Haitao Yan

External Ba2+ Block of the Two-pore Domain Potassium Channel TREK-1 Defines Conformational Transition in Its Selectivity Filter

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba2+ blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba2+ at the assumed K+ binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba2+ was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K+-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K+ and proton.

Involvement of Tissue Transglutaminase in Endothelin 1–Induced Hypertrophy in Cultured Neonatal Rat Cardiomyocytes

A potential link between tissue-type transglutaminase (tTG) and cardiac hypertrophy was suggested recently. However, whether tTG is implicated in hypertrophic agonist-induced cardiac hypertrophy is not yet known. The purpose of this study was to investigate the effects of tTG on cardiomyocyte hypertrophy induced by endothelin (ET) 1. Real-time quantitative RT-PCR and Western blot analysis demonstrated that ET-1 increased the expression of tTG mRNA and protein in cardiomyocytes by activating ETA receptors. ET-1 failed to cause increases in cell size and [3H]leucine uptake, sarcomere reorganization, and gene induction of the atrial natriuretic factor when cardiomyocytes were treated with monodansylcadaverine, a competitive inhibitor of tTG. Furthermore, the effects of ET-1 on multifunctional activities of tTG were determined by evaluating the incorporation of [3H]putrescine into N,N′-dimethylated casein and charcoal absorption, respectively. The results showed that ET-1 did not influence the basal transglutaminase activity of cardiomyocytes but significantly inhibited the 0.1-mmol/L Ca2+-stimulated transglutaminase activity. Otherwise, ET-1 elevated the activity of GTPase in a concentration- and time-dependent manner. In vivo, right ventricular hypertrophy induced by 2 weeks of chronic hypoxia was depressed by the tTG inhibitor cystamine (10 to 30 mg/kg, 2 times per day, IP) in a dose-dependent manner. Taken together, our data strongly supported the notion that tTG may act as a positive regulator of the hypertrophic program in response to ET-1. This is probably attributable to the signaling activity of tTG rather than transglutaminase activity.

Allosteric coupling between proximal C-terminus and selectivity filter is facilitated by the movement of transmembrane segment 4 in TREK-2 channel.

TREK-2, a member of two-pore-domain potassium channel family, regulates cellular excitability in response to diverse stimuli. However, how such stimuli control channel function remains unclear. Here, by characterizing the responses of cytosolic proximal C-terminus deletant (ΔpCt) and transmembrane segment 4 (M4)-glycine hinge mutant (G312A) to 2-Aminoethoxydiphenyl borate (2-APB), an activator of TREK-2, we show that the transduction initiated from pCt domain is allosterically coupled with the conformation of selectivity filter (SF) via the movements of M4, without depending on the original status of SF. Moreover, ΔpCt and G312A also exhibited blunted responses to extracellular alkalization, a model to induce SF conformational transition. These results suggest that the coupling between pCt domain and SF is bidirectional, and M4 movements are involved in both processes. Further mechanistic exploration reveals that the function of Phe316, a residue close to the C-terminus of M4, is associated with such communications. However, unlike TREK-2, M4-hinge of TREK-1 only controls the transmission from pCt to SF, rather than SF conformational changes triggered by pHo changes. Together, our findings uncover the unique gating properties of TREK-2, and elucidate the mechanisms for how the extracellular and intracellular stimuli harness the pore gating allosterically.

A Novel Muscarinic Antagonist R2HBJJ Inhibits Non-Small Cell Lung Cancer Cell Growth and Arrests the Cell Cycle in G0/G1

Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs). In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC) cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC.

The GluN3A subunit exerts a neuroprotective effect in brain ischemia and the hypoxia process.

NMDARs (N-methyl-D-aspartate receptors) mediate the predominantly excitatory neurotransmission in the CNS (central nervous system). Excessive release of glutamate and overactivation of NMDARs during brain ischemia and the hypoxia process are causally linked to excitotoxicity and neuronal damage. GluN3 subunits, the third member of the NMDAR family with two isoforms, GluN3A and GluN3B, have been confirmed to display an inhibitory effect on NMDAR activity. However, the effect of GluN3 subunits in brain ischemia and hypoxia is not clearly understood. In the present study, the influence of ischemia and hypoxia on GluN3 subunit expression was observed by using the 2VO (two-vessel occlusion) rat brain ischemia model and cell OGD (oxygen and glucose deprivation) hypoxia model. It was found that GluN3A protein expression in rat hippocampus and the prefrontal cortex was increased quickly after brain ischemia and remained at a high level for at least 24 h. However, the expression of the GluN3B subunit was not remarkably changed in both the animal and cell models. After OGD exposure, rat hippocampal neurons with GluN3A subunit overexpression displayed more viability than the wild-type neurons. NG108-15 cells overexpressing GluN3A presented pronounced resistance to glutamate insult. Blocking the increase of intracellular Ca2+ concentration may underlie the neuroprotective mechanism of up-regulated GluN3A subunit. Suppressing the generation of hydroxyl radicals and NO (nitric oxide) is probably also involved in the neuroprotection.

