Direct modulation of TRPC ion channels by Gα proteins.

GPCR-Gi protein pathways are involved in the regulation of vagus muscarinic pathway under physiological conditions and are closely associated with the regulation of internal visceral organs. The muscarinic receptor-operated cationic channel is important in GPCR-Gi protein signal transduction as it decreases heart rate and increases GI rhythm frequency. In the SA node of the heart, acetylcholine binds to the M2 receptor and the released Gßγ activates GIRK (I(K,ACh)) channel, inducing a negative chronotropic action. In gastric smooth muscle, there are two muscarinic acetylcholine receptor (mAChR) subtypes, M2 and M3. M2 receptor activates the muscarinic receptor-operated nonselective cationic current (mIcat, NSCC(ACh)) and induces positive chronotropic effect. Meanwhile, M3 receptor induces hydrolysis of PIP2 and releases DAG and IP3. This IP3 increases intracellular Ca2+ and then leads to contraction of GI smooth muscles. The activation of mIcat is inhibited by anti-Gi/o protein antibodies in GI smooth muscle, indicating the involvement of Gαi/o protein in the activation of mIcat. TRPC4 channel is a molecular candidate for mIcat and can be directly activated by constitutively active Gαi QL proteins. TRPC4 and TRPC5 belong to the same subfamily and both are activated by Gi/o proteins. Initial studies suggested that the binding sites for G protein exist at the rib helix or the CIRB domain of TRPC4/5 channels. However, recent cryo-EM structure showed that IYY58-60 amino acids at ARD of TRPC5 binds with Gi3 protein. Considering the expression of TRPC4/5 in the brain, the direct G protein activation on TRPC4/5 is important in terms of neurophysiology. TRPC4/5 channels are also suggested as a coincidence detector for Gi and Gq pathway as Gq pathway increases intracellular Ca2+ and the increased Ca2+ facilitates the activation of TRPC4/5 channels. More complicated situation would occur when GIRK, KCNQ2/3 (IM) and TRPC4/5 channels are co-activated by stimulation of muscarinic receptors at the acetylcholine-releasing nerve terminals. This review highlights the effects of GPCR-Gi protein pathway, including dopamine, µ-opioid, serotonin, glutamate, GABA, on various oragns, and it emphasizes the importance of considering TRPC4/5 channels as crucial players in the field of neuroscience.

Oncogenic role of FOXM1 in human prostate cancer (Review).

Prostate cancer is the leading cause of cancer­related mortality among men worldwide. In particular, castration­resistant prostate cancer presents a formidable clinical challenge and emphasizes the need to develop novel therapeutic strategies. Forkhead box M1 (FOXM1) is a multifaceted transcription factor that is implicated in the acquisition of the multiple cancer hallmark capabilities in prostate cancer cells, including sustaining proliferative signaling, resisting cell death and the activation of invasion and metastasis. Elevated FOXM1 expression is frequently observed in prostate cancer, and in particular, FOXM1 overexpression is closely associated with poor clinical outcomes in patients with prostate cancer. In the present review, recent advances in the understanding of the oncogenic role of deregulated FOXM1 expression in prostate cancer were highlighted. In addition, the molecular mechanisms by which FOXM1 regulates prostate cancer development and progression were described, thereby providing knowledge and a conceptual framework for FOXM1. The present review also provided valuable insight into the inherent challenges associated with translating biomedical knowledge into effective therapeutic strategies for prostate cancer.

Cyclosporin A inhibits prostate cancer growth through suppression of E2F8 transcription factor in a MELK­dependent manner.

The treatment of advanced prostate cancer remains a formidable challenge due to the limited availability of effective treatment options. Therefore, it is imperative to identify promising druggable targets that provide substantial clinical benefits and to develop effective treatment strategies to overcome therapeutic resistance. Cyclosporin A (CsA) showed an anticancer effect on prostate cancer in cultured cell and xenograft models. E2F8 was identified as a master transcription factor that regulated a clinically significant CsA specific gene signature. The expression of E2F8 increased during prostate cancer progression and high levels of E2F8 expression are associated with a poor prognosis in patients with prostate cancer. MELK was identified as a crucial upstream regulator of E2F8 expression through the transcriptional regulatory network and Bayesian network analyses. Knockdown of E2F8 or MELK inhibited cell growth and colony formation in prostate cancer cells. High expression levels of E2F8 and androgen receptor (AR) are associated with a worse prognosis in patients with prostate cancer compared with low levels of both genes. The inhibition of E2F8 improved the response to AR blockade therapy. These results suggested that CsA has potential as an effective anticancer treatment for prostate cancer, while also revealing the oncogenic role of E2F8 and its association with clinical outcomes in prostate cancer. These results provided valuable insight into the development of therapeutic and diagnostic approaches for prostate cancer.

