Mdivi-1: Effective but complex mitochondrial fission inhibitor.

Mdivi-1, Mitochondrial DIVIsion inhibitor 1, has been widely employed in research under the assumption that it exclusively influences mitochondrial fusion, but effects other than mitochondrial dynamics have been underinvestigated. This paper provides transcriptome and DNA methylome-wide analysis for Mdivi-1 treated SH-SY5Y human neuroblastoma cells using RNA sequencing (RNA-seq) and methyl capture sequencing (MC-seq) methods. Gene ontology analysis of RNA sequences revealed that p53 transcriptional gene network and DNA replication initiation-related genes were significantly up and down-regulated, respectively, showing the correlation with the arrest cell cycle in the G1 phase. MC-seq, a powerful sequencing method for capturing DNA methylation status in CpG sites, revealed that although Mdivi-1 does not induce dramatic DNA methylation change, the subtle alterations were concentrated within the CpG island. Integrative analysis of both sequencing data disclosed that the p53 transcriptional network was activated while the Parkinson's disease pathway was halted. Next, we investigated several changes in mitochondria in response to Mdivi-1. Copy number and transcription of mitochondrial DNA were suppressed. ROS levels increased, and elevated ROS triggered mitochondrial retrograde signaling rather than inducing direct DNA damage. In this study, we could better understand the molecular network of Mdivi-1 by analyzing DNA methylation and mRNA transcription in the nucleus and further investigating various changes in mitochondria, providing inspiration for studying nuclear-mitochondrial communications.

Receptor-specific contributions of caveolae, PKC, and Src tyrosine kinase to serotonergic and adrenergic regulation of Kv channels and vasoconstriction.

AIMS: Caveolae are invaginated, Ω-shaped membrane structures. They are now recognized as portals for signal transduction of multiple chemical and mechanical stimuli. Notably, the contribution of caveolae has been reported to be receptor-specific. However, details of how they differentially contribute to receptor signaling remain unclear. MAIN METHODS: Using isometric tension measurements, patch-clamping, and western blotting, we examined the contribution of caveolae and their related signaling pathways to serotonergic (5-HT2A receptor-mediated) and adrenergic (α1-adrenoceptor-mediated) signaling in rat mesenteric arteries. KEY FINDINGS: Disruption of caveolae by methyl-ß-cyclodextrin effectively blocked vasoconstriction mediated by the 5-HT2A receptor (5-HT2AR), but not by the α1-adrenoceptor. Caveolar disruption selectively impaired 5-HT2AR-mediated voltage-dependent K+ channel (Kv) inhibition, but not α1-adrenoceptor-mediated Kv inhibition. In contrast, both serotonergic and α1-adrenergic effects on vasoconstriction, as well as Kv currents, were similarly blocked by the Src tyrosine kinase inhibitor PP2. However, inhibition of protein kinase C (PKC) by either GO6976 or chelerythrine selectively attenuated the effects mediated by the α1-adrenoceptor, but not by 5-HT2AR. Disruption of caveolae decreased 5-HT2AR-mediated Src phosphorylation, but not α1-adrenoceptor-mediated Src phosphorylation. Finally, the PKC inhibitor GO6976 blocked Src phosphorylation by the α1-adrenoceptor, but not by 5-HT2AR. SIGNIFICANCE: 5-HT2AR-mediated Kv inhibition and vasoconstriction are dependent on caveolar integrity and Src tyrosine kinase, but not on PKC. In contrast, α1-adrenoceptor-mediated Kv inhibition and vasoconstriction are not dependent on caveolar integrity, but rather on PKC and Src tyrosine kinase. Caveolae-independent PKC is upstream of Src activation for α1-adrenoceptor-mediated Kv inhibition and vasoconstriction.

Unveiling the impact of lysosomal ion channels: balancing ion signaling and disease pathogenesis.

