LAUSR

In 1961, Julius Axelrod and colleagues (1) described the effects of psychotropic drugs on the uptake of catecholamine neurotransmitters by several tissues, including at the nerve endings (2), that opened the floodgates of investigation into the identity of the [uptake] channels. Such concerted efforts led to the cloning of many transporter genes, including that for neurotransmitter g-aminobutyric acid (GABA), the principal inhibitory neurotransmitter in the central nervous system (3). Subsequently, it became clear that GABA exerts its physiological actions by activating the ligand-gated ionotropic Cl channels, GABAA, or the metabotropic GABAB receptor (4). GABAA activation typically results in membrane hyperpolarization, resulting in the rapid synaptic inhibition of excitatory signals associated with anxiety disorders. Allosteric modulation of GABAA can be achieved by a diverse class of compounds, including barbiturates, alcohol, steroids, and benzodiazepines. Benzodiazepines are of particular interest for general anxiety disorders, owing to an efficacious anxiolytic profile. Most benzodiazepines also promotemuscle relaxation, which led to the subsequent discovery of GABA and associated channels and receptors in peripheral tissues (5), including airway smoothmuscle (ASM) (6, 7). Research by Charles Emala and colleagues over the past 2 decades has advanced our understanding of the physiological function of GABA channels and receptors on ASM, especially GABAA. Activation of GABAA channels results in ASM relaxation (6, 8, 9) and augments b2-adrenoceptor–mediated airway relaxation (10). Mechanistically, GABAA activation on ASM cells inhibits agonist-evoked membrane depolarization and intracellular calcium flux (8, 9). In contrast, the metabotropic GABAB receptors are G protein–coupled receptors (GPCRs) that, on stimulation with agonists such as baclofen, inhibit (by means of Gi protein) adenylyl cyclases (11). Inhibition of adenylyl cyclases reduces the accumulation of cAMP and short-circuits protein kinase A–mediated ASM relaxation (12). Moreover, GABAB activation leads to increased intracellular calcium (comparable with that induced by Gq-coupled GPCRs such as the M3muscarinic acetylcholine receptor) and potentiates ASM contraction stimulated by other contractile agonists (13), thus limiting the utility of GABAB in the treatment of airflow obstruction in asthma. GABAA channels are hetero-oligomeric with multiple possible combinations of distinct subunits, which creates challenges in developing selective agonists. Fortunately, GABAA on human ASM cells is limited to pentameric oligomers of only a4 and a5 subunits (among a-subunit variants) (8). Emala and colleagues have previously demonstrated that selective pharmacological targeting of these subunits evokes airway relaxation (9, 14, 15) and also limits the development of airway hyperresponsiveness and inflammation inmurine models (14, 16). Finally, in vivo studies in murine models of asthma have established that deletion of the GABAA a4 subunit in mice results in exacerbation of airway inflammation, underscoring its relevance to mitigating asthma features beyond airway constriction (17). In this issue of the Journal, Perez-Zoghbi and colleagues (pp. 482–490) report on their studies in which they sought to characterize a recently developed (16) and highly efficacious agonist (PI320) of GABAA that can also improve tissue specificity, thus avoiding nonspecific activation at central sites (18). PI320 is a modified (polyethylene glycol chain) derivative of an existing GABAA ligand, the imidazodiazepineMIDD0301. Using a combination of in vivo (forced oscillation technique, flexiVent; SCIREQ) and ex vivo (murine precision-cut lung slices [PCLS]) approaches, they demonstrate that PI320 can inhibit methacholine-induced airway contraction. Using transgenic mice that express the intracellular Ca sensor GCaMP6f, the authors elegantly show that PI320 inhibits contractile agonist–induced Ca oscillations. To gain mechanistic insight into PI320-mediated inhibition of agonistinduced Ca, they also loaded murine PCLS with membranepermeable caged inositol 1,4,5-trisphosphate (IP3) to examine the contribution of IP3 receptors on the sarcoplasmic reticulum. By combining flash photolysis (using an ultraviolet-light illuminator) to uncage IP3 in PCLS in tandem with monitoring changes in the airway lumen area with phase-contrast microscopy, the authors demonstrate that PI320 significantly inhibits the airway constriction induced by uncaging IP3. Also, PI320 does not inhibit caffeine-induced murine PCLS contraction. Collectively, these experiments demonstrated that PI320 regulates Camobilization mediated through IP3 receptors (and not ryanodine receptors) on the sarcoplasmic reticulum. The physiological approaches applied by the authors are essential toward establishing the relevance of GABAergic functional outcomes in ASM. However, as noted by the authors, preliminary studies suggest that PI320-mediated relaxation (at least in murine tissues) appears to be independent of GABAA. Although this may be partly due to the differential pharmacokinetic and pharmacodynamic behavior of PI320, muscimol (GABAA agonist), and flumazenil (GABAA antagonist) in different species, additional studies with human tissues (e.g., PCLS) are warranted to establish the clinical relevance of targeting GABAA with PI320. These in vivo and ex vivo approaches could also significantly benefit from supplementation with in vitro live-cell micromechanical approaches that allow for the integration of pharmacological and gene-deletion–based approaches to detail various nuances associated with receptor activation and ASM cell function. Further, reductionist approaches aimed at linking the proximal events after GABAA activation to the downstream signaling driving ASM relaxation will provide much needed clarity into the mechanistic action of PI320. Potentiation of GABAA channels