Low concentrations of tricyclic antidepressants stimulate TRPC4 channel activity by acting as an opioid receptor ligand.

Traditionally prescribed for mood disorders, tricyclic antidepressants (TCAs) have shown promising therapeutic effects on chronic neuralgia and irritable bowel syndrome. However, the mechanism by which these atypical effects manifest is unclear. Among the proposed mechanisms is the well-known pain-related inhibitory G-protein coupled receptor, namely the opioid receptor (OR). Here, we confirmed that TCA indeed stimulates OR and regulates the gating of TRPC4, a downstream signaling of the Gi-pathway. In an ELISA to quantify the amount of intracellular cAMP, a downstream product of OR/Gi-pathway, treatment with amitriptyline (AMI) showed a decrease in [cAMP]i similar to that of the µOR agonist. Next, we explored the binding site of TCA by modeling the previously revealed ligand-bound structure of µOR. A conserved aspartate residue of ORs was predicted to participate in salt bridge interaction with the amine group of TCAs, and in aspartate-to-arginine mutation, AMI did not decrease the FRET-based binding efficiency between the ORs and Gαi2. As an alternative way to monitor the downstream signaling of Gi-pathway, we evaluated the functional activity of TRPC4 channel, as it is well known to be activated by Gαi. TCAs increased the TRPC4 current through ORs, and TCA-evoked TRPC4 activation was abolished by an inhibitor of Gαi2 or its dominant-negative mutant. As expected, TCA-evoked activation of TRPC4 was not observed in the aspartate mutants of OR. Taken together, OR could be proclaimed as a promising target among numerous binding partners of TCA, and TCA-evoked TRPC4 activation may help to explain the nonopioid analgesic effect of TCA.NEW & NOTEWORTHY Endogenous opioid systems modulate pain perception, but concerns about opioid-related substance misuse limit their use. This study has raised TRPC4 channel as a candidate target for alternative analgesics, tricyclic antidepressants (TCAs). TCAs have been shown to bind to and activate opioid receptors (ORs), leading to downstream signaling pathways involving TRPC4. The functional selectivity and biased agonism of TCA towards TRPC4 in dependence on OR may provide a better understanding of its efficacy or side effects.

Emerging role of E2F8 in human cancer.

E2F8 is a multifaceted transcription factor that plays a crucial role in mediating the hallmarks of cancer, including sustaining proliferative signaling, resisting cell death, and activating invasion and metastasis. Aberrant E2F8 expression is associated with poor clinical outcomes in most human cancers. However, E2F8 also exhibits tumor-suppressing activity; thus, the role of E2F8 in cell-fate determination is unclear. In this review, we highlight the recent progress in understanding the role of E2F8 in human cancers, which will contribute to building a conceptual framework and broadening our knowledge pertaining to E2F8. This review provides insight into future challenges and perspectives regarding the translation of biological knowledge into therapeutic strategies for the treatment of cancer.

Molecular architecture of the Gαi-bound TRPC5 ion channel.

G-protein coupled receptors (GPCRs) and ion channels serve as key molecular switches through which extracellular stimuli are transformed into intracellular effects, and it has long been postulated that ion channels are direct effector molecules of the alpha subunit of G-proteins (Gα). However, no complete structural evidence supporting the direct interaction between Gα and ion channels is available. Here, we present the cryo-electron microscopy structures of the human transient receptor potential canonical 5 (TRPC5)-Gαi3 complexes with a 4:4 stoichiometry in lipid nanodiscs. Remarkably, Gαi3 binds to the ankyrin repeat edge of TRPC5 ~ 50 Å away from the cell membrane. Electrophysiological analysis shows that Gαi3 increases the sensitivity of TRPC5 to phosphatidylinositol 4,5-bisphosphate (PIP2), thereby rendering TRPC5 more easily opened in the cell membrane, where the concentration of PIP2 is physiologically regulated. Our results demonstrate that ion channels are one of the direct effector molecules of Gα proteins triggered by GPCR activation-providing a structural framework for unraveling the crosstalk between two major classes of transmembrane proteins: GPCRs and ion channels.