Ion homeostasis, which is regulated by ion channels, is crucial for intracellular signaling. These channels are involved in diverse signaling pathways, including cell proliferation, migration, and intracellular calcium dynamics. Consequently, ion channel dysfunction can lead to various diseases. In addition, these channels are present in the plasma membrane and intracellular organelles. However, our understanding of the function of intracellular organellar ion channels is limited. Recent advancements in electrophysiological techniques have enabled us to record ion channels within intracellular organelles and thus learn more about their functions. Autophagy is a vital process of intracellular protein degradation that facilitates the breakdown of aged, unnecessary, and harmful proteins into their amino acid residues. Lysosomes, which were previously considered protein-degrading garbage boxes, are now recognized as crucial intracellular sensors that play significant roles in normal signaling and disease pathogenesis. Lysosomes participate in various processes, including digestion, recycling, exocytosis, calcium signaling, nutrient sensing, and wound repair, highlighting the importance of ion channels in these signaling pathways. This review focuses on different lysosomal ion channels, including those associated with diseases, and provides insights into their cellular functions. By summarizing the existing knowledge and literature, this review emphasizes the need for further research in this field. Ultimately, this study aims to provide novel perspectives on the regulation of lysosomal ion channels and the significance of ion-associated signaling in intracellular functions to develop innovative therapeutic targets for rare and lysosomal storage diseases.

Development of Cellular Signaling Pathways by Bioceramic Heat Treatment (Sintering) in Osteoblast Cells.

Bioceramics are calcium-phosphate-based materials used in medical and dental implants for replacing or repairing damaged bone tissues; however, the effect of bioceramic sintering on the intracellular signaling pathways remains unknown. In order to address this, we analyzed the impact of sintering on the cell signaling pathways of osteoblast cells using sintered and non-sintered hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP). X-ray diffraction indicated that only the morphology of HA was affected by sintering; however, the sintered bioceramics were found to have elevated the calcium concentrations in relation to the non-sintered variants. Both bioceramics inhibited the JNK signaling pathway; the sintered HA exhibited half the value of the non-sintered variant, while the sintered ß-TCP rarely expressed a p-JNK value. The total Src and Raptor protein concentrations were unaffected by the sintering, while the p-Src concentrations were decreased. The p-EGFR signaling pathway was regulated by the non-sintered bioceramics, while the p-p38 concentrations were reduced by both the sintered ß-TCP and HA. All of the bioceramics attenuated the total AKT concentrations, particularly the non-sintered HA, and the AKT phosphorylation concentration, except for the non-sintered ß-TCP. Thus, the sintering of bioceramics affects several intracellular signaling pathways. These findings may elucidate the bioceramic function and expand their application scope as novel substrates in clinical applications.

Multiple proliferation signaling pathways are modulated by octacalcium phosphate in osteoblasts.

Octacalcium phosphate (OCP), a type of bioactive ceramics, may be associated with dentine, tooth apatite, and especially bone generation, and promotes wound healing after fracture. Recently, commercial bone grafting products containing a large amount of OCP material have been released because OCP can be synthesized in large quantities. It is reported to increase cell proliferation, but the interaction between OCP and cell signaling pathways is still unclear. In this study, first, we demonstrated OCP mediated cell signaling pathways with only purified OCP materials. OCP regulated P38, JNK (c-Jun N-terminal kinase), Src, and AKT (protein kinase B) signaling pathways. OCP crystals appeared in the characteristic ribbon shape but varied by several tens of micrometers in size. The X-ray diffraction pattern was the same as previously reported. We studied two concentrations of OCP (10 mg/ml and 20 mg/ml) to understand whether the effect of OCP on cell signaling pathways is dose dependent. We confirmed that OCP treatment affected cell proliferation and alkaline phosphatase and disrupted Src phosphorylation but did not change the total protein level. P38 phosphorylation was activated with OCP treatment and inhibited by SB203580, but P38 total protein level did not change. OCP inhibited JNK phosphorylation signaling, whereas PD98509 inhibited JNK phosphorylation with or without OCP. Interestingly, the AKT total level decreased after OCP treatment, but AKT phosphorylation increased considerably. Our results demonstrate that OCP materials modulate cell signaling pathways and increase cell proliferation.

A growth-factor-activated lysosomal K+ channel regulates Parkinson's pathology.

