Discovery of 'molecular switches' within a GIRK activator scaffold that afford selective GIRK inhibitors.
ABSTRACT: This letter describes a multi-dimensional SAR campaign based on a potent, efficacious and selective GIRK1/2 activator (~10-fold versus GIRK1/4 and inactive on nonGIRK 1-containing GIRKs, GIRK 2 or GIRK2/3). Further chemical optimization through an iterative parallel synthesis effort identified multiple 'molecular switches' that modulated the mode of pharmacology from activator to inhibitor, as well as engendering varying selectivity profiles for GIRK1/2 and GIRK1/4. Importantly, these compounds were all inactive on nonGIRK1 containing GIRK channels. However, SAR was challenging as subtle structural modifications had large effects on both mode of pharmacology and GIRK1/2 and GIRK1/4 channel selectivity.
Project description:The tetrameric G protein-gated K+ channels (GIRKs) mediate inhibitory effects of neurotransmitters that activate Gi/o-coupled receptors. GIRKs are activated by binding of the G?? dimer, via contacts with G?. G? underlies membrane targeting of G??, but has not been implicated in channel gating. We observed that, in Xenopus oocytes, expression of G? alone activated homotetrameric GIRK1* and heterotetrameric GIRK1/3 channels, without affecting the surface expression of GIRK or G?. G? and G? acted interdependently: the effect of G? required the presence of ambient G? and was enhanced by low doses of coexpressed G?, whereas excess of either G? or G? imparted suboptimal activation, possibly by sequestering the other subunit "away" from the channel. The unique distal C-terminus of GIRK1, G1-dCT, was important but insufficient for G? action. Notably, GIRK2 and GIRK1/2 were not activated by G?. Our results suggest that G? regulates GIRK1* and GIRK1/3 channel's gating, aiding G? to trigger the channel's opening. We hypothesize that G? helps to relax the inhibitory effect of a gating element ("lock") encompassed, in part, by the G1-dCT; GIRK2 acts to occlude the effect of G?, either by setting in motion the same mechanism as G?, or by triggering an opposing gating effect.
Project description:G-protein coupled inwardly rectifying potassium channels (GIRKs) are ubiquitously expressed throughout the human body and are an integral part of inhibitory signal transduction pathways. Upon binding of G?? subunits released from G-protein coupled receptors (GPCRs), GIRK channels open and reduce the activity of excitable cells via hyperpolarization. As such, they play a role in cardiac output, the coordination of movement and cognition. Due to their involvement in a multitude of pathways, the precision control of GIRK channels is an important endeavour. Here, we describe the development of the photoswitchable agonist LOGO (the Light Operated GIRK-channel Opener), which activates GIRK channels in the dark and is rapidly deactivated upon exposure to long wavelength UV irradiation. LOGO is the first K+ channel opener and selectively targets channels that contain the GIRK1 subunit. It can be used to optically silence action potential firing in dissociated hippocampal neurons and LOGO exhibits activity in vivo, controlling the motility of zebrafish larvae in a light dependent fashion. We envisage that LOGO will be a valuable research tool to dissect the function of GIRK channels from other GPCR dependent signalling pathways.
Project description:G-protein-gated inwardly rectifying K+ (GIRK) channels are essential effectors of inhibitory neurotransmission in the brain. GIRK channels have been implicated in diseases with abnormal neuronal excitability, including epilepsy and addiction. GIRK channels are tetramers composed of either the same subunit (e.g., homotetramers) or different subunits (e.g., heterotetramers). Compounds that specifically target subsets of GIRK channels in vivo are lacking. Previous studies have shown that alcohol directly activates GIRK channels through a hydrophobic pocket located in the cytoplasmic domain of the channel. Here, we report the identification and functional characterization of a GIRK1-selective activator, termed GiGA1, that targets the alcohol pocket. GiGA1 activates GIRK1/GIRK2 both in vitro and in vivo and, in turn, mitigates the effects of a convulsant in an acute epilepsy mouse model. These results shed light on the structure-based development of subunit-specific GIRK modulators that could provide potential treatments for brain disorders.
