Sahil Talwar

A novel anticonvulsant mechanism via inhibition of complement receptor C5ar1 in murine epilepsy models

The role of complement system-mediated inflammation is of key interest in seizure and epilepsy pathophysiology, but its therapeutic potential has not yet been explored. We observed that the pro-inflammatory C5a receptor, C5ar1, is upregulated in two mouse models after status epilepticus; the pilocarpine model and the intrahippocampal kainate model. The C5ar1 antagonist, PMX53, was used to assess potential anticonvulsant actions of blocking this receptor pathway. PMX53 was found to be anticonvulsant in several acute models (6Hz and corneal kindling) and one chronic seizure model (intrahippocampal kainate model). The effects in the 6Hz model were not found in C5ar1-deficient mice, or with an inactive PMX53 analogue suggesting that the anticonvulsant effect of PMX53 is C5ar1-specific. In the pilocarpine model, inhibition or absence of C5ar1 during status epilepticus lessened seizure power and protected hippocampal neurons from degeneration as well as halved SE-associated mortality. C5ar1-deficiency during pilocarpine-induced status epilepticus also was accompanied by attenuation of TNFα upregulation by microglia, suggesting that C5ar1 activation results in TNFα release contributing to disease. Patch clamp studies showed that C5a-induced microglial K(+) outward currents were also inhibited with PMX53 providing a potential mechanism to explain acute anticonvulsant effects. In conclusion, our data indicate that C5ar1 activation plays a role in seizure initiation and severity, as well as neuronal degeneration following status epilepticus. The widespread anticonvulsant activity of PMX53 suggests that C5ar1 represents a novel target for improved anti-epileptic drug development which may be beneficial for pharmaco-resistant patients.

Phosphorylation of α3 glycine receptors induces a conformational change in the glycine-binding site.

Inflammatory pain sensitization is initiated by prostaglandin-induced phosphorylation of α3 glycine receptors (GlyRs) that are specifically located in inhibitory synapses on spinal pain sensory neurons. Phosphorylation reduces the magnitude of glycinergic synaptic currents, thereby disinhibiting nociceptive neurons. Although α1 and α3 subunits are both expressed on spinal nociceptive neurons, α3 is a more promising therapeutic target as its sparse expression elsewhere implies a reduced risk of side-effects. Here we compared glycine-mediated conformational changes in α1 and α3 GlyRs to identify structural differences that might be exploited in designing α3-specific analgesics. Using voltage-clamp fluorometry, we show that glycine-mediated conformational changes in the extracellular M2-M3 domain were significantly different between the two GlyR isoforms. Using a chimeric approach, we found that structural variations in the intracellular M3-M4 domain were responsible for this difference. This prompted us to test the hypothesis that phosphorylation of S346 in α3 GlyR might also induce extracellular conformation changes. We show using both voltage-clamp fluorometry and pharmacology that Ser346 phosphorylation elicits structural changes in the α3 glycine-binding site. These results provide the first direct evidence for phosphorylation-mediated extracellular conformational changes in pentameric ligand-gated ion channels, and thus suggest new loci for investigating how phosphorylation modulates structure and function in this receptor family. More importantly, by demonstrating that phosphorylation alters α3 GlyR glycine-binding site structure, they raise the possibility of developing analgesics that selectively target inflammation-modulated GlyRs.

Phosphorylation mediated structural and functional changes in pentameric ligand-gated ion channels: implications for drug discovery.

Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes, including fast neurotransmission in the brain. They are targeted by a large number of clinically-important drugs and disruptions to their function are associated with many neurological disorders. The phosphorylation of pLGICs can result in a wide range of functional consequences. Indeed, many neurological disorders result from pLGIC phosphorylation. For example, chronic pain is caused by the protein kinase A-mediated phosphorylation of α3 glycine receptors and nicotine addiction is mediated by the phosphorylation of α4- or α7-containing nicotinic receptors. A recent study demonstrated that phosphorylation can induce a global conformational change in a pLGIC that propagates to the neurotransmitter-binding site. Here we present evidence that phosphorylation-induced global conformational changes may be a universal phenomenon in pLGICs. This raises the possibility of designing drugs to specifically treat disease-modified pLGICs. This review summarizes some of the opportunities available in this area.

Fluorescence-based high-throughput functional profiling of ligand-gated ion channels at the level of single cells

Ion channels are involved in many physiological processes and are attractive targets for therapeutic intervention. Their functional properties vary according to their subunit composition, which in turn varies in a developmental and tissue-specific manner and as a consequence of pathophysiological events. Understanding this diversity requires functional analysis of ion channel properties in large numbers of individual cells. Functional characterisation of ligand-gated channels involves quantitating agonist and drug dose-response relationships using electrophysiological or fluorescence-based techniques. Electrophysiology is limited by low throughput and high-throughput fluorescence-based functional evaluation generally does not enable the characterization of the functional properties of each individual cell. Here we describe a fluorescence-based assay that characterizes functional channel properties at single cell resolution in high throughput mode. It is based on progressive receptor activation and iterative fluorescence imaging and delivers >100 dose-responses in a single well of a 384-well plate, using α1-3 homomeric and αβ heteromeric glycine receptor (GlyR) chloride channels as a model system. We applied this assay with transiently transfected HEK293 cells co-expressing halide-sensitive yellow fluorescent protein and different GlyR subunit combinations. Glycine EC50 values of different GlyR isoforms were highly correlated with published electrophysiological data and confirm previously reported pharmacological profiles for the GlyR inhibitors, picrotoxin, strychnine and lindane. We show that inter and intra well variability is low and that clustering of functional phenotypes permits identification of drugs with subunit-specific pharmacological profiles. As this method dramatically improves the efficiency with which ion channel populations can be characterized in the context of cellular heterogeneity, it should facilitate systems-level analysis of ion channel properties in health and disease and the discovery of therapeutics to reverse pathological alterations.

