Roles of Intramolecular Interactions in the Regulation of TRP Channels.

The transient receptor potential (TRP) channels, classified into six (-A, -V, -P, -C, -M, -ML, -N and -Y) subfamilies, are important membrane sensors and mediators of diverse stimuli including pH, light, mechano-force, temperature, pain, taste, and smell. The mammalian TRP superfamily of 28 members share similar membrane topology with six membrane-spanning helices (S1-S6) and cytosolic N-/C-terminus. Abnormal function or expression of TRP channels is associated with cancer, skeletal dysplasia, immunodeficiency, and cardiac, renal, and neuronal diseases. The majority of TRP members share common functional regulators such as phospholipid PIP2, 2-aminoethoxydiphenyl borate (2-APB), and cannabinoid, while other ligands are more specific, such as allyl isothiocyanate (TRPA1), vanilloids (TRPV1), menthol (TRPM8), ADP-ribose (TRPM2), and ML-SA1 (TRPML1). The mechanisms underlying the gating and regulation of TRP channels remain largely unclear. Recent advances in cryogenic electron microscopy provided structural insights into 19 different TRP channels which all revealed close proximity of the C-terminus with the N-terminus and intracellular S4-S5 linker. Further studies found that some highly conserved residues in these regions of TRPV, -P, -C and -M members mediate functionally critical intramolecular interactions (i.e., within one subunit) between these regions. This review provides an overview on (1) intramolecular interactions in TRP channels and their effect on channel function; (2) functional roles of interplays between PIP2 (and other ligands) and TRP intramolecular interactions; and (3) relevance of the ligand-induced modulation of intramolecular interaction to diseases.

Regulation of PKD2 channel function by TACAN.

Autosomal dominant polycystic kidney disease is caused by mutations in the membrane receptor PKD1 or the cation channel PKD2. TACAN (also termed TMEM120A), recently reported as an ion channel in neurons for mechanosensing and pain sensing, is also distributed in diverse non-neuronal tissues, such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in a complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes, we found that TACAN inhibits the channel activity of PKD2 gain-of-function mutant F604P. TACAN fragments containing the first and last transmembrane domains interacted with the PKD2 C- and N-terminal fragments, respectively. The TACAN N-terminus acted as a blocking peptide, and TACAN inhibited the function of PKD2 by the binding of PKD2 with TACAN. By patch clamping in mammalian cells, we found that TACAN inhibits both the single-channel conductance and the open probability of PKD2 and mutant F604P. PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. Furthermore, we found that TACAN aggravates PKD2-dependent tail curvature and pronephric cysts in larval zebrafish. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechanosensitivity of the PKD2-TACAN channel complex. KEY POINTS: TACAN inhibits the function of PKD2 in vitro and in vivo. TACAN N-terminal S1-containing fragment T160X interacts with the PKD2 C-terminal fragment N580-L700, and its C-terminal S6-containing fragment L296-D343 interacts with the PKD2 N-terminal A594X. TACAN inhibits the function of the PKD2 channel by physical interaction. The complex of PKD2 with TACAN, but not PKD2 alone, confers mechanosensitivity.

Expression and Development of TARP γ-4 in Embryonic Zebrafish.

Fast excitatory synaptic transmission in the CNS is mediated by the neurotransmitter glutamate, binding to and activating AMPA receptors (AMPARs). AMPARs are known to interact with auxiliary proteins that modulate their behavior. One such family of proteins is the transmembrane AMPAR-related proteins, known as TARPs. Little is known about the role of TARPs during development or about their function in nonmammalian organisms. Here, we report on the presence of TARP γ-4 in developing zebrafish. We find that zebrafish express 2 forms of TARP γ-4: γ-4a and γ-4b as early as 12 h post-fertilization. Sequence analysis shows that both γ-4a and γ-4b shows great level of variation particularly in the intracellular C-terminal domain compared to rat, mouse, and human γ-4. RT-qPCR showed a gradual increase in the expression of γ-4a throughout the first 5 days of development, whereas γ-4b levels were constant until day 5 when levels increased significantly. Knockdown of TARP γ-4a and γ-4b via either splice-blocking morpholinos or translation-blocking morpholinos resulted in embryos that exhibited deficits in C-start escape responses, showing reduced C-bend angles. Morphant larvae displayed reduced bouts of swimming. Whole-cell patch-clamp recordings of AMPAR-mediated currents from Mauthner cells showed a reduction in the frequency of mEPCs but no change in amplitude or kinetics. Together, these results suggest that γ-4a and γ-4b are required for proper neuronal development.

