052P Brighton
Winter Meeting December 2007 |
The role of Gln-83 in the GABAρ1 subunit of the GABAA receptor
Katherine Millen, Sarah Lummis
University of Biochemistry, Cambridge, United Kingdom
GABAρ1 is a subunit of the GABAC receptor, a subclass of the GABAA receptor family and a member of the Cys-loop superfamily of ligand gated ion channels which also includes the nicotinic acetylcholine, 5-HT3 and glycine receptors. These receptors share many structural and functional similarities. The GABAC receptor is a homopentamer, with each subunit comprising an extracellular domain (ECD), and a transmembrane region which forms an anion selective pore. As with the other family members the receptor binding site is located between the ECD of two adjacent subunits, and is lined by six binding loops termed A-F. In this study we examined the role of Gln-83, located in the GABAC receptor ECD.
Mutant human GABAρ1 receptor subunits were made using site-directed mutagenesis in pcDNA3.1. GABAρ1 was then subcloned into the pGEMHE vector for mRNA production. Xenopus oocytes were injected with ∼50ng of GABAρ1 receptor subunit mRNA and mutant receptors were functionally assessed using two electrode voltage clamp electrophysiology 24-36 h post-injection. Homology models were created using FUGUE (Shi et al., 2001) aligned sequences and MODELLER 6v2 (Sali et al., 1993) according to the methods described in Harrison et al. (2006).
Substitution of Gln-83 with Ala, Glu and Asn yielded receptors with similar increases (10-30 fold) in GABA EC50 values when compared to wild type receptors (Table 1). These receptors also had lower τoff values, indicating faster off rates (Table 1).
Table 1. * Significantly different from WT, p < 0.05 using one-way ANOVA with Dunnett’s post test
|
pEC50 (mean ± SE) |
EC50 (μM) |
nH (mean ± SE) |
τoff (s) |
Imax (μA) |
n |
| WT |
5.97 ± 0.015 |
1.08 |
1.83 ± 0.10 |
118.3 ± 6.9 |
12.58 ± 1.86 |
20 |
| Q83A |
4.56 ± 0.043* |
27.4 |
1.24 ± 0.14 |
24.2 ± 2.16* |
14.53 ± 2.67 |
6 |
| Q83E |
4.92 ± 0.061* |
12.1 |
1.12 ± 0.12 |
22.7 ± 1.95* |
10.07 ± 3.50 |
6 |
| Q83N |
4.77 ± 0.044* |
16.9 |
1.26 ± 0.14 |
24.4 ± 2.73* |
10.50 ± 2.33 |
6 |
The data shows that GABA responses are sensitive to Gln-83 substitutions, indicating the importance of this residue for receptor function. Our homology model of the GABAC receptor shows that although positioned outside the six binding loops, Gln-83 is located close to the binding site; the backbone oxygen and amide hydrogen could form hydrogen bonds with Tyr-102, which has been reported to play a role in coupling GABA binding to channel opening (Torres et al., 2002). We conclude that Gln-83 plays a role in GABA binding or subsequent receptor gating.
Harrison NJ et al. (2006) J. Mol. Model., 12:317-324
Sali A et al. (1993) J. Mol. Biol., 234:779-815
Shi J et al. (2001) J. Mol. Biol., 310:243-257
Torres VI et al.(2002) J. Biol. Chem. 277:43741-43748
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