Pore residues of transient receptor potential channels canonical 1 and 4 heteromer determine channel properties.

Transient receptor potential channels canonical 1 and 4 (TRPC1 and TRPC4) are proteins belonging to the same TRPC channel family, and the two are known to form a heterotetrameric channel. TRPC4 can form a homotetrameric, nonselective cation channel by itself, but the involvement of the TRPC1 subunit changes several major characteristics of the channel. In this study, we focused on the pore region (selectivity filter, pore helix, and S6 helix) of TRPC1 and TRPC4 as a determinant of the identity and characteristics of a heteromeric TRPC1/4 channel: decreased calcium permeability of the channel and outward-rectifying current-voltage (I-V) curve. Mutants and chimeras of the pore residues were created, and their currents were recorded using whole cell patch clamp. The lower gate mutants of TRPC4 exhibited diminished calcium permeability as measured by GCaMP6 fluorescence. Also, chimeric channels substituting the pore region of TRPC1 to TRPC4 were made to locate the pore region that is critical in the production of an outward-rectifying I-V curve characteristic of TRPC1/4 heteromeric channels.NEW & NOTEWORTHY Heteromer research has been a challenging field due to lack of structural studies. Using chimeras and single mutants, we present evidence that the pore region of TRPC1/4 heteromer contributes to determining the channel's characteristics such as calcium permeability, I-V curve, and conductance.

Negative self-regulation of transient receptor potential canonical 4 by the specific interaction with phospholipase C-δ1.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. It is suggested that TRPC4ß is regulated by phospholipase C (PLC) signaling and is especially maintained by phosphatidylinositol 4,5-bisphosphate (PIP2). In this study, we present the regulation mechanism of the TRPC4 channel with PIP2 hydrolysis which is mediated by a channel-bound PLCδ1 but not by the GqPCR signaling pathway. Our electrophysiological recordings demonstrate that the Ca2+ via an open TRPC4 channel activates PLCδ1 in the physiological range, and it causes the decrease of current amplitude. The existence of PLCδ1 accelerated PIP2 depletion when the channel was activated by an agonist. Interestingly, PLCδ1 mutants which have lost the ability to regulate PIP2 level failed to reduce the TRPC4 current amplitude. Our results demonstrate that TRPC4 self-regulates its activity by allowing Ca2+ ions into the cell and promoting the PIP2 hydrolyzing activity of PLCδ1.

The intracellular Ca2+ channel TRPML3 is a PI3P effector that regulates autophagosome biogenesis.

Autophagy is a multiple fusion event, initiating with autophagosome formation and culminating with fusion with endo-lysosomes in a Ca2+-dependent manner. The source of Ca2+ and the molecular mechanism by which Ca2+ is provided for this process are not known. The intracellular Ca2+ permeable channel transient receptor potential mucolipin 3 (TRPML3) localizes in the autophagosome and interacts with the mammalian autophagy-related protein 8 (ATG8) homolog GATE16. Here, we show that lipid-regulated TRPML3 is the Ca2+ release channel in the phagophore that provides the Ca2+ necessary for autophagy progress. We generated a TRPML3-GCaMP6 fusion protein as a targeted reporter of TRPML3 compartment localization and channel function. Notably, TRPML3-GCaMP6 localized in the phagophores, the level of which increased in response to nutrient starvation. Importantly, phosphatidylinositol-3-phosphate (PI3P), an essential lipid for autophagosome formation, is a selective regulator of TRPML3. TRPML3 interacted with PI3P, which is a direct activator of TRPML3 current and Ca2+ release from the phagophore, to promote and increase autophagy. Inhibition of TRPML3 suppressed autophagy even in the presence of excess PI3P, while activation of TRPML3 reversed the autophagy inhibition caused by blocking PI3P. Moreover, disruption of the TRPML3-PI3P interaction abolished both TRPML3 activation by PI3P and the increase in autophagy. Taken together, these results reveal that TRPML3 is a downstream effector of PI3P and a key regulator of autophagy. Activation of TRPML3 by PI3P is the critical step providing Ca2+ from the phagophore for the fusion process, which is essential for autophagosome biogenesis.

Distribution and Function of Platelet-derived Growth Factor Receptor Alpha-positive Cells and Purinergic Neurotransmission in the Human Colon: Is It Different Between the Right and Left Colon?

