A method for the quantitation of the α1, α2 and α3 isoforms of the GABAA receptor in human brain using competitive PCR

Abstract

The GABAA receptor is the site of action of the inhibitory neurotransmitter GABA, as well as a number of pharmacologically important drugs such as benzodiazepines, barbiturates, and ethanol. The GABAA receptor is a pentameric complex composed of distinct polypeptides, which have been divided into five subunit classes on the basis of sequence homology. To date, 17 isoforms of the receptor have been identified and cloned in mammalian brain, and designated α1–6, β1–4, γ1–4, δ and ρ1–2. In addition, several isoforms exist in alternatively spliced forms (for review see ref. [13]). Studies on recombinant receptors have revealed that receptors constituted from different isoforms exhibit distinct pharmacological properties. For example, the α subunit class appears to be responsible for GABA enhancement of benzodiazepine binding [15]. GABAA receptor function is modulated by benzodiazepine agonists such as flunitrazepam and diazepam, barbiturates, anaesthetics, neurosteroids, and ethanol. Chronic treatment of animals with many of these compounds can bring about profound changes in receptor expression and pharmacology [12]. The RT/PCR assay described here was developed to quantify the α1, α2 and α3 isoforms in the same assay. The amount of each isoform was quantified on the basis of a standard curve generated under identical PCR conditions to the target sequences. In this way it is possible to quantify multiple samples in each RT/PCR assay, thereby reducing inter-assay variability. The assay can be applied to quantify the expression of these isoforms in response to acute and chronic drug administration, or in particular disease states. Altered expression may reflect a corresponding change in protein synthesis, or an alteration of the subtype composition of GABAA receptor.