Lysosomes have fundamental physiological roles and have previously been implicated in Parkinson's disease1-5. However, how extracellular growth factors communicate with intracellular organelles to control lysosomal function is not well understood. Here we report a lysosomal K+ channel complex that is activated by growth factors and gated by protein kinase B (AKT) that we term lysoKGF. LysoKGF consists of a pore-forming protein TMEM175 and AKT: TMEM175 is opened by conformational changes in, but not the catalytic activity of, AKT. The minor allele at rs34311866, a common variant in TMEM175, is associated with an increased risk of developing Parkinson's disease and reduces channel currents. Reduction in lysoKGF function predisposes neurons to stress-induced damage and accelerates the accumulation of pathological α-synuclein. By contrast, the minor allele at rs3488217-another common variant of TMEM175, which is associated with a decreased risk of developing Parkinson's disease-produces a gain-of-function in lysoKGF during cell starvation, and enables neuronal resistance to damage. Deficiency in TMEM175 leads to a loss of dopaminergic neurons and impairment in motor function in mice, and a TMEM175 loss-of-function variant is nominally associated with accelerated rates of cognitive and motor decline in humans with Parkinson's disease. Together, our studies uncover a pathway by which extracellular growth factors regulate intracellular organelle function, and establish a targetable mechanism by which common variants of TMEM175 confer risk for Parkinson's disease.

Intellectual disability-associated UNC80 mutations reveal inter-subunit interaction and dendritic function of the NALCN channel complex.

The sodium-leak channel NALCN forms a subthreshold sodium conductance that controls the resting membrane potentials of neurons. The auxiliary subunits of the channel and their functions in mammals are largely unknown. In this study, we demonstrate that two large proteins UNC80 and UNC79 are subunits of the NALCN complex. UNC80 knockout mice are neonatal lethal. The C-terminus of UNC80 contains a domain that interacts with UNC79 and overcomes a soma-retention signal to achieve dendritic localization. UNC80 lacking this domain, as found in human patients, still supports whole-cell NALCN currents but lacks dendritic localization. Our results establish the subunit composition of the NALCN complex, uncover the inter-subunit interaction domains, reveal the functional significance of regulation of dendritic membrane potential by the sodium-leak channel complex, and provide evidence supporting that genetic variations found in individuals with intellectual disability are the causes for the phenotype observed in patients.

Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation.

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3.

The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signalling. The transient receptor potential mucolipin (TRPML) channel family belongs to the TRP superfamily and is composed of three members: TRPML1-TRPML3. TRPMLs are the major Ca2+-permeable channels on late endosomes and lysosomes (LEL). They regulate the release of Ca2+ from organelles, which is important for various physiological processes, including organelle trafficking and fusion. Loss-of-function mutations in the MCOLN1 gene, which encodes TRPML1, cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV, and a gain-of-function mutation (Ala419Pro) in TRPML3 gives rise to the varitint-waddler (Va) mouse phenotype. Notably, TRPML channels are activated by the low-abundance and LEL-enriched signalling lipid phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), whereas other phosphoinositides such as PtdIns(4,5)P2, which is enriched in plasma membranes, inhibit TRPMLs. Conserved basic residues at the N terminus of the channel are important for activation by PtdIns(3,5)P2 and inhibition by PtdIns(4,5)P2. However, owing to a lack of structural information, the mechanism by which TRPML channels recognize PtdIns(3,5)P2 and increase their Ca2+ conductance remains unclear. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3 channel from the common marmoset (Callithrix jacchus) at an overall resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain that we term the mucolipin domain. Combined with functional studies, these data suggest that the mucolipin domain is responsible for PtdIns(3,5)P2 binding and subsequent channel activation, and that it acts as a 'gating pulley' for lipid-dependent TRPML gating.

GABBR2 mutations determine phenotype in rett syndrome and epileptic encephalopathy.

OBJECTIVE: Rett syndrome (RTT) and epileptic encephalopathy (EE) are devastating neurodevelopmental disorders with distinct diagnostic criteria. However, highly heterogeneous and overlapping clinical features often allocate patients into the boundary of the two conditions, complicating accurate diagnosis and appropriate medical interventions. Therefore, we investigated the specific molecular mechanism that allows an understanding of the pathogenesis and relationship of these two conditions. METHODS: We screened novel genetic factors from 34 RTT-like patients without MECP2 mutations, which account for ∼90% of RTT cases, by whole-exome sequencing. The biological function of the discovered variants was assessed in cell culture and Xenopus tropicalis models. RESULTS: We identified a recurring de novo variant in GABAB receptor R2 (GABBR2) that reduces the receptor function, whereas different GABBR2 variants in EE patients possess a more profound effect in reducing receptor activity and are more responsive to agonist rescue in an animal model. INTERPRETATION: GABBR2 is a genetic factor that determines RTT- or EE-like phenotype expression depending on the variant positions. GABBR2-mediated γ-aminobutyric acid signaling is a crucial factor in determining the severity and nature of neurodevelopmental phenotypes. Ann Neurol 2017;82:466-478.

