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The structure and function of Cys-loop receptors

The structure and function of Cys-loop receptors Tim Hales, Professor of Pharmacology and Physiology thales@gwu.edu 994-3546 URL: http://www.gwumc.edu/pharm/cys-loop.htm. Cation channels. Anion channels. a 6 a 4 a 5 a 3 a 2 a 1 e g 1 g 3 g 2 b 1 a 1 a 3 a 2 r 3 r 2 r 1 p d

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The structure and function of Cys-loop receptors

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  1. The structure and function of Cys-loop receptors Tim Hales, Professor of Pharmacology and Physiology thales@gwu.edu 994-3546 URL: http://www.gwumc.edu/pharm/cys-loop.htm

  2. Cation channels Anion channels a6 a4 a5 a3 a2 a1 e g1 g3 g2 b1 a1 a3 a2 r3 r2 r1 p d q b1 b3 b2 ZAC a6 a3 a4 a2 b3 a5 a b4 b2 g e d b a10 a9 a7 C D B A 5-HT3 nACh GABAA Glycine GABAA

  3. The structure of Cys-loop receptors Agonist Binding Site Cysteine-loop TM2-TM3 loop Hydrophobic Gate Cytoplasmic residues controlling ion conduction Lys → Met mutation in the g2 subunit of the GABAA receptor associated with febrile seizures

  4. Expressing recombinant Cys-loop receptors Subunit cDNA GFP cDNA

  5. a1b2g2 Whole-cell Cell-attached patch ACh GABA a1b2g2(K289M) 2 pA 200 ms 5 4 control MTSEA DTT 3 * Mean open time (ms) ** 2 1 2 pA 0 40 ms a1b2g2 a1b2(K274M)g2 a1b2g2(K289M) a1(K278M)b2g2 MTSEA DTT 2.0 200 pA MTSEA Outside-out patch Inside-out patch 1.5 2 s I (pA) t = 15 a4b2 80 2 pA 1.0 t = 77 ACh 40 ms 70 20 0.5 5-HT 60 0 15 0 30 60 90 120 380 460 540 620 700 10 50 % samples Time (s) ACh-activated current density (pA/pF) 5 40 0 -3 -2 -1 0 1 -4 I (pA) a4(E584R)b2(E439R) 30 20 2 pA 50 ms 10 * 10 8 ** NR 6 % samples 0 4 2 E592 a4b2(E447R) a4(E592R)b2 0 a4(E584R)b2(E439R) a4(F588R)b2(Q443R) a4(E592R)b2(E447R) -4 -3 -2 -1 0 1 I (pA) F588 E584 Ala MTSEA Gln Identifying the function of specific residues in Cys-loop receptors

  6. Projects: • The role of intracellular portals in controlling ion conduction through nACh and 5-HT3 receptors. We use mutant receptors with cysteines introduced into the putative conduction pathway. Using cysteine modifying reagents we test whether the size and/or charge of these residues influence conductance, kinetics and/or ionic selectivity. Ref: Hales et al., 2006 JBC 281:8062-71, Deeb et al., 2006 (submitted). • The functional significance of mutations in the GABAA receptor associated with febrile seizures. How do mutations that cause epilepsy affect receptor function (e.g. reduced expression, reduced open time, reduced conductance etc). Ref: Hales et al., 2006 JBC 281:17034-43. • The identity of receptors that exert tonic and phasic inhibition in hippocampal pyramidal neurons. We use cultured hippocampal neurons to test the properties of receptors involved in synaptic signaling and compare these to those that are tonically active. Ref: McCartney et al., 2006 (submitted). • Funding: NIH DA05010 and NSF 0447156

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