was disrupted by the M3 G329I substitution, both propofol inhibition and enhancement of GluCls were severely impaired (IC50 and EC50 values could not be calculated). Similarly, when the equivalent positions were examined in GlyRs, the M2 S18'I substitution significantly altered the maximum level of enhancement by propofol, and the M3 A288I substitution abolished propofol enhancement. These data are not consistent with separate binding sites for the opposing effects of propofol. Instead, these data suggest that propofol enhancement and inhibition are mediated by binding to a single site in anion-selective pLGICs, and the modulatory effect on channel gating depends on the M2 18' residue.

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.

The impact of human hyperekplexia mutations on glycine receptor structure and function

Hyperekplexia is a rare neurological disorder characterized by neonatal hypertonia, exaggerated startle responses to unexpected stimuli and a variable incidence of apnoea, intellectual disability and delays in speech acquisition. The majority of motor defects are successfully treated by clonazepam. Hyperekplexia is caused by hereditary mutations that disrupt the functioning of inhibitory glycinergic synapses in neuromotor pathways of the spinal cord and brainstem. The human glycine receptor α1 and β subunits, which predominate at these synapses, are the major targets of mutations. International genetic screening programs, that together have analysed several hundred probands, have recently generated a clear picture of genotype-phenotype correlations and the prevalence of different categories of hyperekplexia mutations. Focusing largely on this new information, this review seeks to summarise the effects of mutations on glycine receptor structure and function and how these functional alterations lead to hyperekplexia.

Resveratrol Inhibits Glycine Receptor-Mediated Ion Currents

Resveratrol is found in grapes, red wine, and berries. Resveratrol has been known to have many beneficial health effects, such as anti-cancer, neuroprotective, anti-nociceptive, and life-prolonging effects. However, the single cellular mechanisms by which resveratrol acts are relatively unknown, especially in terms of possible regulation of receptors involved in synaptic transmission. The glycine receptor is an inhibitory ligand-gated ion channel involved in fast synaptic transmission in spinal cord. In the present study, we investigated the effect of resveratrol on human glycine receptor channel activity. Glycine α1 receptors were expressed in Xenopus oocytes and glycine receptor channel activity was measured using a two-electrode voltage clamp technique. Treatment with resveratrol alone had no effect on oocytes injected with H2O or on oocytes injected with glycine α1 receptor cRNA. In the oocytes injected with glycine α1 receptor cRNA, co- or pre-treatment of resveratrol with glycine inhibited the glycine-induced inward peak current (IGly) in a reversible manner. The inhibitory effect of resveratrol on IGly was also concentration dependent, voltage independent, and non-competitive. These results indicate that resveratrol regulates glycine receptor channel activity and that resveratrol-mediated regulation of glycine receptor channel activity is one of several cellular action mechanisms of resveratrol for pain regulation.

New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms

Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated that the dominant mutations p.Q226E, p.V280M, and p.R414H induced spontaneous channel activity, indicating that this is a recurring mechanism in hGlyR pathophysiology. p.Q226E, at the top of TM1, most likely induced tonic activation via an enhanced electrostatic attraction to p.R271 at the top of TM2, suggesting a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated into functional hGlyRs together with unmutated α1 or α1 plus β subunits. These aberrant receptors exhibit significantly reduced glycine sensitivity. To our knowledge, this is the first suggestion that subunits lacking TM4 domains might be incorporated into functional pentameric ligand-gated ion channel receptors.

Open-Channel Structures of the Human Glycine Receptor α1 Full-Length Transmembrane Domain

Glycine receptors play a major role in mediating fastinhibitory neurotransmission in the spinal cord and brain stem, yet their high-resolution structures remain unsolved. We determined open-channel structures of the full-length transmembrane domain (TMD) of the human glycine receptor α1-subunit (hGlyR-α1) using nuclear magnetic resonance (NMR) spectroscopy and electron micrographs. hGlyR-α1 TMD spontaneously forms pentameric Cl(-)-conducting channels, with structures sharing overall topology observed in crystal structures of homologous bacterial and nematode pentameric ligand-gated ion channels (pLGICs). However, the mammalian hGlyR-α1 structures present several distinctive features, including a shorter, pore-lining TM2 helix with helical unwinding near the C-terminal end, a TM3 helical kink at A288 that partially overlaps with the homologous ivermectin-binding site in GluCl, and a highly dynamic segment between S267(15') of TM2 and A288 that likely affects allosteric modulations of channel function. Our structures provide additional templates for identifying potential drug targets in GlyRs and other mammalian pLGICs.

