Abstract
The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.
Highlights
GABA receptors, which bind the neurotransmitter c-aminobutyric acid (GABA) [1], consist of two main families: GABAA and GABAB (GABAC are a subtype of GABAA) [2]
The present study demonstrates the successful isolation of scFvs against GABAA receptor subunits by phage display technology using synthetic peptides
These scFvs bind to their cognate peptide with minimal or no cross-reactivity to peptides of other GABA subunits
Summary
GABA receptors, which bind the neurotransmitter c-aminobutyric acid (GABA) [1], consist of two main families: GABAA and GABAB (GABAC are a subtype of GABAA) [2]. Functioning of membrane receptors and effects of membrane protein activation is studied using monoclonal or polyclonal antibodies Generating these antibodies is a tedious, time consuming [17] and often-unsuccessful process. ScFvs can be expressed as PLOS ONE | www.plosone.org scFvs to GABAA Receptor Subunits monomers or multimers, depending on the size of the linker [24], or through conjugation to dimerization domains such as leucine zippers [25]. As recombinant entities, their affinity and specificity can be engineered [26,27,28]. ScFvs against membrane proteins can be generated to peptide fragments of these proteins, thereby overcoming the need for generating purified protein targets [39]
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