TRIM4 interacts with TRPM8 and regulates its channel function through K423-mediated ubiquitination.

Transient receptor potential melastatin member 8 (TRPM8), a Ca2+ -permeable nonselective cation channel activated by cold and cooling agents, mediates allodynia. Dysfunction or abnormal expression of TRPM8 has been found in several human cancers. The role of ubiquitination in the regulation of TRPM8 function remains poorly understood. Here, we identified the ubiquitin (Ub)-ligase E3, tripartite motif-containing 4 (TRIM4), as a novel interaction partner of TRPM8 and confirmed that the TRIM4-TRPM8 interaction was mediated through the SPRY domain of TRIM4. Patch-clamp assays showed that TRIM4 negatively regulates TRPM8-mediated currents in HEK293 cells. Moreover, TRIM4 reduced the expression of TRPM8 on the cell surface by promoting the K63-linked ubiquitination of TRPM8. Further analyses revealed that the TRPM8 N-terminal lysine residue at 423 was the major ubiquitination site that mediates its functional regulation by TRIM4. A Ub-activating enzyme E1, Ub-like modifier-activating enzyme 1 (UBA1), was also found to interact with TRPM8, thereby regulating its channel function and ubiquitination. In addition, knockdown of UBA1 impaired the regulation of TRPM8 ubiquitination and function by TRIM4. Thus, this study demonstrates that TRIM4 downregulates TRPM8 via K423-mediated TRPM8 ubiquitination and requires UBA1 to regulate TRPM8.

The Fabp5/calnexin complex is a prerequisite for sensitization of mice to experimental autoimmune encephalomyelitis.

We previously showed that calnexin (Canx)-deficient mice are desensitized to experimental autoimmune encephalomyelitis (EAE) induction, a model that is frequently used to study inflammatory demyelinating diseases, due to increased resistance of the blood-brain barrier to immune cell transmigration. We also discovered that Fabp5, an abundant cytoplasmic lipid-binding protein found in brain endothelial cells, makes protein-protein contact with the cytoplasmic C-tail domain of Canx. Remarkably, both Canx-deficient and Fabp5-deficient mice commonly manifest resistance to EAE induction. Here, we evaluated the importance of Fabp5/Canx interactions on EAE pathogenesis and on the patency of a model blood-brain barrier to T-cell transcellular migration. The results demonstrate that formation of a complex comprised of Fabp5 and the C-tail domain of Canx dictates the permeability of the model blood-brain barrier to immune cells and is also a prerequisite for EAE pathogenesis.

The ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2 gene, encoding the polycystic kidney disease protein polycystin-1 and the transient receptor potential channel polycystin-2 (also known as TRPP2), respectively. Polycystin-1 and polycystin-2 form a receptor-ion channel complex located in primary cilia. The function of this complex, especially the role of polycystin-1, is largely unknown due to the lack of a reliable functional assay. In this study, we dissect the role of polycystin-1 by directly recording currents mediated by a gain-of-function (GOF) polycystin-1/polycystin-2 channel. Our data show that this channel has distinct properties from that of the homomeric polycystin-2 channel. The polycystin-1 subunit directly contributes to the channel pore, and its eleven transmembrane domains are sufficient for its channel function. We also show that the cleavage of polycystin-1 at the N-terminal G protein-coupled receptor proteolysis site is not required for the activity of the GOF polycystin-1/polycystin-2 channel. These results demonstrate the ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex, enriching our understanding of this channel and its role in ADPKD.

LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/ß-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes.

BACKGROUND: Pancreatic cancer is one of the most lethal malignancies and has an extremely poor diagnosis and prognosis. The development of resistance to gemcitabine is still a major challenge. The long noncoding RNA PVT1 was reported to be involved in carcinogenesis and chemoresistance; however, the mechanism by which PVT1 regulates the sensitivity of pancreatic cancer to gemcitabine remains poorly understood. METHODS: The viability of pancreatic cancer cells was assessed by MTT assay in vitro and xenograft tumor formation assay in vivo. The expression levels of PVT1 and miR-619-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blotting analysis and qRT-PCR were performed to assess the protein and mRNA levels of Pygo2 and ATG14, respectively. Autophagy was explored via autophagic flux detection under confocal microscopy and autophagic vacuole investigation under transmission electron microscopy (TEM). The functional role and mechanism of PVT1 were further investigated by gain- and loss-of-function assays in vitro. RESULTS: In the present study, we demonstrated that PVT1 was up-regulated in gemcitabine-resistant pancreatic cancer cell lines. Gain- and loss-of-function assays revealed that PVT1 impaired sensitivity to gemcitabine in vitro and in vivo. We further found that PVT1 up-regulated the expression of both Pygo2 and ATG14 and thus regulated Wnt/ß-catenin signaling and autophagic activity to overcome gemcitabine resistance through sponging miR-619-5p. Moreover, we discovered three TCF/LEF binding elements (TBEs) in the promoter region of PVT1, and activation of Wnt/ß-catenin signaling mediated by the up-regulation of Pygo2 increased PVT1 expression by direct binding to the TBE region. Furthermore, PVT1 was discovered to interact with ATG14, thus promoting assembly of the autophagy specific complex I (PtdIns3K-C1) and ATG14-dependent class III PtdIns3K activity. CONCLUSIONS: These data indicate that PVT1 plays a critical role in the sensitivity of pancreatic cancer to gemcitabine and highlight its potential as a valuable target for pancreatic cancer therapy.

