The structure and function of Cys-loop receptors

Tim Hales, Professor of Pharmacology and Physiologythales@gwu.edu

994-3546URL: http://www.gwumc.edu/pharm/cys-loop.htm

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

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

Expressing recombinant Cys-loop receptors

Subunit cDNA

GFP cDNA

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

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