Abstract
Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation.
Highlights
Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity
The strength of synaptic inhibition will depend on many factors, not least the number of g-aminobutyric acid type A receptors (GABAARs) clustered at the postsynaptic membrane, and the mean probability of GABA channel opening
The latter approaches, extremely useful, require the expression and monitoring of recombinant receptor protein expressed in native cells, and the behaviour of native GABAARs can only be ascertained by inference
Summary
Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. Of equal importance for effective synaptic inhibition is the potential for different GABAAR isoforms with their attendant differences in physiological and pharmacological properties, to be targeted to specific domains (inhibitory synapses) in the same cell[12,13] To understand how this exquisite targeting of GABAARs to specific membrane domains in single cells relates to their impact on neural activity requires a method to modulate, irreversibly inactivate and/or to track the movement of such receptors. This can be partly achieved with fixed tissue by using receptor subtypespecific antibodies. These ligands have two major advantages over prior methods: first, we can track native GABAARs in situ without the need for recombinant receptor expression in neurons, and second, by choosing a ligand that occludes the GABA binding site, we can inactivate populations of GABAARs in particular areas thereby gaining valuable insight into their function and trafficking, in addition to revealing the importance of membrane delimited inhibition
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