Site of action of urea based activators of GIRK channels

Abstract

G-protein coupled Inwardly Rectifying Potassium channels (GIRK) channels particularly GIRK1/2 are predominantly expressed in neuronal tissues. They are involved in regulating the resting membrane potential across all cells they are expressed in. In the brain they are responsible for neurotransmitter induced GIRK mediated neuronal inhibition. GIRK (or Kir3) channels form a subfamily of homo- and hetero-tetrameric channels within the Kir channel family characterized by two-transmembrane helices per subunit that form the basic structural features of a permeation pathway among K+-conducting ion channels. GAT1508, a derivative of the urea based GIRK channel activator ML297, was found to be selective for GIRK1/2 mainly expressed in neuronal cells. Phosphatidylinositol (4,5) bis-phosphate (PIP2) has been found to be absolutely necessary for channel gating controlling directly or indirectly the two channel gates. Various other gating molecules that regulate the opening of the channel such as G, Na+, alcohol and the urea-based drugs show synergies in activating GIRK channels. G and alcohol are known to bind at the cleft formed between the LM and DE loops of two adjacent subunits of the GIRK channel, while urea-based compounds were reported to act between the TM1 and TM2 helices of the GIRK1 subunit [1]. A paper also published in 2020 by Yulin Zhao and colleagues [2] reported a urea-based compound termed G-Protein Independent GIRK channel Activator 1 (GiGA1) to be acting at the alcohol pocket of the GIRK channel, in conflict with the report by Xu and colleagues (2020). The main aim of this Masters thesis work was to test whether the urea compounds ML297 and GiGA1 bind at the alcohol or the TM1-TM2 pockets. We do this by exploiting the concept of synergy and competitive inhibition and by making use of the fact that compounds acting on the same site do not synergize with each other. METHODS: Xenopus Oocytes are injected with 2ng each of GIRK1 and GIRK2 (in case of 1-Propanol, GiGA1, GAT1508 experiments) or 2ng each of GIRK1, GIRK2, GABAB1a and GABAB2 receptors (in case of the baclofen-related experiments). Injected oocytes were incubated for 36hrs after which the GIRK currents were recorded at Low K, High K solutions and different drug concentration solutions using TEVC. In the end 3mM Barium chloride was used to apply block to the channel and cause channel inhibition. RESULTS:Dose-response relationship experiments of the compounds estimates the Minimum effective concentration (MEC) to be 100nM for GAT1508 and Baclofen and 9mM for GiGA1. Baclofen and GAT1508 show synergism when given together at their MECs (32.57 % increase from the baseline when compared to 6.57% with 100nM baclofen and 18.30% with 100nM GAT1508). 1M Baclofen had no effect on the EC50 of 1-Propanol whereas GiGA1 and GAT1508 decreased the EC50 of 1-Propanol from 84mM to 45mM and 54mM respectively. CONCLUSION:Baclofen that activates GIRK channels via G did not synergize with 1-Propanol, whereas each of the GiGA1 and GAT1508 showed synergism with 1-Propanol, Just as GAT1508 had been shown to exhibit synergism with Baclofen. These results suggest that the urea compounds do not bind at the alcohol pocket of GIRK channels. --Author's abstract