Satinder Singh, PhD
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Research Summary
Signal transduction in the brain is a complex, highly regulated process. The transmission and regulation of nerve impulses between neurons are mediated by a number of proteins such as ion channels, G-protein coupled receptors, protein kinases, protein phosphatases, and neurotransmitter transporters. Importantly, many of these proteins are the target of potent psychoactive substances and antiepileptic drugs. Furthermore, their dysfunction has been implicated in the development of multiple debilitating neuropsychiatric and neurological diseases.
My lab is interested in elucidating the atomic mechanism by which these signaling proteins work, how disease-associated polymorphisms disrupt activity and regulation, and the mechanism by which therapeutic and illicit compounds exert their effects. We are currently focusing our efforts on neurotransmitter transporters. To achieve our objectives, we use a broad array of complementary biochemical and biophysical techniques, including X-ray crystallography, radioligand binding, and flux assays. Our ultimate goal is to help pave the road toward rational, structure-based drug design efforts and to shed light on the molecular underpinnings of disease-associated polymorphisms.
Specialized Terms: Chemical neurotransmission; Neuropsychiatric disease; Epilepsy; Neuropharmacology; Biogenic amine; Gamma-aminobutyric acid; Neurotransmitter transporter; Antidepressant; Structural neurobiology; X-ray crystallography; Transporter kinetics; hydrogen-deuterium exchange mass spectrometry (HDX-MS); nanodisc; liposome
Extensive Research Description
Signal transduction in the human brain is a complex, highly regulated process. The transmission and regulation of nerve impulses among neurons are mediated by a number of proteins, including ion channels, G-protein coupled receptors, protein kinases, protein phosphatases, and neurotransmitter transporters, to name only a few. Significantly, many of these proteins are the target of potent psychoactive substances and antiepileptic drugs. Furthermore, their dysfunction has been implicated in the development of multiple debilitating neuropsychiatric and neurological diseases such as obsessive-compulsive disorder (OCD), autism, depression, schizophrenia, Parkinson’s disease, Tourette’s syndrome, and epilepsy.
Little is known about the molecular basis of these illnesses, but their underlying neural circuitry is gradually being revealed by a combination of functional neuroimaging, genome-wide association, transgenics, optogenetics, and neuropsychopharmacology. For instance, in OCD, an illness marked by intense intrusive thoughts and ritualistic behavior, hyperactivity in the circuit connecting the orbitofrontal cortex, cingulate gyrus, striatum, and caudate nucleus (the orbitofrontal corticostriatal circuit) has been correlated with symptom severity (Graybiel & Rauch, 2000). These brain regions receive extensive input from serotonergic and dopaminergic neurons, and both serotonin and dopamine have been implicated in the pathogenesis of OCD. Indeed, some of more effective treatments for OCD target the serotonin transporter (SERT) and include the tricyclic antidepressant (TCA) clomipramine and a few of the selective serotonin reuptake inhibitors (SSRIs). Although prescribed less frequently, atypical antipsychotics (dopamine D2 / serotonin 5HT2a receptor antagonists) are used as augmenting agents in treatment refractory cases.
My lab seeks to ascertain how signaling proteins in these neural circuits function at the atomic level, how disease-associated polymorphisms disrupt activity and how therapeutic and illicit compounds exert their effects. To achieve our objectives, we employ a broad range of complementary biochemical and biophysical tools such as X-ray crystallography, steady-state flux/binding kinetics, nanodisc technology, and hydrogen-deuterium exchange mass spectrometry (HDX-MS).
We are presently focusing our efforts on the plasma membrane neurotransmitter transporters for the biogenic amines serotonin (SERT) and dopamine (DAT). These molecular machines work by coupling preexisting sodium and chloride electrochemical gradients to the energetically unfavorable movement of the respective neurotransmitter from the synaptic cleft back into neuronal and glial cytoplasms. Because they function primarily after neurotransmitters have been released from the presynaptic neuron and activated postsynaptic receptors, these integral membrane proteins play a crucial role in terminating synaptic transmission and thus in shaping the duration and magnitude of synaptic signaling.
We are examining the substrate/ion specificity of these symporters and the dynamic conformational changes that occur during the transport cycle. We are also attempting to pinpoint antagonist binding sites and to elucidate the atomic mechanism by which psychoactive substances such as TCAs, SSRIs, cocaine, and amphetamine, modulate transport. Our ultimate goal is to help pave the road toward rational, structure-based drug design efforts and to shed light on the molecular underpinnings of disease-associated polymorphisms and drug resistance.
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Research Interests
Antidepressive Agents; Depression; Dopamine; Epilepsy; gamma-Aminobutyric Acid; Glycine; Lipid Bilayers; Neurobiology; Neuropharmacology; Obsessive-Compulsive Disorder; Schizophrenia; Serotonin; Synaptic Vesicles; Antipsychotic Agents; X-Ray Diffraction; Biogenic Monoamines; Protein Structure, Tertiary; Neurotransmitter Transport Proteins; Neuropsychiatry
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Selected Publications
- A Novel Bromine-Containing Paroxetine Analogue Provides Mechanistic Clues for Binding Ambiguity at the Central Primary Binding Site of the Serotonin Transporter.Slack RD, Abramyan AM, Tang H, Meena S, Davis BA, Bonifazi A, Giancola JB, Deschamps JR, Naing S, Yano H, Singh SK, Newman AH, Shi L. A Novel Bromine-Containing Paroxetine Analogue Provides Mechanistic Clues for Binding Ambiguity at the Central Primary Binding Site of the Serotonin Transporter. ACS Chemical Neuroscience 2019, 10: 3946-3952. PMID: 31424193, PMCID: PMC8272913, DOI: 10.1021/acschemneuro.9b00375.