Two pools of IRE1α in cardiac and skeletal muscle cells.

The endoplasmic reticulum (ER) plays a central role in cellular stress responses via mobilization of ER stress coping responses, such as the unfolded protein response (UPR). The inositol-requiring 1α (IRE1α) is an ER stress sensor and component of the UPR. Muscle cells also have a well-developed and highly subspecialized membrane network of smooth ER called the sarcoplasmic reticulum (SR) surrounding myofibrils and specialized for Ca2+ storage, release, and uptake to control muscle excitation-contraction coupling. Here, we describe 2 distinct pools of IRE1α in cardiac and skeletal muscle cells, one localized at the perinuclear ER and the other at the junctional SR. We discovered that, at the junctional SR, calsequestrin binds to the ER luminal domain of IRE1α, inhibiting its dimerization. This novel interaction of IRE1α with calsequestrin, one of the highly abundant Ca2+ handling proteins at the junctional SR, provides new insights into the regulation of stress coping responses in muscle cells.-Wang, Q., Groenendyk, J., Paskevicius, T., Qin, W., Kor, K. C., Liu, Y., Hiess, F., Knollmann, B. C., Chen, S. R. W., Tang, J., Chen, X.-Z., Agellon, L. B., Michalak, M. Two pools of IRE1α in cardiac and skeletal muscle cells.

Unveiling the Distinct Mechanisms by which Disease-Causing Mutations in the Kelch Domain of KLHL3 Disrupt the Interaction with the Acidic Motif of WNK4 through Molecular Dynamics Simulation.

Kelch-like 3 (KLHL3) is a substrate adaptor of an E3 ubiquitin ligase complex that regulates the degradation of its substrates, including with-no-lysine [K] kinase 4 (WNK4). Mutations in KLHL3 are associated with pseudohypoaldosteronism type II (PHAII), a hereditary form of hypertension. Many PHAII-causing mutations are located in the Kelch domain of KLHL3 that binds with WNK4; however, detailed mechanisms by which these mutations disrupt the binding are not well-understood. In the present study we use molecular dynamics simulations and Western blot analyses to examine the effects of these mutations on the interaction between the Kelch domain of KLHL3 and the acidic motif (AM) of WNK4. The simulation results correlated well with those from Western blot analyses with the exception of the L387P mutation, which led to deregulation of AM degradation by KLHL3 but not recapitulated by simulations. On the basis of the simulation results, a mutation on the binding surface of the Kelch domain affected the Kelch-AM interaction through two major mechanisms: altering the electrostatic potential of the AM binding site and disrupting the Kelch-AM hydrogen bonds. The mutations buried inside the Kelch domain were predicted by our simulations to have no or modest effects on the Kelch-AM interaction. Buried mutations R384Q and S410L disrupted intramolecular hydrogen bonds within the Kelch domain and affected the Kelch-AM interaction indirectly. No significant effect of buried mutation A340V or A494T on the AM degradation or Kelch-AM interaction was observed, implying these mutations may disrupt mechanisms other than Kelch-AM interaction.

Purification and functional characterization of the vacuolar malate transporter tDT from Arabidopsis.