X-Ray Structures of an Interfacial Potentiating Site for Alcohols and Anesthetics in a Pentameric Ligand-Gated Ion Channel

Alcohol consumption produces a variety of undesirable behavioural and physiological effects in animals and humans and can eventually lead to addiction. Converging evidences suggest that its molecular mechanisms of action involve specific protein targets. Among these, pentameric ligand-gated ion channels (pLGICs) and especially GABA-A Receptor have been shown to be one of the main targets of ethanol in the central nervous system. Here we report the first atomic-resolution structure at 2.8 A of ethanol bound to a member of the pLGIC family, the pH-gated prokaryotic homolog GLIC variant F14'A. This GLIC variant is potentiated by concentrations of ethanol similar to the ones effective in the vertebrate Glycine and GABA-A receptors (R. Howard et al., 2012). Comparison the ethanol-bound and apo structures of GLIC F14'A gives a rational and simple explanation to the potentiating effect of ethanol on these receptors by stabilizing the open form. Multiple-sequence alignments and homology structure models suggest that the ethanol binding-pocket identified in GLIC is also present in human Glycine and GABA-A receptors.

Stoichiometry of the Human Glycine Receptor Revealed by Direct Subunit Counting

The subunit stoichiometry of heteromeric glycine-gated channels determines fundamental properties of these key inhibitory neurotransmitter receptors; however, the ratio of α1- to β-subunits per receptor remains controversial. We used single-molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2β. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations.

A Novel Dominant Hyperekplexia Mutation Y705C Alters Trafficking and Biochemical Properties of the Presynaptic Glycine Transporter GlyT2

Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [(3)H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311-Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H(+) and Zn(2+) dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo.

Mutations in the GlyT2 Gene (SLC6A5) Are a Second Major Cause of Startle Disease

Hereditary hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, leading to hypertonia and apnea episodes. Missense, nonsense, frameshift, splice site mutations, and large deletions in the human glycine receptor α1 subunit gene (GLRA1) are the major known cause of this disorder. However, mutations are also found in the genes encoding the glycine receptor β subunit (GLRB) and the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of SLC6A5 in 93 new unrelated human hyperekplexia patients revealed 20 sequence variants in 17 index cases presenting with homozygous or compound heterozygous recessive inheritance. Five apparently unrelated cases had the truncating mutation R439X. Genotype-phenotype analysis revealed a high rate of neonatal apneas and learning difficulties associated with SLC6A5 mutations. From the 20 SLC6A5 sequence variants, we investigated glycine uptake for 16 novel mutations, confirming that all were defective in glycine transport. Although the most common mechanism of disrupting GlyT2 function is protein truncation, new pathogenic mechanisms included splice site mutations and missense mutations affecting residues implicated in Cl(-) binding, conformational changes mediated by extracellular loop 4, and cation-π interactions. Detailed electrophysiology of mutation A275T revealed that this substitution results in a voltage-sensitive decrease in glycine transport caused by lower Na(+) affinity. This study firmly establishes the combination of missense, nonsense, frameshift, and splice site mutations in the GlyT2 gene as the second major cause of startle disease.

Thermodynamic studies of ligand binding to the human homopentameric glycine receptor using isothermal titration calorimetry

In this work, we describe a process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor. Subsequent purification yielded functional, uniform protein with expression yields of up to 5 mg per liter cell culture. As the wild-type protein is prone to proteolytic degradation, the labile sites were removed by mutagenesis resulting in an intracellular loop 2 deletion mutant with N-terminal modifications. This variant of the receptor is both stable during purification and storage on ice for up to a week as a complex with an antagonist. The quality of the protein is suitable for biophysical characterization and structural studies. The interaction of the agonist glycine and the antagonist strychnine with purified protein was analyzed by isothermal titration calorimetry. Strychnine binding is driven enthalpically with a K(D) of 138 ± 55 nM, a ΔH of -9708 ± 1195 cal/mol and a ΔS of -1.0 ± 4.1 cal/mol/K, whereas glycine binding is driven by entropy with a K(D) of 3.2 ± 0.8 μM, a ΔH of -2228 ± 1012 cal/mol and ΔS of 17.7 ± 2.8 cal/mol/K. Strychnine and glycine binding is competitive with a stoichiometry of one ligand molecule to one pentameric glycine receptor.

