Project description:Mutations in the KCNJ5 gene, encoding one of the major subunits of cardiac G-protein-gated inwardly rectifying K+ (GIRK) channels, have been recently linked to inherited forms of sinus node dysfunction. Here, the pathogenic mechanism of the W101C KCNJ5 mutation underlying sinus bradycardia in a patient-derived cellular disease model of sinus node dysfunction (SND) was investigated. A human-induced pluripotent stem cell (hiPSCs) line of a mutation carrier was generated, and CRISPR/Cas9-based gene targeting was used to correct the familial mutation as a control line. Both cell lines were further differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed GIRK channels which underly the acetylcholine-regulated K+ current (IK,ACh). hiPSC-CMs with the W101C KCNJ5 mutation (hiPSCW101C-CM) had a constitutively active IK,ACh under baseline conditions; the application of carbachol was able to increase IK,ACh, further indicating that not all available cardiac GIRK channels were open at baseline. Additionally, hiPSCW101C-CM had a more negative maximal diastolic potential (MDP) and a slower pacing frequency confirming the bradycardic phenotype. Of note, the blockade of the constitutively active GIRK channel with XAF-1407 rescued the phenotype. These results provide further mechanistic insights and may pave the way for the treatment of SND patients with GIRK channel dysfunction.
Project description:Ca(2+) influx through L-type Ca(2+) channels (LTCCs) influences numerous physiological processes ranging from contraction in muscle and memory in neurons to gene expression in many cell types. However, the spatiotemporal organization of functional LTCCs has been nearly impossible to investigate because of methodological limitations. Here, we examined LTCC function with high temporal and spatial resolution using evanescent field fluorescence microscopy. Surprisingly, we found that LTCCs operated in functionally organized clusters, not necessarily as individual proteins. Furthermore, LTCC function in these clusters does not appear to be controlled by simple stochastic gating but instead by a PKC-dependent switch mechanism. This work suggests that resting intracellular free calcium concentration in arterial myocytes is predominantly controlled by this process in combination with rare voltage-dependent openings of individual LTCCs. We propose that Ca(2+) influx via persistent LTCCs may be an important mechanism regulating steady-state local and global Ca(2+) signals.
Project description:A hallmark of the GABA projection neurons of the substantia nigra pars reticulata (SNr), a key basal ganglia output nucleus, is its depolarized membrane potential and rapid spontaneous spikes that encode the basal ganglia output. Parkinsonian movement disorders are often associated with abnormalities in SNr GABA neuron firing intensity and/or pattern. A fundamental question remains regarding the molecular identity of the ion channels that drive these neurons to a depolarized membrane potential. We show here that SNr GABA projection neurons selectively express type 3 canonical transient receptor potential (TRPC3) channels. These channels are tonically active and mediate an inward, Na+-dependent current, leading to a substantial depolarization in these neurons. Inhibition of TRPC3 channels induces hyperpolarization, decreases firing frequency, and increases firing irregularity. These data demonstrate that TRPC3 channels play important roles in ensuring the appropriate firing intensity and pattern in SNr GABA projection neurons that are crucial to movement control.
Project description:The increase in dopamine (DA) neurotransmission stimulated by in vivo cocaine exposure is tempered by G protein-dependent inhibitory feedback mechanisms in DA neurons of the ventral tegmental area (VTA). G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels mediate the direct inhibitory effect of GABAB receptor (GABABR) and D2 DA receptor (D2R) activation in VTA DA neurons. Here we examined the effect of the DA neuron-specific loss of GIRK channels on D2R-dependent regulation of VTA DA neuron excitability and on cocaine-induced, reward-related behaviors. Selective ablation of Girk2 in DA neurons did not alter the baseline excitability of VTA DA neurons but significantly reduced the magnitude of D2R-dependent inhibitory somatodendritic currents and blunted the impact of D2R activation on spontaneous activity and neuronal excitability. Mice lacking GIRK channels in DA neurons exhibited increased locomotor activation in response to acute cocaine administration and an altered locomotor sensitization profile, as well as increased responding for and intake of cocaine in an intravenous self-administration test. These mice, however, showed unaltered cocaine-induced conditioned place preference. Collectively, our data suggest that feedback inhibition to VTA DA neurons, mediated by GIRK channel activation, tempers the locomotor stimulatory effect of cocaine while also modulating the reinforcing effect of cocaine in an operant-based self-administration task.