The regulation of transient receptor potential canonical 4 (TRPC4) channel by phosphodiesterase 5 inhibitor via the cyclic guanosine 3'5'-monophosphate.

The transient receptor potential (TRP) protein superfamily consists of a diverse group of cation channels that bear structural similarities to the fruit fly Drosophila TRP. The TRP superfamily is distinct from other groups of ion channels in displaying a large diversity in ion selectivity, modes of activation, and physiological functions. Classical TRP (transient receptor potential canonical (TRPC)) channels are activated by stimulation of Gq-PLC-coupled receptors and modulated by phosphorylation. The cyclic guanosine monophosphate (cGMP)-PKG pathway is involved in the regulation of TRPC3 and TRPC6 channels. Phosphodiesterase (PDE) 5 inhibitor induced muscle relaxation in corporal smooth muscle cells and was used to treat erectile dysfunction by inhibiting cGMP degradation. Here, we report the functional relationship between TRPC4 and cGMP. In human embryonic kidney (HEK) 293 cells overexpressing TRPC4, cGMP selectively activated TRPC4 channels and increased cytosolic calcium level through TRPC4 channel. We investigated phosphorylation sites in TRPC4 channels and identified S688 as an important phosphorylation site for the cGMP-PKG pathway. Cyclic GMP also activated TRPC4-like current with doubly rectifying current-voltage relationship in prostate smooth muscle cell lines. Taken together, these results show that TRPC4 is phosphorylated by the cGMP-PKG pathway and might be an important target for modulating prostate function by PDE5 inhibitors.

Menthol Modulates Pacemaker Potentials through TRPA1 Channels in Cultured Interstitial Cells of Cajal from Murine Small Intestine.

BACKGROUND/AIMS: ICCs are the pacemaker cells responsible for slow waves in gastrointestinal (GI) smooth muscle, and generate periodic pacemaker potentials in current-clamp mode. METHODS: The effects of menthol on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) from mouse small intestine were studied using the whole cell patch clamp technique. RESULTS: Menthol (1 - 10 µM) was found to induce membrane potential depolarization in a concentration-dependent manner. The effects of various TRP channel antagonists were examined to investigate the receptors involved. The addition of the TRPM8 antagonist, AMTB, did not block menthol-induced membrane potential depolarizations, but TRPA1 antagonists (A967079 or HC-030031) blocked the effects of menthol, as did intracellular GDPßS. Furthermore, external and internal Ca2+ levels were found to depolarize menthol-induced membrane potentials, whereas external Na+ was not. Y-27632 (a Rho kinase inhibitor), SC-560 (a selective COX 1 inhibitor), NS-398 (a selective COX 2 inhibitor), ozagrel (a thromboxane A2 synthase inhibitor) and SQ-29548 (highly selective thromboxane receptor antagonist) were used to investigate the involvements of Rho-kinase, cyclooxygenase (COX), and the thromboxane pathway in menthol-induced membrane potential depolarizations, and all inhibitors were found to block the effect of menthol. CONCLUSIONS: These results suggest that menthol-induced membrane potential depolarizations occur in a G-protein-, Ca2+-, Rho-kinase-, COX-, and thromboxane A2-dependent manner via TRPA1 receptor in cultured ICCs in murine small intestine. The study shows ICCs are targeted by menthol and that this interaction can affect intestinal motility.

The Roles of Rasd1 small G proteins and leptin in the activation of TRPC4 transient receptor potential channels.

TRPC4 is important regulators of electrical excitability in gastrointestinal myocytes, pancreatic ß-cells and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Gαi/o and Gαq protein coupled receptor or epidermal growth factor and leptin in particular. However, our understanding of the roles of small G proteins and leptin on TRPC4 channels is still rudimentary. We discuss potential roles for Rasd1 small G protein and leptin in channel activation in addition to their known role in cellular signaling.