The α1K276E Startle Disease Mutation Reveals Multiple Intermediate States in the Gating of Glycine Receptors

Loss-of-function mutations in human glycine receptors cause hyperekplexia, a rare inherited disease associated with an exaggerated startle response. We have studied a human disease mutation in the M2-M3 loop of the glycine receptor α1 subunit (K276E) using direct fitting of mechanisms to single-channel recordings with the program HJCFIT. Whole-cell recordings from HEK293 cells showed the mutation reduced the receptor glycine sensitivity. In single-channel recordings, rat homomeric α1 K276E receptors were barely active, even at 200 mM glycine. Coexpression of the β subunit partially rescued channel function. Heteromeric mutant channels opened in brief bursts at 300 μM glycine (a concentration that is near-maximal for wild type) and reached a maximum one-channel open probability of about 45% at 100 mm glycine (compared to 96% for wild type). Distributions of apparent open times contained more than one component in high glycine and, therefore, could not be described by mechanisms with only one fully liganded open state. Fits to the data were much better with mechanisms in which opening can also occur from more than one fully liganded intermediate (e.g., "primed" models). Brief pulses of glycine (∼3 ms, 30 mM) applied to mutant channels in outside-out patches activated currents with a slower rise time (1.5 ms) than those of wild-type channels (0.2 ms) and a much faster decay. These features were predicted reasonably well by the mechanisms obtained from fitting single-channel data. Our results show that, by slowing and impairing channel gating, the K276E mutation facilitates the detection of closed reaction intermediates in the activation pathway of glycine channels.

Activated G Protein αsSubunits Increase the Ethanol Sensitivity of Human Glycine Receptors

It is well known that ethanol modulates the function of the Cys loop ligand-gated ion channels, which include the inhibitory glycine receptors (GlyRs). Previous studies have consistently shown that transmembrane and extracellular sites are essential for ethanol actions in GlyRs. In addition, recent evidence has shown that the ethanol modulation of GlyRs is also affected by G protein activation through Gβγ subunits. However, more specific roles of G protein α subunits on ethanol actions are unknown. Here, we show that the allosteric effect of ethanol on the human α(1) GlyR is selectively enhanced by the expression of Gα(s) Q-L. For example, constitutively active Gα(s), but not Gα(q) or Gα(i), was able to displace the alcohol sensitivity of GlyRs toward low millimolar concentrations (17 ± 4 versus 48 ± 5% at 100 mM). Experiments under conditions that increased cAMP and protein kinase A (PKA)-mediated signaling, on the contrary, did not produce the same enhancement in sensitivity, suggesting that the Gα(s) Q-L effect was not dependent on cAMP/PKA-dependent signaling. On the other hand, the effect of Gα(s) Q-L was blocked by a Gβγ scavenger (9 ± 3% of control). Furthermore, two mutant receptors previously shown to have impaired interactions with Gβγ were not affected by Gα(s) Q-L, suggesting that Gβγ is needed for enhancing ethanol sensitivity. These results support the conclusion that activated Gα(s) can facilitate the Gβγ interaction with GlyRs in presence of ethanol, independent of increases in cAMP signaling. Thus, these data indicate that the activated form of Gα(s) is able to positively influence the effect of ethanol on a type of inhibitory receptor important for motor control, pain, and respiration.

The K276E Startle Disease Mutation in the Glycine Receptor: Effects on Channel Gating

Glycine receptors mediate inhibitory synaptic currents that are essential for motor control. Loss-of-function mutations in human glycine receptors cause hyperekplexia, a rare inherited disease associated with an exaggerated startle response. We have studied a human disease mutation in the M2-M3 loop of the glycine receptor alpha1 subunit (K276E) using direct fitting of mechanisms to single-channel recordings with the program HJCFIT. Whole-cell recordings from HEK 293 cells suggested that mutant receptors, both homomeric alpha1 and heteromeric alpha1beta, were much less sensitive to glycine than their wild-type counterparts. Single-channel recordings showed that homomeric alpha1 K276E receptors are barely active, even at 200 mM glycine. In contrast, heteromeric channels show brief bursts of openings at 300 µM glycine (∼EC95 for wild-type) and reach a maximum one-channel open probability of about 45% at 100 mM glycine (cf. 96% for wild-type). Distributions of apparent open times contained more than one component at high concentrations, and so mechanisms that allowed only one open state in the fully liganded receptor were extremely poor at describing the observed single-channel behaviour. Instead, mechanisms in which opening can also occur from more than one fully liganded intermediate state (e.g. ‘primed’ models) gave a much better description of the data. Brief pulses of glycine (0.5-2 ms, 30 mM) applied to outside-out patches activated currents with a slower rise time (1.3 ms) than wild-type channels (0.2 ms), and a much faster decay. These features of the macroscopic currents were well predicted by the mechanisms obtained from fitting single-channel data. Our results show that the mutation K276E impairs the gating of the receptor, demonstrate an unforeseen role for the beta subunit in promoting channel opening, and give further evidence of reaction intermediates in the activation pathway.

