Locking the dimeric GABA(B) G-protein-coupled receptor in its active state

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• G-protein-coupled receptors (GPCRs) are crucial for cell communication in the central nervous system (CNS).
• GPCRs exist in dynamic inactive and active conformations, typically stabilized by agonists.
• The role of individual protomers in the activation of dimeric GPCRs remains largely unknown.

Purpose of the Study:

• To investigate the role of the GABA(B1) subunit's binding domain conformation in the activation of the heterodimeric GABA(B) receptor.
• To determine if a GPCR can be locked into a constitutively active state.

Main Methods:

• Site-directed mutagenesis to introduce cysteines in the GABA(B1) subunit.
• Formation of a disulfide bridge to stabilize the binding domain in a closed conformation.
• Assessment of receptor activity, agonist binding, and antagonist inhibition.

Main Results:

• A disulfide bridge successfully locked the heterodimeric GABA(B) receptor in a constitutively active state.
• The locked receptor exhibited sustained activity independent of agonists.
• Reversibility was achieved via reduction with dithiothreitol, restoring normal agonist and antagonist responses.

Conclusions:

• The closed conformation of the GABA(B1) binding domain is sufficient to constitutively activate the heterodimeric GABA(B) receptor.
• This study provides the first evidence of locking a GPCR into a stable active conformation.
• Understanding GPCR conformational dynamics offers new therapeutic targets for CNS disorders.