Background/Aims: Platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells function in the purinergic regulation of gastrointestinal motility, and purines are reportedly inhibitory neurotransmitters in the enteric nervous system. We explore the distribution and function of PDGFRα+ cells related to purinergic inhibitory neurotransmission in human right and left colons. Methods: Human colonic segments were prepared with mucosa and submucosa intact, and the circular muscle tension and longitudinal muscle tension were recorded. Purinergic neurotransmitters were administered after recording the regular contractions. Immunohistochemistry was performed on the circular muscle layers. Intracellular recording was performed on the colonic muscular layer. SK3, P2RY1, and PDGFR-α mRNA expression was tested by quantitative real-time polymerase chain reaction (qPCR). Results: Adenosine triphosphate (ATP) treatment significantly decreased the frequency and area under the curve (AUC) of the segmental contraction in right and left colons. Beta-nicotinamide adenine dinucleotide (ß-NAD) decreased the frequency in the right colon and the amplitude, frequency and AUC in the left colon. Apamin significantly increased frequency and AUC in the left colon, and after apamin pretreatment, ATP and ß-NAD did not change segmental contractility. Through intracellular recordings, a resting membrane potential decrease occurred after ATP administration; however, the degree of decrease between the right and left colon was not different. PDGFRα+ cells were distributed evenly in the circular muscle layers of right and left colons. SK3, P2RY1, and PDGFRα expression was not different between the right and left colon. Conclusion: Purines reduce right and left colon contractility similarly, and purinergic inhibitory neurotransmission can be regulated by PDGFRα+ cells in the human colon.

Crizotinib attenuates cancer metastasis by inhibiting TGFß signaling in non-small cell lung cancer cells.

Crizotinib is a clinically approved tyrosine kinase inhibitor for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) harboring EML4-ALK fusion. Crizotinib was originally developed as an inhibitor of MET (HGF receptor), which is involved in the metastatic cascade. However, little is known about whether crizotinib inhibits tumor metastasis in NSCLC cells. In this study, we found that crizotinib suppressed TGFß signaling by blocking Smad phosphorylation in an ALK/MET/RON/ROS1-independent manner in NSCLC cells. Molecular docking and in vitro enzyme activity assays showed that crizotinib directly inhibited the kinase activity of TGFß receptor I through a competitive inhibition mode. Cell tracking, scratch wound, and transwell migration assays showed that crizotinib simultaneously inhibited TGFß- and HGF-mediated NSCLC cell migration and invasion. In addition, in vivo bioluminescence imaging analysis showed that crizotinib suppressed the metastatic capacity of NSCLC cells. Our results demonstrate that crizotinib attenuates cancer metastasis by inhibiting TGFß signaling in NSCLC cells. Therefore, our findings will help to advance our understanding of the anticancer action of crizotinib and provide insight into future clinical investigations.

Inhibition of TRPC4 channel activity in colonic myocytes by tricyclic antidepressants disrupts colonic motility causing constipation.

Tricyclic antidepressants (TCAs) have been used to treat depression and were recently approved for treating irritable bowel syndrome (IBS) patients with severe or refractory IBS symptoms. However, the molecular mechanism of TCA action in the gastrointestinal (GI) tract remains poorly understood. Transient receptor potential channel canonical type 4 (TRPC4), which is a Ca2+ -permeable nonselective cation channel, is a critical regulator of GI excitability. Herein, we investigated whether TCA modulates TRPC4 channel activity and which mechanism in colonic myocytes consequently causes constipation. To prove the clinical benefit in patients with diarrhoea caused by TCA treatment, we performed mechanical tension recording of repetitive motor pattern (RMP) in segment, electric field stimulation (EFS)-induced and spontaneous contractions in isolated muscle strips. From these recordings, we observed that all TCA compounds significantly inhibited contractions of colonic motility in human. To determine the contribution of TRPC4 to colonic motility, we measured the electrical activity of heterologous or endogenous TRPC4 by TCAs using the patch clamp technique in HEK293 cells and murine colonic myocytes. In TRPC4-overexpressed HEK cells, we observed TCA-evoked direct inhibition of TRPC4. Compared with TRPC4-knockout mice, we identified that muscarinic cationic current (mIcat ) was suppressed through TRPC4 inhibition by TCA in isolated murine colonic myocytes. Collectively, we suggest that TCA action is responsible for the inhibition of TRPC4 channels in colonic myocytes, ultimately causing constipation. These findings provide clinical insights into abnormal intestinal motility and medical interventions aimed at IBS therapy.