- Computation-guided analysis of paroxetine binding to hSERT reveals functionally important structural elements and dynamics.Abramyan AM, Slack RD, Meena S, Davis BA, Newman AH, Singh SK, Shi L. Computation-guided analysis of paroxetine binding to hSERT reveals functionally important structural elements and dynamics. Neuropharmacology 2019, 161: 107411. PMID: 30391505, PMCID: PMC6494725, DOI: 10.1016/j.neuropharm.2018.10.040.
- Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer.Adhikary S, Deredge DJ, Nagarajan A, Forrest LR, Wintrode PL, Singh SK. Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer. Proceedings Of The National Academy Of Sciences Of The United States Of America 2017, 114: E1786-E1795. PMID: 28223522, PMCID: PMC5347597, DOI: 10.1073/pnas.1613293114.
- Mechanism of Paroxetine (Paxil) Inhibition of the Serotonin Transporter.Davis BA, Nagarajan A, Forrest LR, Singh SK. Mechanism of Paroxetine (Paxil) Inhibition of the Serotonin Transporter. Scientific Reports 2016, 6: 23789. PMID: 27032980, PMCID: PMC4817154, DOI: 10.1038/srep23789.
- Biophysical Approaches to the Study of LeuT, a Prokaryotic Homolog of Neurotransmitter Sodium Symporters.Singh SK, Pal A. Biophysical Approaches to the Study of LeuT, a Prokaryotic Homolog of Neurotransmitter Sodium Symporters. Methods In Enzymology 2015, 557: 167-98. PMID: 25950965, PMCID: PMC4818570, DOI: 10.1016/bs.mie.2015.01.002.
- Cryo-EM: Spinning the Micelles Away.Singh SK, Sigworth FJ. Cryo-EM: Spinning the Micelles Away. Structure (London, England : 1993) 2015, 23: 1561. PMID: 26331455, PMCID: PMC4901521, DOI: 10.1016/j.str.2015.08.001.
- Structure and regulatory interactions of the cytoplasmic terminal domains of serotonin transporter.Fenollar-Ferrer C, Stockner T, Schwarz TC, Pal A, Gotovina J, Hofmaier T, Jayaraman K, Adhikary S, Kudlacek O, Mehdipour AR, Tavoulari S, Rudnick G, Singh SK, Konrat R, Sitte HH, Forrest LR. Structure and regulatory interactions of the cytoplasmic terminal domains of serotonin transporter. Biochemistry 2014, 53: 5444-60. PMID: 25093911, PMCID: PMC4147951, DOI: 10.1021/bi500637f.
- Functionally important carboxyls in a bacterial homologue of the vesicular monoamine transporter (VMAT).Yaffe D, Vergara-Jaque A, Shuster Y, Listov D, Meena S, Singh SK, Forrest LR, Schuldiner S. Functionally important carboxyls in a bacterial homologue of the vesicular monoamine transporter (VMAT). The Journal Of Biological Chemistry 2014, 289: 34229-40. PMID: 25336661, PMCID: PMC4256354, DOI: 10.1074/jbc.M114.607366.
- Radioligand binding to nanodisc-reconstituted membrane transporters assessed by the scintillation proximity assay.Nasr ML, Singh SK. Radioligand binding to nanodisc-reconstituted membrane transporters assessed by the scintillation proximity assay. Biochemistry 2014, 53: 4-6. PMID: 24344975, PMCID: PMC4062192, DOI: 10.1021/bi401412e.
- Crystal structure and association behaviour of the GluR2 amino-terminal domain.Jin R, Singh SK, Gu S, Furukawa H, Sobolevsky AI, Zhou J, Jin Y, Gouaux E. Crystal structure and association behaviour of the GluR2 amino-terminal domain. The EMBO Journal 2009, 28: 1812-23. PMID: 19461580, PMCID: PMC2699365, DOI: 10.1038/emboj.2009.140.
- LeuT: a prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure.Singh SK. LeuT: a prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure. Channels (Austin, Tex.) 2008, 2: 380-9. PMID: 19066470, DOI: 10.4161/chan.2.5.6904.
- A competitive inhibitor traps LeuT in an open-to-out conformation.Singh SK, Piscitelli CL, Yamashita A, Gouaux E. A competitive inhibitor traps LeuT in an open-to-out conformation. Science (New York, N.Y.) 2008, 322: 1655-61. PMID: 19074341, PMCID: PMC2832577, DOI: 10.1126/science.1166777.
- Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.Singh SK, Yamashita A, Gouaux E. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 2007, 448: 952-6. PMID: 17687333, DOI: 10.1038/nature06038.
- Subunit arrangement and function in NMDA receptors.Furukawa H, Singh SK, Mancusso R, Gouaux E. Subunit arrangement and function in NMDA receptors. Nature 2005, 438: 185-92. PMID: 16281028, DOI: 10.1038/nature04089.
- Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters.Yamashita A, Singh SK, Kawate T, Jin Y, Gouaux E. Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 2005, 437: 215-23. PMID: 16041361, DOI: 10.1038/nature03978.
- Photoactive Analogs of Farnesyl Pyrophosphate Containing Benzoylbenzoate Esters: Synthesis and Application to Photoaffinity Labeling of Yeast Protein Farnesyltransferase.Gaon I, Turek TC, Weller VA, Edelstein RL, Singh SK, Distefano MD. Photoactive Analogs of Farnesyl Pyrophosphate Containing Benzoylbenzoate Esters: Synthesis and Application to Photoaffinity Labeling of Yeast Protein Farnesyltransferase. The Journal Of Organic Chemistry 1996, 61: 7738-7745. PMID: 11667728, DOI: 10.1021/jo9602736.