Project description:G protein-gated, inwardly rectifying, potassium (GIRK) channels are important regulators of cellular excitability throughout the body. GIRK channels are heterotetrameric and homotetrameric combinations of the Kir3.1-4 (GIRK1-4) subunits. Different subunit combinations are expressed throughout the central nervous system (CNS) and the periphery, and most of these combinations contain a GIRK1 subunit. For example, the predominance of GIRK channels in the CNS are composed of GIRK1 and GIRK2 subunits, while the GIRK channels in cardiac atrial myocytes are made up mostly of GIRK1 and GIRK4 subunits. Although the vast majority of GIRK channels contain a GIRK1 subunit, discrete populations of cells that express non-GIRK1-containing GIRK (non-GIRK1/X) channels do exist. For instance, dopaminergic neurons in the ventral tegmental area of the brain, associated with addiction and reward, do not express the GIRK1 subunit. Targeting these non-GIRK1/X channels with subunit-selective pharmacological probes could lead to important insights into how GIRK channels are involved in reward and addiction. Such insights may, in turn, reveal therapeutic opportunities for the treatment or prevention of addiction. Previously, our laboratory discovered small molecules that can specifically modulate the activity of GIRK1-containing GIRK channels. However, efforts to generate compounds active on non-GIRK1/X channels from these scaffolds have been unsuccessful. Recently, ivermectin was shown to modulate non-GIRK1/X channels, and historically, ivermectin is known to modulate a wide variety of neuronal channels and receptors. Further, ivermectin is a complex natural product, which makes it a challenging starting point for development of more selective, effective, and potent compounds. Thus, while ivermectin provides proof-of-concept as a non-GIRK1/X channel activator, it is of limited utility. Therefore, we sought to discover a synthetic small molecule that would serve as a starting point for the development of non-GIRK1/X channel modulators. To accomplish this, we used a high-throughput thallium flux assay to screen a 100?000-compound library in search of activators of homomeric GIRK2 channels. Using this approach, we discovered VU0529331, the first synthetic small molecule reported to activate non-GIRK1/X channels, to our knowledge. This discovery represents the first step toward developing potent and selective non-GIRK1/X channel probes. Such molecules will help elucidate the role of GIRK channels in addiction, potentially establishing a foundation for future development of therapies utilizing targeted GIRK channel modulation.
Project description:The G-protein activated, inward-rectifying potassium (K(+)) channels, "GIRKs", are a family of ion channels (Kir3.1-Kir3.4) that has been the focus of intense research interest for nearly two decades. GIRKs are comprised of various homo- and heterotetrameric combinations of four different subunits. These subunits are expressed in different combinations in a variety of regions throughout the central nervous system and in the periphery. The body of GIRK research implicates GIRK in processes as diverse as controlling heart rhythm, to effects on reward/addiction, to modulation of response to analgesics. Despite years of GIRK research, very few tools exist to selectively modulate GIRK channels' activity and until now no tools existed that potently and selectively activated GIRKs. Here we report the development and characterization of the first truly potent, effective, and selective GIRK activator, ML297 (VU0456810). We further demonstrate that ML297 is active in two in vivo models of epilepsy, a disease where up to 40% of patients remain with symptoms refractory to present treatments. The development of ML297 represents a truly significant advancement in our ability to selectively probe GIRK's role in physiology as well as providing the first tool for beginning to understand GIRK's potential as a target for a diversity of therapeutic indications.
Project description:To investigate the effects of various chemical classes of antipsychotic drugs: haloperidol, thioridazine, pimozide and clozapine, on the G-protein-activated inwardly rectifying K(+) (GIRK) channels, we carried out Xenopus oocyte functional assays with GIRK1 and GIRK2 mRNAs or GIRK1 and GIRK4 mRNAs. In oocytes co-injected with GIRK1 and GIRK2 mRNAs, application of each of the various antipsychotic drugs immediately caused a reduction of inward currents through the basally active GIRK channels. These responses were not observed in the presence of 3 mM Ba(2+), which blocks the GIRK channels. In addition, in uninjected oocytes, none of the drugs tested produced any significant current response. These results indicate that all the antipsychotic drugs tested inhibited the brain-type GIRK1/2 heteromultimeric channels. Furthermore, similar results were obtained in oocytes co-injected with GIRK1 and GIRK4 mRNAs, indicating that the antipsychotic drugs also inhibited the cardiac-type GIRK1/4 heteromultimeric channels. All the drugs tested inhibited, in a concentration-dependent manner, both types of GIRK channels with varying degrees of potency and effectiveness at micromolar concentrations. Only pimozide caused slight inhibition of these channels at nanomolar concentrations. We conclude that the various antipsychotic drugs acted as inhibitors at the brain-type and cardiac-type GIRK channels. Our results suggest that inhibition of both types of GIRK channels by these drugs underlies some of the side effects, in particular seizures and sinus tachycardia, observed in clinical practice.