Plasma Membrane Localized GCaMP-MS4A12 by Orai1 Co-Expression Shows Thapsigargin- and Ca2+-Dependent Fluorescence Increases.

Uniquely expressed in the colon, MS4A12 exhibits store-operated Ca2+ entry (SOCE) activity. However, compared to MS4A1 (CD20), a Ca2+ channel and ideal target for successful leukaemia immunotherapy, MS4A12 has rarely been studied. In this study, we investigated the involvement of MS4A12 in Ca2+ influx and expression changes in MS4A12 in human colonic malignancy. Fluorescence of GCaMP-fused MS4A12 (GCaMP-M12) was evaluated to analyse MS4A12 activity in Ca2+ influx. Plasma membrane expression of GCaMP-M12 was achieved by homo- or hetero-complex formation with no-tagged MS4A12 (nt-M12) or Orai1, respectively. GCaMP-M12 fluorescence in plasma membrane increased only after thapsigargin-induced depletion of endoplasmic reticulum Ca2+ stores, and this fluorescence was inhibited by typical SOCE inhibitors and siRNA for Orai1. Furthermore, GCaMP-MS4A12 and Orai1 co-transfection elicited greater plasma membrane fluorescence than GCaMP-M12 co-transfected with nt-M12. Interestingly, the fluorescence of GCaMP-M12 was decreased by STIM1 over-expression, while increased by siRNA for STIM1 in the presence of thapsigargin and extracellular Ca2+. Moreover, immunoprecipitation assay revealed that Orai1 co-expression decreased protein interactions between MS4A12 and STIM1. In human colon tissue, MS4A12 was expressed in the apical region of the colonic epithelium, although its expression was dramatically decreased in colon cancer tissues. In conclusion, we propose that MS4A12 contributes to SOCE through complex formation with Orai1, but does not cooperate with STIM1. Additionally, we discovered that MS4A12 is expressed in the apical membrane of the colonic epithelium and that its expression is decreased with cancer progression.

Transcriptome Analysis of the Anti-TGFß Effect of Schisandra chinensis Fruit Extract and Schisandrin B in A7r5 Vascular Smooth Muscle Cells.

Schisandra chinensis fruit extract (SCE) has been used as a traditional medicine for treating vascular diseases. However, little is known about how SCE and schisandrin B (SchB) affect transcriptional output-a crucial factor for shaping the fibrotic responses of the transforming growth factor ß (TGFß) signaling pathways in in vascular smooth muscle cells (VSMC). In this study, to assess the pharmacological effect of SCE and SchB on TGFß-induced transcriptional output, we performed DNA microarray experiments in A7r5 VSMCs. We found that TGFß induced distinctive changes in the gene expression profile and that these changes were considerably reversed by SCE and SchB. Gene Set Enrichment Analysis (GSEA) with Hallmark signature suggested that SCE or SchB inhibits a range of fibrosis-associated biological processes, including inflammation, cell proliferation and migration. With our VSMC-specific transcriptional interactome network, master regulator analysis identified crucial transcription factors that regulate the expression of SCE- and SchB-effective genes (i.e., TGFß-reactive genes whose expression are reversed by SCE and SchB). Our results provide novel perspective and insight into understanding the pharmacological action of SCE and SchB at the transcriptome level and will support further investigations to develop multitargeted strategies for the treatment of vascular fibrosis.

The agonistic action of URO-K10 on Kv7.4 and 7.5 channels is attenuated by co-expression of KCNE4 ancillary subunit.

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC50, the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the ß ancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 µM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQspecific agonist in the tissue.

Ca2+/calmodulin-dependent regulation of polycystic kidney disease 2-like-1 by binding at C-terminal domain.

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin-L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM N-lobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu-593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

TRP Channels as Emerging Therapeutic Targets for Neurodegenerative Diseases.