Project description:G protein-gated inwardly rectifying potassium (GIRK) channels are essential regulators of cell excitability in the brain. While they are implicated in a variety of neurological diseases in both human and animal model studies, their therapeutic potential has been largely untapped. Here, we review recent advances in the development of small molecule compounds that specifically modulate GIRK channels and compare them with first-generation compounds that exhibit off-target activity. We describe the method of discovery of these small molecule modulators, their chemical features, and their effects in vivo. These studies provide a promising outlook on the future development of subunit-specific GIRK modulators to regulate neuronal excitability in a brain region-specific manner.
Project description: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:We recently found a constitutively active mutant of natriuretic peptide receptor 2 (caNPR2; V883M), which synthesizes larger amounts of cyclic guanosine monophosphate (cGMP) intracellularly without any ligand stimulation than existing drugs. The aim of this study was to investigate the therapeutic effects of gene transduction using caNPR2 for pulmonary arterial hypertension (PAH). In vitro gene transduction into human pulmonary arterial smooth muscle cells using Sendai virus (SeV) vectors carrying caNPR2 induced 10,000-fold increases in the synthesis of cGMP without ligand stimulation, and the proliferation of caNPR2-expressing cells was significantly attenuated. The PAH model rats generated by hypoxia and the administration of SU5416 were then treated with SeV vectors through a direct injection into the left pulmonary artery. Right ventricular systolic pressure was significantly decreased 2 weeks after the treatment, while systemic blood pressure remained unchanged. Histological analyses revealed that the medial wall thickness and occlusion rate of pulmonary arterioles were significantly improved in caNPR2-treated lungs. Neither the systemic integration of virus vectors nor side effects were observed. The massive stimulation of cGMP synthesis by gene therapy with caNPR2 was safe and effective in a PAH rat model and, thus, has potential as a novel therapy for patients with severe progressive PAH.
Project description:Alcohol abuse disorders are associated with dysfunction of frontal cortical areas including the orbitofrontal cortex (OFC). The OFC is extensively innervated by monoamines, and drugs that target monoamine receptors have been used to treat a number of neuropsychiatric diseases, including alcoholism. However, little is known regarding how monoamines affect OFC neuron excitability or whether this modulation is altered by chronic exposure to ethanol. In this study, we examined the effect of dopamine, norepinephrine, and serotonin on lOFC neuronal excitability in naive mice and in those exposed to chronic intermittent ethanol (CIE) treatment. All three monoamines decreased current-evoked spike firing of lOFC neurons and this action required Giα-coupled D2, α2-adrenergic, and 5HT1A receptors, respectively. Inhibition of firing by dopamine or the D2 agonist quinpirole, but not norepinephrine or serotonin, was prevented by the GABAA receptor antagonist picrotoxin. GABA-mediated tonic current was enhanced by dopamine or the D1 agonist SKF81297 but not quinpirole, whereas the amplitude of spontaneous IPSCs was increased by quinpirole but not dopamine. Spiking was also inhibited by the direct GIRK channel activator ML297, whereas blocking these channels with barium increased firing and eliminated the inhibitory actions of monoamines. In the presence of ML297 or the G-protein blocker GDP-β-S, DA induced a further decrease in spike firing, suggesting the involvement of a non-GIRK channel mechanism. In neurons from CIE-treated mice, spike frequency was nearly doubled and inhibition of firing by monoamines or ML297 was lost. These effects occurred in the absence of significant changes in expression of Gi/o or GIRK channel proteins. Together, these findings show that monoamines are important modulators of lOFC excitability and suggest that disruption of this process could contribute to various deficits associated with alcohol dependence.
Project description:Ion channels of the DEG/ENaC family can induce neurodegeneration under conditions in which they become hyperactivated. The Caenorhabditis elegans DEG/ENaC channel MEC-4(d) encodes a mutant channel with a substitution in the pore domain that causes swelling and death of the six touch neurons in which it is expressed. Dominant mutations in the C. elegans DEG/ENaC channel subunit UNC-8 result in uncoordinated movement. Here we show that this unc-8 movement defect is correlated with the selective death of cholinergic motor neurons in the ventral nerve cord. Experiments in Xenopus laevis ooctyes confirm that these mutant proteins, UNC-8(G387E) and UNC-8(A586T), encode hyperactivated channels that are strongly inhibited by extracellular calcium and magnesium. Reduction of extracellular divalent cations exacerbates UNC-8(G387E) toxicity in oocytes. We suggest that inhibition by extracellular divalent cations limits UNC-8 toxicity and may contribute to the selective death of neurons that express UNC-8 in vivo.
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.