Akash Pandhare

Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment*

Background: The lipid sensitivity of the prokaryotic pentameric ligand-gated ion channel (pLGIC), GLIC, is poorly characterized. Results: GLIC is more thermally stable and does not exhibit the same propensity to adopt an uncoupled conformation as the Torpedo nAChR. Conclusion: GLIC is less sensitive to its surrounding membrane environment. Significance: The GLIC and nAChR structures suggest molecular features governing the lipid sensitivity of pLGICs. Although the activity of the nicotinic acetylcholine receptor (nAChR) is exquisitely sensitive to its membrane environment, the underlying mechanisms remain poorly defined. The homologous prokaryotic pentameric ligand-gated ion channel, Gloebacter ligand-gated ion channel (GLIC), represents an excellent model for probing the molecular basis of nAChR sensitivity because of its high structural homology, relative ease of expression, and amenability to crystallographic analysis. We show here that membrane-reconstituted GLIC exhibits structural and biophysical properties similar to those of the membrane-reconstituted nAChR, although GLIC is substantially more thermally stable. GLIC, however, does not possess the same exquisite lipid sensitivity. In particular, GLIC does not exhibit the same propensity to adopt an uncoupled conformation where agonist binding is uncoupled from channel gating. Structural comparisons provide insight into the chemical features that may predispose the nAChR to the formation of an uncoupled state.

[3H]Epibatidine photolabels non-equivalent amino acids in the agonist binding Site of torpedo and α4β2 nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α2βγδ and expressed human α4β2 nAChRs with [3H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr198 in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu109 and γTyr117 in Segment E of the complementary (−) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [3H]epibatidine photolabeled α4Tyr195 (equivalent to Torpedo αTyr190) in Segment C as well as β2Val111 and β2Ser113 in Segment E (equivalent to Torpedo γLeu109 and γTyr111, respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the α-γ transmitter binding site, whereas it binds in two distinct orientations in the α4β2 nAChR.

Photoaffinity Labeling the Agonist Binding Domain of α4β4 and α4β2 Neuronal Nicotinic Acetylcholine Receptors with [125I]Epibatidine and 5[125I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human alpha4beta2 and rat alpha4beta4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [(125)I]epibatidine and 5[(125)I]A-85380. Our results show that both agonists photoincorporated into the beta4 subunit with little or no labeling of the beta2 and alpha4 subunits respectively. [(125)I]epibatidine labeling in the beta4 subunit was mapped to two overlapping proteolytic fragments that begin at beta4V102 and contain Loop E (beta4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that beta4Q117 in Loop E is the principal site of [(125)I]epibatidine labeling. This was accomplished by substituting residues in the beta2 subunit with the beta4 homologs and finding [(125)I]epibatidine labeling in beta4 and beta2F119Q subunits with little, if any, labeling in alpha4, beta2, or beta2S113R subunits. Finally, functional studies established that the beta2F119/beta4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.

Hydrophobic Photolabeling Studies Identify the Lipid-Protein Interface of the 5-HT3A Receptor

A HEK-293 cell line that stably expresses mouse 5-HT(3A)Rs containing a C-terminal extension that confers high-affinity binding of alpha-bungarotoxin (alphaBgTx) was established (alphaBgTx-5-HT(3A)Rs) and used to purify alphaBgTx-5-HT(3A)Rs in a lipid environment for use in structural studies using photoaffinity labeling. alphaBgTx-5-HT(3A)Rs were expressed robustly (60 pmol of [(3)H]BRL-43694 binding sites (approximately 3 microg of receptor) per milligram of protein) and displayed the same functional properties as wild-type receptors (serotonin EC(50) = 5.3 +/- 0.04 microM). While [(125)I]alphaBgTx bound to the alphaBgTx-5-HT(3A)Rs with high affinity (K(d) = 11 nM), application of nonradioactive alphaBgTx (up to 300 microM) had no effect on serotonin-induced current responses. alphaBgTx-5-HT(3A)Rs were purified on an alphaBgTx-derivatized affinity column from detergent extracts in milligram quantities and at approximately 25% purity. The hydrophobic photolabel 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) was used to identify the amino acids at the lipid-protein interface of purified and lipid-reconstituted alphaBgTx-5-HT(3A)Rs. [(125)I]TID photoincorporation into the alphaBgTx-5-HT(3A)R subunit was initially mapped to subunit proteolytic fragments of 8 kDa, containing the M4 transmembrane segment and approximately 60% of incorporated (125)I, and 17 kDa, containing the M1-M3 transmembrane segments. Within the M4 segment, [(125)I]TID labeled Ser(451), equivalent to the [(125)I]TID-labeled residue Thr(422) at the lipid-exposed face of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha1M4 alpha-helix. These results provide a first definition of the surface of the 5-HT(3A)R M4 helix that is exposed to lipid and establish that this surface is equivalent to the surface exposed to lipid in the Torpedo nAChR.