The exact transport characteristics of the vacuolar dicarboxylate transporter tDT from Arabidopsis are elusive. To overcome this limitation, we combined a range of experimental approaches comprising generation/analysis of tDT overexpressors, 13CO2 feeding and quantification of 13C enrichment, functional characterization of tDT in proteoliposomes, and electrophysiological studies on vacuoles. tdt knockout plants showed decreased malate and increased citrate concentrations in leaves during the diurnal light-dark rhythm and after onset of drought, when compared with wildtypes. Interestingly, under the latter two conditions, tDT overexpressors exhibited malate and citrate levels opposite to tdt knockout plants. Highly purified tDT protein transports malate and citrate in a 1:1 antiport mode. The apparent affinity for malate decreased with decreasing pH, whereas citrate affinity increased. This observation indicates that tDT exhibits a preference for dianion substrates, which is supported by electrophysiological analysis on intact vacuoles. tDT also accepts fumarate and succinate as substrates, but not α-ketoglutarate, gluconate, sulfate, or phosphate. Taking tDT as an example, we demonstrated that it is possible to reconstitute a vacuolar metabolite transporter functionally in proteoliposomes. The displayed, so far unknown counterexchange properties of tDT now explain the frequently observed reciprocal concentration changes of malate and citrate in leaves from various plant species. tDT from Arabidopsis is the first member of the well-known and widely present SLC13 group of carrier proteins, exhibiting an antiport mode of transport.

Identification and characterization of hydrophobic gate residues in TRP channels.

Transient receptor potential (TRP) channels, subdivided into 6 subfamilies in mammals, have essential roles in sensory physiology. They respond to remarkably diverse stimuli, comprising thermal, chemical, and mechanical modalities, through opening or closing of channel gates. In this study, we systematically substituted the hydrophobic residues within the distal fragment of pore-lining helix S6 with hydrophilic residues and, based on Xenopus oocyte and mammalian cell electrophysiology and a hydrophobic gate theory, identified hydrophobic gates in TRPV6/V5/V4/C4/M8. We found that channel activity drastically increased when TRPV6Ala616 or Met617 or TRPV5Ala576 or Met577, but not any of their adjacent residues, was substituted with hydrophilic residues. Channel activity strongly correlated with the hydrophilicity of the residues at those sites, suggesting that consecutive hydrophobic residues TRPV6Ala616-Met617 and TRPV5Ala576-Met577 form a double-residue gate in each channel. By the same strategy, we identified a hydrophobic single-residue gate in TRPV4Iso715, TRPC4Iso617, and TRPM8Val976. In support of the hydrophobic gate theory, hydrophilic substitution at the gate site, which removes the hydrophobic gate seal, substantially increased the activity of TRP channels in low-activity states but had little effect on the function of activated channels. The double-residue gate channels were more sensitive to small changes in the gate's hydrophobicity or size than single-residue gate channels. The unconventional double-reside gating mechanism in TRP channels may have been evolved to respond especially to physiologic stimuli that trigger relatively small gate conformational changes.-Zheng, W., Hu, R., Cai, R., Hofmann, L., Hu, Q., Fatehi, M., Long, W., Kong, T., Tang, J., Light, P., Flockerzi, V., Cao, Y., Chen, X.-Z. Identification and characterization of hydrophobic gate residues in TRP channels.

Regulation of TRPP3 Channel Function by N-terminal Domain Palmitoylation and Phosphorylation.

Transient receptor potential polycystin-3 (TRPP3) is a cation channel activated by calcium and proton and is involved in hedgehog signaling, intestinal development, and sour tasting. How TRPP3 channel function is regulated remains poorly understood. By N-terminal truncation mutations, electrophysiology, and Xenopus oocyte expression, we first identified fragment Asp-21-Ser-42 to be functionally important. We then found that deletion mutant Δ1-36 (TRPP3 missing fragment Met-1-Arg-36) has a similar function as wild-type TRPP3, whereas Δ1-38 is functionally dead, suggesting the importance of Val-37 or Cys-38. Further studies found that Cys-38, but not Val-37, is functionally critical. Cys-38 is a predicted site of palmitoylation, and indeed TRPP3 channel activity was inhibited by palmitoylation inhibitor 2-bromopalmitate and rescued by palmitoylation substrate palmitic acid. The TRPP3 N terminus (TRPP3NT, Met-1-Leu-95) localized along the plasma membrane of HEK293 cells but stayed in the cytoplasm with 2-bromopalmitate treatment or C38A mutation, indicating that TRPP3NT anchors to the surface membrane through palmitoylation at Cys-38. By acyl-biotin exchange assays, we showed that TRPP3, but not mutant C38A, is indeed palmitoylated. When putative phosphorylation sites near Cys-38 were mutated to Asp or Glu to mimic phosphorylation, only T39D and T39E reduced TRPP3 function. Furthermore, TRPP3NT displayed double bands in which the upper band was abolished by λ phosphatase treatment or T39A mutation. However, palmitoylation at Cys-38 and phosphorylation at Thr-39 independently regulated TRPP3 channel function, in contrast to previous reports about correlated palmitoylation with a proximate phosphorylation. Palmitoylation at Cys-38 represents a novel mechanism of functional regulation for TRPP3.