Involvement of HCN Channel in Muscarinic Inhibitory Action on Tonic Firing of Dorsolateral Striatal Cholinergic Interneurons

The striatum is the most prominent nucleus in the basal ganglia and plays an important role in motor movement regulation. The cholinergic interneurons (ChIs) in striatum are involved in the motion regulation by releasing acetylcholine (ACh) and modulating the output of striatal projection neurons. Here, we report that muscarinic ACh receptor (M receptor) agonists, ACh and Oxotremorine (OXO-M), decreased the firing frequency of ChIs by blocking the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Scopolamine (SCO), a nonselective antagonist of M receptors, abolished the inhibition. OXO-M exerted its function by activating the Gi/o cAMP signaling cascade. The single-cell reverse transcription polymerase chain reaction (scRT-PCR) revealed that all the five subtypes of M receptors and four subtypes of HCN channels were expressed on ChIs. Among them, M2 receptors and HCN2 channels were the most dominant ones and expressed in every single studied cholinergic interneuron (ChI).Our results suggest that ACh regulates not only the output of striatal projection neurons, but also the firing activity of ChIs themselves by activating presynaptic M receptors in the dorsal striatum. The activation of M2 receptors and blockage of HCN2 channels may play an important role in ACh inhibition on the excitability of ChIs. This finding adds a new G-protein coupled receptor mediated regulation on ChIs and provides a cellular mechanism for control of cholinergic activity and ACh release in the dorsal striatum.

ZC88, a novel 4-amino piperidine analog, inhibits the growth of neuroblastoma cells through blocking hERG potassium channel.

Many studies have provided convincing evidence for hERG as an important diagnostic and prognostic factor in human cancers, as well as a useful target for antineoplastic therapy. Our previous study also revealed that knockdown of herg gene expression by shRNA interference inhibited the growth of neuroblastoma cells in vitro and in vivo. In the experiment, a novel 4-amino piperidine analog, ZC88, was examined for its effect on hERG potassium channels and its antitumor potency was observed in vitro and in vivo. The results showed that ZC88 could block hERG1 and hERG1b channels expressed in Xenopus oocytes in a concentration-dependent manner. ZC88 displayed significant antiproliferative activity in several tumor cell lines and the tumor cells with higher expression of hERG presented higher sensitivity to ZC88. The mitotic progression of tumor cells was markedly suppressed in the presence of ZC88 through arresting cells in G0/G1 phase. ZC88 significantly inhibited the tumor growth in nude mice at a dosage with slight influence on the cardiac QT interval. The antitumor effect of ZC88 was correlated at least partly with its blockage of hERG channels, which implicated a positive role of hERG potassium channel in tumor cell proliferation.

Aquaporin 4 Forms a Macromolecular Complex with Glutamate Transporter 1 and Mu Opioid Receptor in Astrocytes and Participates in Morphine Dependence

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes and provides a mechanism by which water permeability of the plasma membrane can be regulated. Evidence suggests that AQP4 is associated with glutamate transporter-1 (GLT-1) for glutamate clearance and contributes to morphine dependence. Previous studies show that AQP4 deficiency changed the mu opioid receptor expression and opioid receptors’ characteristics as well. In this study, we focused on whether AQP4 could form macromolecular complex with GLT-1 and mu opioid receptor (MOR) and participates in morphine dependence. By using immunofluorescence staining, fluorescence resonance energy transfer, and co-immunoprecipitation, we demonstrated that AQP4 forms protein complexes with GLT-1 and MOR in both brain tissue and primary cultured astrocytes. We then showed that the C-terminus of AQP4 containing the amino acid residues 252 to 323 is the site of interaction with GLT-1. Protein kinase C, activated by morphine, played an important role in regulating the expression of these proteins. These findings may help to reveal the mechanism that AQP4, GLT-1, and MOR form protein complex and participate in morphine dependence, and deeply understand the reason that AQP4 deficiency maintains extracellular glutamate homeostasis and attenuates morphine dependence, moreover emphasizes the function of astrocyte in morphine dependence.

Sigma-1 receptor agonist increases axon outgrowth of hippocampal neurons via voltage-gated calcium ions channels

Sigma‐1 receptors (Sig‐1Rs) are unique endoplasmic reticulum proteins that have been implicated in both neurodegenerative and ischemic diseases, such as Alzheimers disease and stroke. Accumulating evidence has suggested that Sig‐1R plays a role in neuroprotection and axon outgrowth. The underlying mechanisms of Sig‐1R‐mediated neuroprotection have been well elucidated. However, the mechanisms underlying the effects of Sig‐1R on axon outgrowth are not fully understood.

Intersubunit Concerted Cooperative and cis-Type Mechanisms Modulate Allosteric Gating in Two-Pore-Domain Potassium Channel TREK-2.

In response to diverse stimuli, two-pore-domain potassium channel TREK-2 regulates cellular excitability, and hence plays a key role in mediating neuropathic pain, mood disorders and ischemia through. Although more and more input modalities are found to achieve their modulations via acting on the channel, the potential role of subunit interaction in these modulations remains to be explored. In the current study, the deletion (lack of proximal C-terminus, ΔpCt) or point mutation (G312A) was introduced into TREK-2 subunits to limit K+ conductance and used to report subunit stoichiometry. The constructs were then combined with wild type (WT) subunit to produce concatenated dimers with defined composition, and the gating kinetics of these channels to 2-Aminoethoxydiphenyl borate (2-APB) and extracellular pH (pHo) were characterized. Our results show that combination of WT and ΔpCt/G312A subunits reserves similar gating properties to that of WT dimmers, suggesting that the WT subunit exerts dominant and positive effects on the mutated one, and thus the two subunits controls channel gating via a concerted cooperative manner. Further introduction of ΔpCt into the latter subunit of heterodimeric channel G312A-WT or G312A-G312A attenuated their sensitivity to 2-APB and pHo alkalization, implicating that these signals were transduced by a cis-type mechanism. Together, our findings elucidate the mechanisms for how the two subunits control the pore gating of TREK-2, in which both intersubunit concerted cooperative and cis-type manners modulate the allosteric regulations induced by 2-APB and pHo alkalization.