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 ZAC a6 a3 a4 a2 b3 a5 a b4 b2 g e d b a10 a9 a7 C D B A nACh 5-HT3 Anion channels a1 g1 g3 g2 b1 r3 r2 r1 p q GABAA Glycine
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
Expressing recombinant Cys-loop receptors Subunit cDNA GFP cDNA
Identifying the function of specific residues in Cys-loop receptors a1b2g2 200 ms 2 pA a1b2g2(K289M) 1 2 3 4 5 a1b2(K274M)g2 a1(K278M)b2g2 Mean open time (ms) ** * 2 s 200 pA a4(F588R)b2(Q443R) a4(E584R)b2(E439R) a4(E592R)b2(E447R) a4(E592R)b2 a4b2(E447R) 10 20 30 40 50 60 70 80 NR ACh-activated current density (pA/pF) ** * ACh Whole-cell Cell-attached patch GABA Inside-out patch 5-HT MTSEA ACh Outside-out patch 40 ms 2 pA a4(E584R)b2(E439R) 50 ms 2 4 6 8 10 -4 -3 -2 -1 1 % samples I (pA) a4b2 20 5 15 40 ms 2 pA control MTSEA DTT I (pA) 30 60 90 120 0.5 1.0 1.5 2.0 380 460 540 620 700 t = 15 t = 77 Time (s) Ala Gln MTSEA E592 F588 E584
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