Novel Regulatory Site within the TM3–4 Loop of Human Recombinant α3 Glycine Receptors Determines Channel Gating and Domain Structure

Glycine receptors are Cys loop ligand-gated ion channels that mediate fast inhibitory synaptic transmission in the mammalian central nervous system. The functionally distinct splice variants alpha3L and alpha3K of the human glycine receptor differ by a 15-amino acid insert within the long intracellular TM3-4 loop, a region of high intersubunit diversity. In a mutational study, effects of the insert on ion channel function and secondary structure of the TM3-4 loop were investigated. Whole cell current responses and protein surface expression data indicated that the major effect of mutations within the insert was on channel gating. Changes in channel gating correlated with the distribution of charged residues about the splice region. Analysis of complex molecular weight indicated that recombinant TM3-4 loops of alpha3L and alpha3K associated into oligomers of different stoichiometry. Secondary structure analysis suggested that the insert stabilized the overall fold of the large cytoplasmic domain of alpha3L subunits. The absence of the insert resulted in a channel that was still functional, but the TM3-4 cytoplasmic domain appeared not stably folded. Thus, our data identified the spliced insert within the large TM 3-4 loop of alpha3 Gly receptors as a novel regulatory motif that serves a 2-fold role: (i) the presence of the insert stabilizes the overall spatial structure of the domain, and (ii) the insert presents a control unit that regulates gating of the receptor ion channel.

Structure and Dynamics of TM Domains of Human Glycine Receptor in LPPG Micelles

The structure and backbone dynamics of the entire four transmembrane domains of human glycine receptor α1 subunit (GlyRTM1234) were studied in LPPG micelle. Using triple-resonance multi-dimensional NMR methods, over 80% residues were assigned. The chemical shift index clearly showed that most residues in the TM region were in helical conformation, consistent with a four-helix-bundle TM protein. We have collected sufficient amount of distance, H-bond, and dihedral angle restraints from NOE and chemical shift data, as well as long-range distance restraints from paramagnetic relaxation enhancement measurements. The structure model, determined using the available restraints, showed a kink between Trp72 and Cys76 in the beginning of TM3 domain. Moreover, the end of TM2 domain, S53 to S56, is more flexible compared to the rest of TM2. In contrast, part of TM23 loop, V63 to V66, showed a helical secondary structure. Backbone dynamics measurements indicated the existence of significant internal motions of TM1234 in LPPG micelles. The relaxation data also resulted in an overall rotational correlation time of ∼33.4 ns, estimated based on the non-flexible helical residues. This overall tumbling time corresponds to a 66.8 kDa protein-LPPG complex with ∼98 LPPG molecules per GlyRTM1234. The size is confirmed by the dynamic light scattering measurement. We suggest that the flexible end of TM2 and the beginning of TM3 can better coordinate the coupling between the TM2-TM3 loop and the extracellular domain. This coupling is thought to be important for mediating channel gating. In addition, it is confirmed that the early termination of TM2 helix in GlyR is an intrinsic property and is independent of the presence or absence of other TM domains (Funded by NIH R37GM049202 & R01GM069766).

Residual dipolar coupling measurements of transmembrane proteins using aligned low-q bicelles and high-resolution magic angle spinning NMR spectroscopy.

Bicelles are a major medium form to produce weak alignment of soluble proteins for residual dipolar coupling (RDC) measurements. The obstacle to using the same type of bicelles for transmembrane proteins with solution-state NMR spectroscopy is the loss of signals due to the adhesion or penetration of the proteins into large bicelles, resulting in slow protein tumbling. In this study, weak alignment of the second and third transmembrane domains (TM23) of the human glycine receptor (GlyR) was achieved in low-q bicelles (q = DMPC/DHPC). Although protein-free bicelles with such low q would likely show isotropic properties, the insertion of TM23 induced weakly preferred orientations so that the RDC of the embedded protein can be measured. The extent of the alignment increased but the TM23 signal intensity decreased when q was varied from 0.19 to 0.60. A q of 0.50 was found to be an optimal compromise between alignment and the signal-to-noise ratio. In each pair of NMR experiments for RDC measurements, the same sample and pulse sequence were used, with one being performed at high-resolution magic-angle spinning to obtain pure J-couplings without RDC. A meaningful structure refinement in bicelles was possible by iteratively fitting the experimental RDCs to the back-calculated RDCs using the high-resolution NMR structure of GlyR TM23 in trifluoroethanol as the starting template. Combination of this method with the conventional high-resolution NMR in membrane mimicking mixtures of water and organic solvents offers an attractive way to derive structural information for membrane proteins in their native environment.