Extracellular disulfide bridges stabilize TRPC5 dimerization, trafficking, and activity.

Crucial cysteine residues can be involved in the modulation of protein activity via the modification of thiol (-SH) groups. Among these reactions, disulfide bonds (S-S) play a key role in the folding, stability, and activity of membrane proteins. However, the regulation of extracellular cysteines in classical transient receptor potential (TRPC) channels remains controversial. Here, we examine the functional importance of the extracellular disulfide bond in TRPC5 in modulating channel gating and trafficking. Specifically, we investigated TRPC5 activity in transiently transfected HEK293 cells with wild-type (WT) or cysteine (C553 and C558) mutants in the pore loop. Using reducing agents, we determined that a disulfide linkage mediates the tetrameric formation of the TRPC5 channel. By measuring the TRPC5 current, we observed that C553S or C558S mutants completely lose channel activity induced by lanthanides or receptor stimulation. Co-expression of TRPC5 (WT) with mutants demonstrated a dominant-negative function in mutants, which inhibited the activity of TRPC5 (WT). We generated TRPC5-TRPC5 dimers and observed reduced activity of WT-mutant (C553S or C558S) dimers compared to WT-WT dimers. When pretreated with reducing agents for 12 h, the TRPC5 current decreased due to a reduction in membrane TRPC5 distribution. In addition, we identified a reduced expression of C553S mutant in plasma membrane. We analyzed a dimeric interaction of wild-type and mutant TRPC5 using co-immunoprecipitation and FRET method, indicating a weak interaction between dimeric partners. These results indicated that the disulfide bond between conserved extracellular cysteines, especially C553, is essential for functional TRPC5 activity by channel multimerization and trafficking.

Dexamethasone activates transient receptor potential canonical 4 (TRPC4) channels via Rasd1 small GTPase pathway.

Canonical transient receptor potential 4 (TRPC4) channels are calcium-permeable, nonselective cation channels that are widely distributed in mammalian cells. It is generally speculated that TRPC4 channels are activated by Gq/11-PLC pathway or directly activated by Gi/o proteins. Although many mechanistic studies regarding TRPC4 have dealt with heterotrimeric G proteins, here, we first report the functional relationship between TRPC4 and small GTPase, Rasd1. Rasd1 selectively activated TRPC4 channels, and it was the only Ras protein among Ras protein family that can activate TRPC4 channels. For this to occur, it was found that certain population of functional Gαi1 and Gαi3 proteins are essential. Meanwhile, dexamethasone, a synthetic glucocorticoid and anti-inflammatory drug was known to increase messenger RNA (mRNA) level of Rasd1 in pancreatic ß-cells. We have found that dexamethasone triggers TRPC4-like cationic current in INS-1 cells via increasing protein expression level of Rasd1. This relationship among dexamethasone, Rasd1, and TRPC4 could suggest a new therapeutic agent for hospitalized diabetes mellitus (DM) patients with prolonged dexamethasone prescription.

Characteristics of gintonin-mediated membrane depolarization of pacemaker activity in cultured interstitial cells of Cajal.

BACKGROUND/AIMS: Ginseng regulates gastrointestinal (GI) motor activity but the underlying components and molecular mechanisms are unknown. We investigated the effect of gintonin, a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, on the pacemaker activity of the interstitial cells of Cajal (ICC) in murine small intestine and GI motility. MATERIALS AND METHODS: Enzymatic digestion was used to dissociate ICC from mouse small intestines. The whole-cell patch-clamp configuration was used to record pacemaker potentials and currents from cultured ICC in the absence or presence of gintonin. In vivo effects of gintonin on gastrointestinal (GI) motility were investigated by measuring the intestinal transit rate (ITR) of Evans blue in normal and streptozotocin (STZ)-induced diabetic mice. RESULTS: We investigated the effects of gintonin on pacemaker potentials and currents in cultured ICC from mouse small intestine. Gintonin caused membrane depolarization in current clamp mode but this action was blocked by Ki16425, an LPA1/3 receptor antagonist, and by the addition of GDPßS, a GTP-binding protein inhibitor, into the ICC. To study the gintonin signaling pathway, we examined the effects of U-73122, an active PLC inhibitor, and chelerythrine and calphostin, which inhibit PKC. All inhibitors blocked gintonin actions on pacemaker potentials, but not completely. Gintonin-mediated depolarization was lower in Ca(2+)-free than in Ca(2+)-containing external solutions and was blocked by thapsigargin. We found that, in ICC, gintonin also activated Ca(2+)-activated Cl(-) channels (TMEM16A, ANO1), but not TRPM7 channels. In vivo, gintonin (10-100 mg/kg, p.o.) not only significantly increased the ITR in normal mice but also ameliorated STZ-induced diabetic GI motility retardation in a dose-dependent manner. CONCLUSIONS: Gintonin-mediated membrane depolarization of pacemaker activity and ANO1 activation are coupled to the stimulation of GI contractility through LPA1/3 receptor signaling pathways in cultured murine ICC. Gintonin might be a ingredient responsible for ginseng-mediated GI tract modulations, and could be a novel candidate for development as a prokinetic agent that may prevent or alleviate GI motility dysfunctions in human patients.