Xenopus borealis as an alternative source of oocytes for biophysical and pharmacological studies of neuronal ion channels.

For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we report the first use of oocytes from the closely related species Xenopus borealis as an alternative expression system for neuronal ion channels. Using the two-electrode voltage-clamp technique, we show that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either X. laevis or X. borealis oocytes. Potential advantages of the X. borealis oocytes include a smaller endogenous chloride current and the ability to produce more intense fluorescence signals when studied with voltage-clamp fluorometry. Scanning electron microscopy revealed a difference in vitelline membrane structure between the two species, which may be related to the discrepancy in fluorescence signals observed. We demonstrate that X. borealis oocytes are a viable heterologous system for expression of neuronal ion channels with some potential advantages over X. laevis oocytes for certain applications.

The relative orientation of the TM3 and TM4 domains varies between α1 and α3 glycine receptors.

Glycine receptors (GlyRs) are anion-conducting members of the pentameric ligand-gated ion channel family. We previously showed that the dramatic difference in glycine efficacies of α1 and α3 GlyRs is largely attributable to their nonconserved TM4 domains. Because mutation of individual nonconserved TM4 residues had little effect, we concluded that the efficacy difference was a distributed effect of all nonconserved TM4 residues. We therefore hypothesized that the TM4 domains of α1 and α3 GlyRs differ in structure, membrane orientation, and/or molecular dynamic properties. Here we employed voltage-clamp fluorometry to test whether their TM4 domains interact differently with their respective TM3 domains. We found a rhodamine fluorophore covalently attached to a homologous TM4 residue in each receptor interacts differentially with a conserved TM3 residue. We conclude that the α1 and α3 GlyR TM4 domains are orientated differently relative to their TM3 domains. This may underlie their differential ability to influence glycine efficacy.

Rigor, reproducibility, and in vitro cerebrospinal fluid assays: The devil in the details

Rigor, Reproducibility, and In Vitro Cerebrospinal Fluid Assays: The Devil in the Details Olivia A. Moody, BA, Sahil Talwar, PhD, Meagan A. Jenkins, PhD, Amanda A. Freeman, PhD, Lynn Marie Trotti, MD, MSc, Paul S. Garc ıa, MD, PhD, Donald Bliwise, PhD, Joseph W. Lynch, Brad Cherson, RPh, Eric M. Hernandez, MD, PhD, Neil Feldman, MD, Prabhjyot Saini, MSc, David B. Rye, MD, PhD, and Andrew Jenkins, PhD

Glycine Receptor Drug Discovery

Postsynaptic glycine receptor (GlyR) chloride channels mediate inhibitory neurotransmission in the spinal cord and brain stem, although presynaptic and extrasynaptic GlyRs are expressed more widely throughout the brain. In humans, GlyRs are assembled as homo- or heteromeric pentamers of α1-3 and β subunits. GlyR malfunctions have been linked to a range of neurological disorders including hyperekplexia, temporal lobe epilepsy, autism, breathing disorders, and chronic inflammatory pain. Although it is possible that GlyRs may eventually be clinically targeted for a variety of neurological disorders, most research to date has focused on developing GlyR-targeted treatments for chronic pain. Inflammatory pain sensitization is caused by inflammatory mediators downregulating the magnitude of α3 GlyR-mediated inhibitory postsynaptic currents in spinal nociceptive neurons. Consistent with this paradigm, it is now well established that the selective enhancement of α3 GlyR current magnitude is effective in alleviating inflammatory pain. In this review, we briefly describe the physiological roles and pharmacological properties of GlyRs. We then outline the methods commonly used to discover new GlyR-active compounds and review recent progress, in our laboratory and elsewhere, in developing GlyR-targeted analgesics. We conclude that the eventual development of an α3 GlyR-targeted analgesic is an eminently feasible goal. However, in selecting or designing new therapeutic leads, we caution against the automatic exclusion of compounds with potentiating effects on α1 GlyRs. Also, as GlyRs are strongly potentiated by Zn2+ at nanomolar concentrations, we also caution against the identification of false positives caused by contaminating Zn2+ in otherwise pure compound samples.

Rigor, Reproducibility and in vitro CSF assays: The Devil in the Details

Rigor, Reproducibility, and In Vitro Cerebrospinal Fluid Assays: The Devil in the Details Olivia A. Moody, BA, Sahil Talwar, PhD, Meagan A. Jenkins, PhD, Amanda A. Freeman, PhD, Lynn Marie Trotti, MD, MSc, Paul S. Garc ıa, MD, PhD, Donald Bliwise, PhD, Joseph W. Lynch, Brad Cherson, RPh, Eric M. Hernandez, MD, PhD, Neil Feldman, MD, Prabhjyot Saini, MSc, David B. Rye, MD, PhD, and Andrew Jenkins, PhD