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Mutagenesis of the GABA rho 1 receptor alters agonist affinity and channel gating
T Kusama1, J B Wang, C E Spivak
1Molecular Neurobiology Branch, National Institute on Drug Abuse, Baltimore, MD 21224.
Abstract:
Seventeen site-directed mutations were constructed in the GABA rho 1 receptor with the aim of finding agonist binding domains common to rho 1 and rho 2 receptors but distinct from those identified in members of the family of homologous, ligand gated ion channels. Mutated cDNAs were expressed in Xenopus oocytes and tested by voltage clamp experiments. Five of the mutations abolished responsiveness to GABA. Mutation Q189H, in the conserved cysteine loop, diminished apparent GABA affinity to about 1/10 of wild type values in a manner consistent with decreased allosteric cooperativity among agonist recognition sites. Mutation R316A, located in the extracellular loop between transmembrane domains II and III, increased the Hill coefficient to 3.9 in a fashion consistent with enhanced open probability of a receptor multimer.
Insights
Researchers identified key mutations in GABA rho 1 receptors, revealing distinct agonist binding domains. These findings advance understanding of ligand-gated ion channel function and GABA receptor pharmacology.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- GABA receptors are crucial ligand-gated ion channels.
- Understanding specific binding domains is key to receptor function.
- GABA rho receptors share similarities with other homologous channels.
Purpose of the Study:
- To identify agonist binding domains in GABA rho 1 receptors.
- To find domains common to GABA rho 1 and rho 2 receptors.
- To distinguish these domains from those in other ligand-gated ion channels.
Main Methods:
- Site-directed mutagenesis of the GABA rho 1 receptor.
- Expression of mutated cDNAs in Xenopus oocytes.
- Voltage clamp electrophysiology experiments to assess receptor function.
Main Results:
- Five mutations abolished GABA responsiveness.
- Mutation Q189H reduced GABA affinity and allosteric cooperativity.
- Mutation R316A enhanced the Hill coefficient, suggesting increased open probability.
Conclusions:
- Specific mutations alter GABA rho 1 receptor function and ligand binding.
- The identified mutations provide insights into agonist binding site characteristics.
- This research contributes to the understanding of GABA receptor subtypes and their pharmacology.