Reciprocal positive regulation between TRPV6 and NUMB in PTEN-deficient prostate cancer cells.

Calcium acts as a second messenger and plays a crucial role in signaling pathways involved in cell proliferation. Recently, calcium channels related to calcium influx into the cytosol of epithelial cells have attracted attention as a cancer therapy target. Of these calcium channels, TRPV6 is overexpressed in prostate cancer and is considered an important molecule in the process of metastasis. However, its exact role and mechanism is unclear. NUMB, well-known tumor suppressor gene, is a novel interacting partner of TRPV6. We show that NUMB and TRPV6 have a reciprocal positive regulatory relationship in PC-3 cells. We repeated this experiment in two other prostate cancer cell lines, DU145 and LNCaP. Interestingly, there were no significant changes in TRPV6 expression following NUMB knockdown in DU145. We revealed that the presence or absence of PTEN was the cause of NUMB-TRPV6 function. Loss of PTEN caused a positive correlation of TRPV6-NUMB expression. Collectively, we determined that PTEN is a novel interacting partner of TRPV6 and NUMB. These results demonstrated a novel relationship of NUMB-TRPV6 in prostate cancer cells, and show that PTEN is a novel regulator of this complex.

Isoform- and receptor-specific channel property of canonical transient receptor potential (TRPC)1/4 channels.

Transient receptor potential canonical (TRPC) 1, the first mammalian homologue of Drosophila trp gene, is distributed widely in mammalian cells and is involved in many physiological functions. TRPC1 is reported to be functional following heteromeric formation with other TRPC channels such as TRPC4 or TRPC5. It is known that the composition of this widely distributed TRPC1 is far from simple; functionality of such channels has been highly controversial. Furthermore, TRPC1 gene is known to have two splicing variants; one encodes long (TRPC1α) and the other encodes short (TRPC1ß) TRPC1 isoforms, respectively. In this study, we examined the functionality of TRPC1/4 channels using various activation systems. Gq/11-coupled receptor (e.g., M1 or M3 receptors) stimulation significantly increased TRPC1α/4 currents but induced mild activation of TRPC1ß/4. In addition, when expressed with TRPC4, TRPC1α acted as a pore-constituting subunit and not a ß ancillary subunit. Multimerized with TRPC4, TRPC1α also generated strong pore field strength. We also found that Gi/o-coupled receptor (e.g., M2 receptor) stimulation was insufficient to activate TRPC1α/4 and TRPC1ß/4 channels but selectively activated TRPC4 homomeric channels. These findings demonstrate that TRPC1/4 channel shows dynamic gating property depending on TRPC1 isoform subtypes and receptor stimulation system. Therefore, careful discrimination of the specificity of TRPC1 isoforms and upstream activation system is important in thorough understanding of TRPC1 and TRPC1/4 channels.

Activation of TRPC4ß by Gαi subunit increases Ca2+ selectivity and controls neurite morphogenesis in cultured hippocampal neuron.

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels. TRPC channels are activated by stimulation of Gαq-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gαi. We studied the essential role of Gαi subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gαi2 subunit. Activation of TRPC4 by Gαi2 increased Ca2+ permeability and Ca2+ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca2+ influx. Moreover, both TRPC4ß and the TRPC4ß-Gαi2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gαi2(Q205L)-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gαi proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.