Functional Chimeras of GLIC Obtained by Adding the Intracellular Domain of Anion- and Cation-Conducting Cys-Loop Receptors

Pentameric ligand-gated ion channels (pLGICs), also called Cys-loop receptors in eukaryotic superfamily members, play diverse roles in neurotransmission and serve as primary targets for many therapeutic drugs. Structural studies of full-length eukaryotic pLGICs have been challenging because of glycosylation, large size, pentameric assembly, and hydrophobicity. X-ray structures of prokaryotic pLGICs, including the Gloeobacter violaceus LGIC (GLIC) and the Erwinia chrysanthemi LGIC (ELIC), and truncated eukaryotic pLGICs have significantly improved and complemented the understanding of structural details previously obtained with acetylcholine-binding protein and Torpedo nicotinic acetylcholine receptors. Prokaryotic pLGICs share their overall structural features with eukaryotic pLGICs for the ligand-binding extracellular and channel-lining transmembrane domains. The large intracellular domain (ICD) is present only in eukaryotic members and is characterized by a low level of sequence conservation and significant variability in length (50-250 amino acids), making the ICD a potential target for the modulation of specific pLGIC subunits. None of the structures includes a complete ICD. Here, we created chimeras by adding the ICD of cation-conducting (nAChR-α7) and anion-conducting (GABAρ1, Glyα1) eukaryotic homopentamer-forming pLGICs to GLIC. GLIC-ICD chimeras assemble into pentamers to form proton-gated channels, as does the parent GLIC. Additionally, the sensitivity of the chimeras toward modulation of functional maturation by chaperone protein RIC-3 is preserved as in those of the parent eukaryotic channels. For a previously described GLIC-5HT3A-ICD chimera, we now provide evidence of its successful large-scale expression and purification to homogeneity. Overall, the chimeras provide valuable tools for functional and structural studies of eukaryotic pLGIC ICDs.

The antidepressant bupropion is a negative allosteric modulator of serotonin type 3A receptors.

&NA; The FDA‐approved antidepressant and smoking cessation drug bupropion is known to inhibit dopamine and norepinephrine reuptake transporters, as well as nicotinic acetylcholine receptors (nAChRs) which are cation‐conducting members of the Cys‐loop superfamily of ion channels, and more broadly pentameric ligand‐gated ion channels (pLGICs). In the present study, we examined the ability of bupropion and its primary metabolite hydroxybupropion to block the function of cation‐selective serotonin type 3A receptors (5‐HT3ARs), and further characterized bupropions pharmacological effects at these receptors. Mouse 5‐HT3ARs were heterologously expressed in HEK‐293 cells or Xenopus laevis oocytes for equilibrium binding studies. In addition, the latter expression system was utilized for functional studies by employing two‐electrode voltage‐clamp recordings. Both bupropion and hydroxybupropion inhibited serotonin‐gated currents from 5‐HT3ARs reversibly and dose‐dependently with inhibitory potencies of 87 &mgr;M and 112 &mgr;M, respectively. Notably, the measured IC50 value for hydroxybupropion is within its therapeutically‐relevant concentrations. The blockade by bupropion was largely non‐competitive and non‐use‐dependent. Unlike its modulation at cation‐selective pLGICs, bupropion displayed no significant inhibition of the function of anion‐selective pLGICs. In summary, our results demonstrate allosteric blockade by bupropion of the 5‐HT3AR. Importantly, given the possibility that bupropions major active metabolite may achieve clinically relevant concentrations in the brain, our novel findings delineate a not yet identified pharmacological principle underlying its antidepressant effect. Graphical abstract Figure. No caption available. HighlightsBupropion non‐competitively blocked function of 5‐HT3ARs in X. laevis oocytes.Bupropion inhibition at these receptors was non‐use‐dependent.The metabolite hydroxybupropion also inhibited function of 5‐HT3ARs in oocytes.Our study establishes the 5‐HT3AR as a hitherto unidentified molecular target of bupropion.The study further provides a novel pharmacological basis for bupropions antidepressant effect.

