The small molecule GAT1508 activates brain-specific GIRK1/2 channel heteromers and facilitates conditioned fear extinction in rodents

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.

PIP2 regulation of TRPC5 channel activation and desensitization

“…Next, we probed the effect of PKC-mediated effects on the strength of channel–PIP 2 interaction by comparing the kinetics of inhibition due to dephosphorylation of PIP 2 before and after the channel was treated with PMA. The 5’-ptaseOCRL system has been used effectively to compare the influence of different channel modulators on the strength of channel–PIP 2 interactions ( 27 ). After the PMA-induced inhibition of TRPC5 currents reached a steady state ( Fig.…”

Next-generation inward rectifier potassium channel modulators: discovery and molecular pharmacology

“…Although F137 and D173 are required for ML297's ability to activate GIRK1-containing GIRK channels, it remains to be determined conclusively whether these amino acids interact directly with ML297. Computer modeling, mutagenesis, and medicinal chemistry studies have begun to shed more light on the role of these and other amino acid residues in activation of GIRK by ML297 and the closely structurally related compound, GAT1508 (93). These studies implicated additional residues as important for the preference for some compounds, like GAT1508 GIRK1/GIRK2 heteromers compared to GIRK1/GIRK4 heteromers.…”

A benzopyran with antiarrhythmic activity is an inhibitor of Kir3.1-containing potassium channels

“…The pore helix (F137) residue has been shown to be a critical Kir3.1 determinant for current potentiation of heteromeric Kir3 currents and also for ML297- and GAT1508-induced current potentiation ( 6 , 7 , 8 , 9 ). In addition, the M2 helix residue D173 was also shown to be critical for ML297- and GAT1508-induced current stimulation ( Fig.…”

“…Previous studies showed that residues F137 and D179 in the Kir3.1 channel were critical for activation by ML297 and GAT1508. The double mutant Kir3.2:S148F/N184D + Kir3.2 WT could be activated by ML297 and GAT1508 ( 8 , 9 ), demonstrating that these two residues of Kir3.1 are necessary and sufficient for activation by these drugs. We tested the single Kir3.4:S143F, N179D and double Kir3.4:S143F/N179D mutants coexpressed with Kir3.4 and found that the Kir3.1 (F137) was necessary and sufficient for the selective inhibition of heteromeric GIRK channels by the BP-G1 compound ( Fig.…”

“…Mutation of Kir3.1(F137) to the corresponding Ser residue found in Kir3.4 produces functional Kir3.1(F137S) channels although their trapping in the ER continues ( 6 , 7 ). This active Kir3.1 mutant has been an invaluable tool to assessing the role of Kir3.1 subunits in the regulation of heteromeric channel activity, but in addition, it has been found to be a critical determinant of activity in the action of urea-based channel activators ( 8 , 9 ). Kir3 channel activity is strongly inwardly rectifying.…”