Daryl L. Davies, Ph.D.

Transcription

1 Using the OpusXpress to Investigate Ligand- Gated Ion Channels and Transporters in an Academic Laboratory Daryl L. Davies, Ph.D. Director of the Alcohol and Brain Research Lab Research Assistant Professor Molecular Pharmacology and Toxicology School of Pharmacy University of Southern California Los Angeles, CA USA

2 The Lab

3 Why Study Alcohol? Alcohol is the major drug abuse problem in the US Alcoholism affects over 18 million people in the United States alone (cocaine addicts = 6 million) Alcohol Related Deaths 1, annually Cirrhosis Leading Cause of Accidental Deaths in Year Olds Alcohol related birth defects Estimated 1:3 live births (FAS) Leading cause of birth defects with mental retardation 1% Preventable Economic Impact: $185 Billion Per Year ($638 per person) Lost Production Medical Costs Vehicle Accidents Violent Crimes Social Responses Fire Loss

4 Research Questions - Alcohol How does alcohol change brain function? Where does alcohol act in the brain? What neurochemical systems represent initial sites (targets) for ethanol that cause behavioral change? What are the cellular and molecular targets within these systems? Why are these targets sensitive to ethanol? What are the cellular and molecular bases for differences in sensitivity to ethanol between systems? What are the genetic bases for differences in sensitivity to ethanol? How can we use this knowledge to treat alcoholism? Development of alcohol antagonists

5 Using Pressure to Study Molecular Mechanisms/Targets for Ethanol Key Question--What are the molecular targets for ethanol on ligand-gated ion channels? We have developed techniques for using pressure in combination with molecular biology to address this question OpusXpress An automated voltage clamp workstation Uses two-electrode voltage clamp Medium throughput device Provides a method of manipulating the molecular structure of receptors and testing the resultant effects on ethanol (and other drug action) and sensitivity to pressure antagonism Our initial investigations focused on GlyR GlyRs are member of the Cys-loop superfamily of the ligand-gated ion channels

6 Hyperbaric TEVC and the OpusXpress Hyperbaric two-electrode voltage clamp system High Throughput TEVC System to exploit novel sites identified by Hyperbaric TEVC

7 Pressure Significantly Antagonizes High but not Low Concentrations of Ethanol in α1 GlyR Pressure antagonized the effects of 4 to 2 mm ethanol using EC2 glycine Pressure did not antagonize the effects of 1 or 25 mm ethanol using EC2 glycine in the same oocytes that were sensitive to antagonism of higher concentrations These data suggest that there are two sites of action for ethanol in α1 GlyR Pressure antagonism insensitive Pressure antagonism sensitive Davies et al J. Neurochemistry, 24

8 A52S mutation in α1 GlyR The A52S substitution in α1 GlyR is characterized by normal glycine binding properties, with an incerease in the glycine EC5 A52S defines part of a site involved in receptor activation rather than receptor binding An alanine-to-serine exchange at position 52 in the α1 GlyR subunit is responsible for the spasmodic phenotype in mice The mutation at A52S of the α1glyr converts the pressure antagonism sensitive α1glyr to be pressure antagonism insensitive. Ethanol may act through the A52 region by potentiating glycine-induced activation and that pressure antagonizes ethanol by blocking ethanol potentiation of glycine activation in the A52 region. The α2glyr subunit contains a threonine residue at a position equivalent to alanine 52 of the α1 wild-type. Both serine and threonine are polar residues -- Side chain differences may play a role in ethanol sensitivity/pressure antagonism.

9 Molecular Model for Ethanol Sites of Action in α1glyr Loop 7 is Orange Loop 2 is Yellow Enlarged view of the interface between the ligand-binding and transmembrane domains. Important amino acid residues in Loop 7 and the Cterminus of the ligand-binding domain that could interact with A52 are shown. The designation of loops is from the crystal structure of the acetylcholine binding protein

10 WT α1 Glycine CRC 125 Glycine Response (% of maximal current) Concentration (µm) 5 Glycine Conc 1-3 µm Perfuse 2 ml/min V h = -7 mv N=6 I 3 (µa) Glycine EC5 = 153 µm Hill Slope = I 7 (µa) Time (min) Sweep:1 Visible:1 of 1

11 Ethanol Potentiation 4 I 2 (µa) -4 I 5 (µa) -1 I 6 (µa) -1 I 7 (µa) Ethanol Potentiation of Glycine EC 1 Glyeince Induced Cl - Currents % Potentiation 2 Time (min) Sweep:1 Visible:1 of Glycine EtOH Gly + EtOH Glycine EtOH Gly + EtOH

12 NH 3 + OH - Proton coupled transporter Broad substrate specificity Great target for drugs and prodrugs to increase oral bioavailability CO

13 PEPT1 Transporter The GOAL of this project was to gain critical insights into the structure-function relationships of hpept1 by identifying the transmembrane segments that line the substrate translocation pathway and determining the amino acids that play a critical role in substratebinding, proton-binding, and mechanism of transport.

14 PEPT1 Transporter Studies PEPT1 Protocol (Feb3) 5 Episodic Stimulation Step Protocol mV Increament -2mV 2 I 5 (µa) Membrane Potential (mv) mV mV Gly-Sar I-V (Jan25'5#1) Time (msec) I 7 (µa) Voltage dependence of the Gly-Sar-evoked current for hpept1. Current evoked by 2 mm Gly-Sar in a single oocyte expressing the human intestinal oligopeptide transporter hpept Time (ms) Sweep:1 Visible:11 of 11 Current (ua) I Membrane Potential (mv) I/V Relationship Curve

15 Practical Issues

16 Alcohol and Brain Research LAB Directors Ronald Alkana Daryl Davies Collaborators Roberta Brinton James Trudell John Mihic John Woodward Jiang Ye Ian Haworth Consultants R. Adron Harris Neil Harrison Mike Quick Tina Machu Brian King Students Graduate Dan Crawford Ash Kulkarni Yi Shen Post BS Sacha Kuo USC UG Bo Wang Peter Wong Rahul Shaw STAR Program Julio Chong Yu-Chieh Phan Claribel Sanchez Jenny Martinez Sandra Hamada Technicians Miriam Fine Daya Iyer Research Scientist Kiaxun Li Post Doc Andrei Kochegarov Financial Support NSF ABMRF USC Sch of Pharmacy NIAAA