Receptor system response kinetics reveal functional subtypes of native murine and recombinant human GABAA receptors

Results from studies using in situ hybridization, immunocytochemistry and electrophysiological techniques have indicated regional variability in receptor affinity and pharmacology, and have provided evidence that a diversity of ligand-gated ion channels exists in vivo and in vitro. The extent of this diversity is unknown, especially for the amino acid receptors, which are major sources of both excitatory and inhibitory control in the central nervous system (see Roberts, 1986, for review). Given the possibilities of different combinations of subunit isoforms, subunit isoform splice variants and post-transcriptional modifications, there are a vast number of potential receptor subtypes (see Whiting et al. 1995, for review). Heterologous expression of cloned receptors has provided evidence that subunit composition can play an important role in the functional characteristics of the resultant receptor ion channel subtype (Levitan et al. 1988; Pritchett et al. 1989; Verdoorn et al. 1990; Wafford et al. 1993). Thus, there exists the potential for an overwhelming complexity of functional receptor subtypes that may be difficult to separate and study individually. Whole-cell and single channel electrophysiological methods have been used to identify and classify receptor subtypes based upon functional ion channel properties. They have provided considerable information at least about agonist affinity, pharmacological sensitivity, ion channel conductance and single channel kinetics. However, receptor channels are generally studied at or near equilibrium agonist conditions that do not reflect the brief, transient agonist exposure that occurs at the synapse. Additionally, these approaches are time intensive and thus multiple agonist or drug concentrations frequently cannot be completed in the same cell or patch recording. Thus, efforts to identify and study functional characteristics of receptors isolated in a particular recording have been limited. We used rapid ligand exchange techniques to characterize directly intrinsic kinetic properties of receptors in excised patches. Rapid exposure of an excised multichannel patch to a brief pulse of agonist can evoke a transient current response defined here as the ‘receptor system response’. Computer modelling indicated that similar to the engineering concept of the impulse system response, stimulation of a ligand-gated receptor with a brief pulse of agonist (‘impulse’ input) can produce a current response (output response) that defines the system in terms of its frequency properties. The duration of ligand exposure defining the ‘impulse’ is dependent upon receptor properties (see below). The limit can be estimated as a duration sufficient for ligand binding of some receptors but more brief than the time required for maximal channel activation (approximately 80 % of the current rise time; see below). Under these conditions, the kinetics of the receptor system response are uniquely determined by the maximal activation and deactivation properties of the receptor population that are undistorted by slow ligand application or by slow exit from receptor desensitization. Characteristics of the receptor system response allow determination of two important intrinsic gating properties of ligand-gated receptors: maximal ion channel activation rate (approximately equal to maximal opening rate) and receptor deactivation rate. We applied this approach to study structure-function relationships of the GABA type A (GABAA) receptor. In this study, computer-modelling techniques were used to evaluate the properties of transient and equilibrium receptor activation. The results indicate that for at least some ligand-gated ion channels, maximal activation and deactivation kinetic rates can be determined by simulating synaptic conditions of agonist exposure to produce a receptor system response. It was also found that this information is sufficient for functional receptor subtype identification or characterization. Data are presented showing the consistency in receptor subtype between transfected cells and between some cultured neurons. This suggested that the receptor system response may provide a means for functional phenotyping of native receptor subtypes. Finally, these results are compared with known miniature IPSC properties to elucidate synaptic determinants of these characteristic responses to transient agonist exposure.