the structure and function of cys-loop receptors
DESCRIPTION
The structure and function of Cys-loop receptors Tim Hales, Professor of Pharmacology and Physiology [email protected] 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 - PowerPoint PPT PresentationTRANSCRIPT
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The structure and function of Cys-loop receptors
Tim Hales, Professor of Pharmacology and [email protected]
994-3546URL: http://www.gwumc.edu/pharm/cys-loop.htm
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Cation channels
ZA
C 6
3
4
2 3 5 4 2
1
0
9
7 C D B A
nACh 5-HT3
Anion channels
6
4
5
3
2
1
1 3 2 1 1
3
2 3 2 1 1 3 2
GABAA GABAAGlycine
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Agonist Binding Site
Cysteine-loop
TM2-TM3 loop
Hydrophobic Gate
The structure of Cys-loop receptors
Lys → Met mutation in the 2 subunit of the GABAA receptor associated with febrile seizures
Cytoplasmic residues controlling ion conduction
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Expressing recombinant Cys-loop receptors
Subunit cDNA
GFP cDNA
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Identifying the function of specific residues in Cys-loop receptors
ACh
Whole-cellCell-attached patch
GABA
AChOutside-out patch
200 ms
2 pA
(K289M)
0
1
2
3
4
5
(K
274M)
(K278M)
(K289M)
Mea
n op
en t
ime
(ms)
**
*
2 s
200 pA
(F588R)
(Q443R)
E584R)
(E439R)
(E592R)
(E447R)
(E592R)
(E447R)0
10
20
30
40
50
60
70
80
NR
AC
h-ac
tivat
ed c
urre
nt d
ensi
ty (
pA/p
F)
***
Inside-out patch
5-HT MTSEA
40 ms2 pA
control MTSEA DTT
I (p
A)
0 30 60 90 1200
0.5
1.0
1.5
2.0
380 460 540 620 700
MTSEA DTT
= 15
= 77
Time (s)
Ala
GlnMTSEA
40 ms
2 pA
4(E584R)2(E439R)
50 ms
0
2
4
6
8
10
-4 -3 -2 -1 0 1
% s
amp
les
I (pA)
42 2 pA
20
-3 -2 -1 0 10
5
10
15
-4%
sam
ple
sI (pA)
E592
F588
E584
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Projects:
1. 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).
2. 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.
3. 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