1. The structure and function of Cys-loop receptors
Tim Hales, Professor of Pharmacology and Physiology
URL:

2. 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

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. 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

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: , 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:
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