Direct interaction of the resistance to inhibitors of cholinesterase type 3 protein with the serotonin receptor type 3A intracellular domain.

Pentameric ligand‐gated ion channels (pLGIC) are expressed in both excitable and non‐excitable cells that are targeted by numerous clinically used drugs. Assembly from five identical or homologous subunits yields homo‐ or heteromeric pentamers, respectively. The protein known as Resistance to Inhibitors of Cholinesterase (RIC‐3) was identified to interfere with assembly and functional maturation of pLGICs. We have shown previously for serotonin type 3A homopentamers (5‐HT3A) that the interaction with RIC‐3 requires the intracellular domain (ICD) of this pLGIC. After expression in Xenopus laevis oocytes RIC‐3 attenuated serotonin‐induced currents in 5‐HT3A wild‐type channels, but not in functional 5‐HT3AglvM3M4 channels that have the 115‐amino acid ICD replaced by a heptapeptide. In complementary experiments we have shown that engineering the Gloeobacter violaceus ligand‐gated ion channel (GLIC) to contain the 5‐HT3A‐ICD confers sensitivity to RIC‐3 in oocytes to otherwise insensitive GLIC. In this study, we identify endogenous RIC‐3 protein expression in X. laevis oocytes. We purified RIC‐3 to homogeneity after expression in Echericia coli. By using heterologously over‐expressed and purified RIC‐3 and the chimera consisting of the 5‐HT3A‐ICD and the extracellular and transmembrane domains of GLIC in pull‐down experiments, we demonstrate a direct and specific interaction between the two proteins. This result further underlines that the domain within 5‐HT3AR that mediates the interaction with RIC‐3 is the ICD. Importantly, this is the first experimental evidence that the interaction between 5‐HT3AR‐ICD and RIC‐3 does not require other proteins. In addition, we demonstrate that the pentameric assembly of the GLIC‐5‐HT3A‐ICD chimera interacts with RIC‐3.

Pentameric quaternary structure of the intracellular domain of serotonin type 3A receptors

In spite of extensive efforts over decades an experimentally-derived structure of full-length eukaryotic pentameric ligand-gated ion channels (pLGICs) is still lacking. These pharmaceutically highly-relevant channels contain structurally well-conserved and characterized extracellular and transmembrane domains. The intracellular domain (ICD), however, has been orphaned in structural studies based on the consensus assumption of being largely disordered. In the present study, we demonstrate for the first time that the serotonin type 3A (5-HT3A) ICD assembles into stable pentamers in solution in the absence of the other two domains, thought to be the drivers for oligomerization. Additionally, the soluble 5-HT3A-ICD construct interacted with the protein RIC-3 (resistance to inhibitors of cholinesterase). The interaction provides evidence that the 5-HT3A-ICD is not only required but also sufficient for interaction with RIC-3. Our results suggest the ICD constitutes an oligomerization domain. This novel role significantly adds to its known contributions in receptor trafficking, targeting, and functional fine-tuning. The innate diversity of the ICDs with sizes ranging from 50 to 280 amino acids indicates new methodologies need to be developed to determine the structures of these domains. The use of soluble ICD proteins that we report in the present study constitutes a useful approach to address this gap.

