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J Biol Chem
2020 Mar 13;29511:3614-3634. doi: 10.1074/jbc.RA119.011527.
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The small molecule GAT1508 activates brain-specific GIRK1/2 channel heteromers and facilitates conditioned fear extinction in rodents.
Xu Y, Cantwell L, Molosh AI, Plant LD, Gazgalis D, Fitz SD, Dustrude ET, Yang Y, Kawano T, Garai S, Noujaim SF, Shekhar A, Logothetis DE, Thakur GA.
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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 brainGIRK1/2 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 brainGIRK1/2 channel subunits, directly or allosterically activates GIRK1/2 channels in the BLA, and facilitates fear extinction in a rodent model.
Figure 6. Specific activation by GAT compounds increases brain GIRK2 but not cardiac GIRK4 channel–PIP2 interactions. A and B, normalized salt-bridge formation between the head group PIP2 and positively-charged channel residues are calculated during the last 25 ns of the 100-ns MD simulation runs in the absence of ligand or with ML297 and GAT1508 bound in the GIRK2/2FD (blue) and GIRK4/4FD (red) systems. C, GIRK1/2 channel NPo was assessed by diC8–PIP2 concentration–response curves using inside-out macropatches from Xenopus oocytes in the presence and absence of 10 μm ML297 or GAT1508; data are means ± S.E. for 5–6 patches per condition. When studied under control conditions (black solid circle), GIRK1/2 channels showed an apparent affinity to diC8–PIP2 of 36 ± 7 μm and an EMAX of 5.7 ± 0.3 (100%). ML297 (brown solid triangle) increases the apparent affinity to 23 ± 5 μm and the EMAX to 7.5 ± 0.4 (100%). GAT1508 (olive green inverted solid triangle) increased the apparent affinity and EMAX further to 5.5 ± 7 and 10.5 ± 0.3 μm (184%), respectively. D, under control conditions, GIRK1/4 channels had an apparent affinity for diC8-PIP2 of 48.4 ± 8 μm and an EMAX of 4.4 ± 0.2 (100%). ML297 increased the apparent affinity to 17.4 ± 2 μm and raised the EMAX to 5.4 ± 0.2 (123% of the control). In contrast, treatment with GAT1508 did not change the apparent affinity for diC8-PIP2 from control (46 ± 7.4 μm) or the efficacy (98.6 and 103%, respectively.
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