Project description:G protein-gated inwardly-rectifying K(+) (GIRK/family 3 of inwardly-rectifying K(+) ) channels are coupled to neurotransmitter action and can play important roles in modulating neuronal excitability. We investigated the temporal and spatial expression of GIRK1, GIRK2 and GIRK3 subunits in the developing and adult brain of mice and rats using biochemical, immunohistochemical and immunoelectron microscopic techniques. At all ages analysed, the overall distribution patterns of GIRK1-3 were very similar, with high expression levels in the neocortex, cerebellum, hippocampus and thalamus. Focusing on the hippocampus, histoblotting and immunohistochemistry showed that GIRK1-3 protein levels increased with age, and this was accompanied by a shift in the subcellular localization of the subunits. Early in development (postnatal day 5), GIRK subunits were predominantly localized to the endoplasmic reticulum in the pyramidal cells, but by postnatal day 60 they were mostly found along the plasma membrane. During development, GIRK1 and GIRK2 were found primarily at postsynaptic sites, whereas GIRK3 was predominantly detected at presynaptic sites. In addition, GIRK1 and GIRK2 expression on the spine plasma membrane showed identical proximal-to-distal gradients that differed from GIRK3 distribution. Furthermore, although GIRK1 was never found within the postsynaptic density (PSD), the level of GIRK2 in the PSD progressively increased and GIRK3 did not change in the PSD during development. Together, these findings shed new light on the developmental regulation and subcellular diversity of neuronal GIRK channels, and support the contention that distinct subpopulations of GIRK channels exert separable influences on neuronal excitability. The ability to selectively target specific subpopulations of GIRK channels may prove effective in the treatment of disorders of excitability.
Project description:Activation of G protein-gated inwardly-rectifying K(+) (GIRK or Kir3) channels by metabotropic gamma-aminobutyric acid (B) (GABA(B)) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABA(B) receptors in cerebellar Purkinje cells at post- and pre-synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co-immunoprecipitation analysis demonstrated that GIRK subunits are co-assembled with GABA(B) receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, post-synaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The post-synaptic association of GIRK subunits with GABA(B) receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At pre-synaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABA(B) receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABA(B) receptors. The association of GIRK channels and GABA(B) receptors with excitatory synapses at both post- and pre-synaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum.
Project description:ML297 was recently identified as a potent and selective small molecule agonist of G-protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels. Here, we show ML297 selectively activates recombinant neuronal GIRK channels containing the GIRK1 subunit in a manner that requires phosphatidylinositol-4,5-bisphosphate (PIP2), but is otherwise distinct from receptor-induced, G-protein-dependent channel activation. Two amino acids unique to the pore helix (F137) and second membrane-spanning (D173) domain of GIRK1 were identified as necessary and sufficient for the selective activation of GIRK channels by ML297. Further investigation into the behavioral effects of ML297 revealed that in addition to its known antiseizure efficacy, ML297 decreases anxiety-related behavior without sedative or addictive liabilities. Importantly, the anxiolytic effect of ML297 was lost in mice lacking GIRK1. Thus, activation of GIRK1-containing channels by ML297 or derivatives may represent a new approach to the treatment of seizure and/or anxiety disorders.
Project description:Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical G betagamma effector G protein-activated K(+) channel (GIRK; Kir3) physically interacts with G betagamma but also with G alpha(i/o). Whether and how G alpha(i/o) subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, G alpha(i3) and G betagamma. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that G betagamma and G alpha(i3) distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that G betagamma greatly enhances the binding of GIRK1 subunit to G alpha(i3)(GDP) and, unexpectedly, to G alpha(i3)(GTP). i-FRET showed that both G alpha(i3) and G betagamma induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a "constitutively active" G alpha(i3) mutant and G betagamma together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either G alpha(i3) or G betagamma. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-G alpha-G betagamma signaling complex and mediates allosteric interactions between G alpha(i)(GTP) and G betagamma. Our findings imply that G alpha(i/o) and the G alpha(i) betagamma heterotrimer can regulate a G betagamma effector both before and after activation by neurotransmitters.