Bacterial resistance in India: Studying plasma antibiotic levels

Escalating bacterial resistance to modern antibiotics has been posing a great challenge while treating patients with serious infections in hospitals. The dreaded problem is often further compounded by sub-therapeutic dosages and/or indiscriminate overuse of antibiotics in developing as well as developed countries. As per the estimates made available by the Center for Disease Control and Prevention, over 2 million people are sick due to bacterial infections resistant to one or more antibiotics, and that 23,000 individuals die from drug resistance in the United States. Ironically, 40% of the worlds antibiotic drugs are produced in India, and that over 58,000 babies succumbed to death in 1 year as a direct result of infection with highly-resistant bacteria transmitted from their mothers.[1] A simulation by the Rand Corporation estimated that resistant micro-organisms could kill about 10 million individuals worldwide in 2050, which is greater than cancer deaths. However, we are yet unaware of the full impact in the absence of an efficient global tracking system overseeing the issue. Bacterial resistance evolves as a result of mutations in these organisms as well as selection pressure exerted by unrestrained antibiotic use which provides a competitive advantage to such mutated strains; thereby decreasing overall effectiveness of antibiotics in treating even common infections. In addition, suboptimal use of antibiotic agents further allows to foster stepwise selection of resistance. Thus, mutations, in part, herald resistance genes which may now become transmissible via extrachromosomal elements and/or bacterial plasmids, which are DNA structures that mediate the transfer of resistance genes between bacteria. The resultant resistant clones are amenable to rapid worldwide spread which is facilitated by interspecies gene transmission, poor sanitation and hygiene in communities and hospitals, and the ever increasing global travel, trade, and disease transmission.[1] One of the infamous examples of this is the New Delhi matallo-beta-lactamase-1 (NDM-1) resistance gene discovered by Dr. Timothy Walsh and a team of collaborators.[2] It was first discovered in the United Kingdom in patients returning from India, detected in the wastewater of New Delhi, and hence named “NDM-1.” NDM-1 has been disseminated to 18 countries including the United States and the European countries over the span of 1 year. Such are the epidemiological consequences of the resistant gene travel in our modern “close-knit” world. In response to the emerging antibiotic resistance threat, healthcare providers are increasingly being pushed toward the use of newer and higher antibiotics. Not surprisingly, for more than two decades, life-saving broad spectrum antibiotics such as carbapenems are favorably being administered to critically ill patients in hospitals. The carbapenems belong to the beta-lactam family of antibiotics with an exceptional activity against a broad spectrum of microbes, and with a long history of safety and efficacy for serious infections.[3] They are beta-lactams of choice for the treatment of infections caused by multi-drug resistant organisms.[3] However, unfortunately, now they also have met with resistant bacteria, for example, the arrival of untreatable strains of carbapenem-resistant Enterobacteriaceae. An older carbapenem, imipenem, is degraded by a renal tubular enzyme, dehydropeptidase-1 (DHP-1), and requires co-administration of cilastatin, a DHP-1 inhibitor. However, newer carbapenems such as doripenem, ertapenem, and meropenem are relatively immune to the DHP-1 degradation.[4] Resistant bacteria acquire or develop various strategies to render carbapenems ineffective including structural changes within penicillin-binding proteins, production of metallo-beta-lactamases, and altered membrane permeability due to loss of specific outer membrane proteins, for example, porins.[4] Toward developing effective interventions to address the emergence of microbial resistance as well as understanding the underlying mechanism (s) of resistance, multi-pronged remedial measures are required to put in place. Of these, a major contributing arm is basic sciences as well as clinical research. In this issue of IJCCM the article by Abhilash et al.[6] exemplifies such a contribution to research in India, albeit at a smaller scale, and is a welcome step forward. Here, authors investigate the plasma concentration of imipenem at different loci of infection and conclude that loci variability does not constitute a major factor in affecting the drug plasma concentration within their sample. They further recommend imipenem dosing regimen revisions for the treatment of infection with recalcitrant organisms. Imipenem, indeed, is a tricky antibiotic to use in the case of critically ill patients as compared to that in other patient populations owing to its variable pharmacokinetic activity in the former. Moreover, a very weak correlation exists between its dosage and serum concentration.[5] Importantly, as discussed in the article, a large polymerase chain reaction-based gene screen of isolated bacteria would potentially provide valuable insights into the current/emerging mechanisms of bacterial resistance. Therefore, additional large-scale clinical and microbial gene studies in India are warranted to formulate effective antibiotic dosing regimens, and to deal with the menace of bacterial resistance thereof.

A modified clear-native polyacrylamide gel electrophoresis technique to investigate the oligomeric state of MBP-5-HT3A-intracellular domain chimeras

The main principles of higher-order protein oligomerization are elucidated by many structural and biophysical studies. An astonishing number of proteins self-associate to form dimers or higher-order quaternary structures which further interact with other biomolecules to elicit complex cellular responses. In this study, we describe a simple and convenient approach to determine the oligomeric state of purified protein complexes that combines implementation of a novel form of clear-native gel electrophoresis and size exclusion chromatography in line with multi-angle light scattering. Here, we demonstrate the accuracy of this ensemble approach by characterizing the previously established pentameric state of the intracellular domain of serotonin type 3A (5-HT3A) receptors.