The development of treatment for neurodegenerative diseases (NDs) such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis is facing medical challenges due to the increasingly aging population. However, some pharmaceutical companies have ceased the development of therapeutics for NDs, and no new treatments for NDs have been established during the last decade. The relationship between ND pathogenesis and risk factors has not been completely elucidated. Herein, we review the potential involvement of transient receptor potential (TRP) channels in NDs, where oxidative stress and disrupted Ca2+ homeostasis consequently lead to neuronal apoptosis. Reactive oxygen species (ROS) -sensitive TRP channels can be key risk factors as polymodal sensors, since progressive late onset with secondary pathological damage after initial toxic insult is one of the typical characteristics of NDs. Recent evidence indicates that the dysregulation of TRP channels is a missing link between disruption of Ca2+ homeostasis and neuronal loss in NDs. In this review, we discuss the latest findings regarding TRP channels to provide insights into the research and quests for alternative therapeutic candidates for NDs. As the structures of TRP channels have recently been revealed by cryo-electron microscopy, it is necessary to develop new TRP channel antagonists and reevaluate existing drugs.

Characterization and functional roles of KCNQ-encoded voltage-gated potassium (Kv7) channels in human corpus cavernosum smooth muscle.

The group of KCNQ-encoded voltage-gated potassium (Kv7) channels includes five family members (Kv7.1-7.5). We examined the molecular expression and functional roles of Kv7 channels in corporal smooth muscle (CSM). Isolated rabbit CSM strips were mounted in an organ bath system to characterize Kv7 channels during CSM relaxation. Intracellular Ca2+ levels were measured in the CSM using the Ca2+ dye Fluo-4 AM. The expression of the KCNQ1-5 (the encoding genes for Kv7.1-7.5) and KCNE1-5 subtypes was determined by quantitative real-time PCR. Electrophysiological recordings and an in situ proximity ligation assay (PLA) were also performed. ML213 (a Kv7.2/7.4/7.5 activator) exhibited the most potent relaxation effect. XE911 (a Kv7.1-7.5 blocker) significantly inhibited the relaxation caused by ML213. Removal of the endothelium from the CSM did not affect the relaxation effect of ML213. H-89 (a protein kinase A inhibitor) and ESI-09 (an exchange protein directly activated by cAMP inhibitor) significantly inhibited ML213-induced relaxation (H-89: 31.3%; ESI-09: 52.7%). XE991 significantly increased basal [Ca2+]i in hCSM cells. KCNQ4 (the Kv7.4-encoding gene) and KCNE4 in CSM were the most abundantly expressed subtypes in humans and rats, respectively. KCNQ4 and KCNE4 expression was significantly decreased in diabetes mellitus rats. ML213 significantly increased the outward current amplitude. XE991 inhibited the ML213-induced outward currents. ML213 hyperpolarized the hCSM cell membrane potential. Subsequent addition of XE991 completely reversed the ML213-induced hyperpolarizing effects. A combination of Kv7.4 and Kv7.5 antibodies generated a strong PLA signal. We found that the Kv7.4 channel is a potential target for ED treatment.

Analysis of interaction between intracellular spermine and transient receptor potential canonical 4 channel: multiple candidate sites of negatively charged amino acids for the inward rectification of transient receptor potential canonical 4.

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.

Altered expression of fucosylation pathway genes is associated with poor prognosis and tumor metastasis in non­small cell lung cancer.

Fucosylation is a post­translational modification that attaches fucose residues to protein­ or lipid­bound oligosaccharides. Certain fucosylation pathway genes are aberrantly expressed in several types of cancer, including non­small cell lung cancer (NSCLC), and this aberrant expression is associated with poor prognosis in patients with cancer. However, the molecular mechanism by which these fucosylation pathway genes promote tumor progression has not been well­characterized. The present study analyzed public microarray data obtained from NSCLC samples. Multivariate analysis revealed that altered expression of fucosylation pathway genes, including fucosyltransferase 1 (FUT1), FUT2, FUT3, FUT6, FUT8 and GDP­L­fucose synthase (TSTA3), correlated with poor survival in patients with NSCLC. Inhibition of FUTs by 2F­peracetyl­fucose (2F­PAF) suppressed transforming growth factor ß (TGFß)­mediated Smad3 phosphorylation and nuclear translocation in NSCLC cells. In addition, wound­healing and Transwell migration assays demonstrated that 2F­PAF inhibited TGFß­induced NSCLC cell migration and invasion. Furthermore, in vivo bioluminescence imaging analysis revealed that 2F­PAF attenuated the metastatic capacity of NSCLC cells. These results may help characterize the oncogenic role of fucosylation in NSCLC biology and highlight its potential for developing cancer therapeutics.