PHOSPHORYLATION-DEPENDENT REGULATION OF GABA TYPE A RECEPTORS: IMPLICATIONS FOR RECEPTOR FUNCTION AND SENSITIVITY TO ALLOSTERIC MODULATORS

Fast inhibitory transmission in the brain is mediated primarily by gamma-aminobutyric acid type A receptors (GABA_ARs), with the α1β2γ2 subtype being predominant in adults. The functional pentamer consists of five subunits arranged around a chloride-permeable pore, and each subunit contains an extracellular domain (ECD), a transmembrane domain (TMD), and a large intracellular domain (ICD). Their activity can be modulated by various protein kinases that target serine residues within the ICD. Although multiple studies have reported that phosphorylation decreases the maximal currents of these receptors, the precise molecular mechanism remains under debate. A leading hypothesis is that this effect results from a reduction in the channel’s unitary conductance. To test whether phosphorylation of the GABA_ARs ICD by PKA affects the function of the α1β2γ2 receptor, we performed whole-cell and single-channel electrophysiological recordings. Under phosphorylation conditions induced by 8-bromo-cAMP, the concentration–response curve showed no significant shift in the EC₅₀, current densities or maximal currents recorded in whole-cell configuration. At the single-channel level, however, the introduction of a phosphomimetic mutant produced a marked reduction in the unitary conductance of the receptor. Remarkably, the application of well-known positive allosteric modulators (PAMs), such as propofol and allopregnanolone, partially restored the reduced unitary conductance. These findings support the critical role of phosphorylation in modulating GABA_ARs function, consistent with observations in other members of the pentameric ligand-gated ion channels (pLGICs) family. This work aims to provide further insight into the intracellular regulation of these receptors, as many molecular, biophysical, and structural aspects remain unclear.