No abstract

G-protein–gated inwardly-rectifying K+ (GIRK) channels are targets of Gi/o-protein–signaling systems that inhibit cell excitability. GIRK channels exist as homotetramers (GIRK2 and GIRK4) or heterotetramers with nonfunctional homomeric subunits (GIRK1 and GIRK3). Although they have been implicated in multiple conditions, the lack of selective GIRK drugs that discriminate among the different GIRK channel subtypes has hampered investigations into their precise physiological relevance and therapeutic potential. Here, we report on a highly-specific, potent, and efficacious activator of brain GIRK1/2 channels. Using a chemical screen and electrophysiological assays, we found that this activator, the bromothiophene-substituted small molecule GAT1508, is specific for brain-expressed GIRK1/2 channels rather than for cardiac GIRK1/4 channels. Computational models predicted a GAT1508-binding site validated by experimental mutagenesis experiments, providing insights into how urea-based compounds engage distant GIRK1 residues required for channel activation. Furthermore, we provide computational and experimental evidence that GAT1508 is an allosteric modulator of channel–phosphatidylinositol 4,5-bisphosphate interactions. Through brain-slice electrophysiology, we show that subthreshold GAT1508 concentrations directly stimulate GIRK currents in the basolateral amygdala (BLA) and potentiate baclofen-induced currents. Of note, GAT1508 effectively extinguished conditioned fear in rodents and lacked cardiac and behavioral side effects, suggesting its potential for use in pharmacotherapy for post-traumatic stress disorder. In summary, our findings indicate that the small molecule GAT1508 has high specificity for brain GIRK1/2 channel subunits, directly or allosterically activates GIRK1/2 channels in the BLA, and facilitates fear extinction in a rodent model.

No abstract

Homomeric a7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by a7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to a7 at the 159 position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L159M) in heteromeric nAChR receptors containing a4 and b2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the a7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c] quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3yl)-urea. The presence of the mutation in the b2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the a4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant b subunit, regardless of the position of the mutant b subunit within the pentameric complex. Similar results were obtained when b2L159M was coexpressed with a2 or a3 and when the L159M mutation was placed in b4 and coexpressed with a2, a3, or a4. Functional receptors were not observed when b1L159M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the a4b2L159M receptor compared with a7 and the availability of high-resolution a4b2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of a7 subunits but are insensitive to PAMs, which greatly increase the open probability of a7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in a7. Specifically, the mutation of the TM2 159 leucine to methionine in a subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal b subunits allows heteromeric receptors to respond to select a7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.