Far Upstream Element-Binding Protein 1 Binds the 3' Untranslated Region of PKD2 and Suppresses Its Translation.

Autosomal dominant polycystic kidney disease pathogenesis can be recapitulated in animal models by gene mutations in or dosage alterations of polycystic kidney disease 1 (PKD1) or PKD2, demonstrating that too much and too little PKD1/PKD2 are both pathogenic. Gene dosage manipulation has become an appealing approach by which to compensate for loss or gain of gene function, but the mechanisms controlling PKD2 expression remain incompletely characterized. In this study, using cultured mammalian cells and dual-luciferase assays, we found that the 3' untranslated region (3'UTR) of PKD2 mRNA inhibits luciferase protein expression. We then identified nucleotides 691-1044, which we called 3FI, as the 3'UTR fragment necessary for repressing the expression of luciferase or PKD2 in this system. Using a pull-down assay and mass spectrometry we identified far upstream element-binding protein 1 (FUBP1) as a 3FI-binding protein. In vitro overexpression of FUBP1 inhibited the expression of PKD2 protein but not mRNA. In embryonic zebrafish, FUBP1 knockdown (KD) by morpholino injection increased PKD2 expression and alleviated fish tail curling caused by morpholino-mediated KD of PKD2. Conversely, FUBP1 overexpression by mRNA injection significantly increased pronephric cyst occurrence and tail curling in zebrafish embryos. Furthermore, FUBP1 binds directly to eukaryotic translation initiation factor 4E-binding protein 1, indicating a link to the translation initiation complex. These results show that FUBP1 binds 3FI in the PKD2 3'UTR to inhibit PKD2 translation, regulating zebrafish disease phenotypes associated with PKD2 KD.

Critical Roles of Two Hydrophobic Residues within Human Glucose Transporter 9 (hSLC2A9) in Substrate Selectivity and Urate Transport.

High blood urate levels (hyperuricemia) have been found to be a significant risk factor for cardiovascular diseases and inflammatory arthritis, such as hypertension and gout. Human glucose transporter 9 (hSLC2A9) is an essential protein that mainly regulates urate/hexose homeostasis in human kidney and liver. hSLC2A9 is a high affinity-low capacity hexose transporter and a high capacity urate transporter. Our previous studies identified a single hydrophobic residue in trans-membrane domain 7 of class II glucose transporters as a determinant of fructose transport. A mutation of isoleucine 335 to valine (I355V) in hSLC2A9 can reduce fructose transport while not affecting glucose fluxes. This current study demonstrates that the I335V mutant transports urate similarly to the wild type hSLC2A9; however, Ile-335 is necessary for urate/fructose trans-acceleration exchange to occur. Furthermore, Trp-110 is a critical site for urate transport. Two structural models of the class II glucose transporters, hSLC2A9 and hSLC2A5, based on the crystal structure of hSLC2A1 (GLUT1), reveal that Ile-335 (or the homologous Ile-296 in hSLC2A5) is a key component for protein conformational changes when the protein translocates substrates. The hSLC2A9 model also predicted that Trp-110 is a crucial site that could directly interact with urate during transport. Together, these studies confirm that hSLC2A9 transports both urate and fructose, but it interacts with them in different ways. Therefore, this study advances our understanding of how hSLC2A9 mediates urate and fructose transport, providing further information for developing pharmacological agents to treat hyperuricemia and related diseases, such as gout, hypertension, and diabetes.

Co-chaperone Specificity in Gating of the Polypeptide Conducting Channel in the Membrane of the Human Endoplasmic Reticulum.

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic polypeptide-conducting channel, the heterotrimeric Sec61 complex. Previous work has characterized the Sec61 complex as a potential ER Ca(2+) leak channel in HeLa cells and identified ER lumenal molecular chaperone immunoglobulin heavy-chain-binding protein (BiP) as limiting Ca(2+) leakage via the open Sec61 channel by facilitating channel closing. This BiP activity involves binding of BiP to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344. Of note, the Y344H mutation destroys the BiP binding site and causes pancreatic ß-cell apoptosis and diabetes in mice. Here, we systematically depleted HeLa cells of the BiP co-chaperones by siRNA-mediated gene silencing and used live cell Ca(2+) imaging to monitor the effects on ER Ca(2+) leakage. Depletion of either one of the ER lumenal BiP co-chaperones, ERj3 and ERj6, but not the ER membrane-resident co-chaperones (such as Sec63 protein, which assists BiP in Sec61 channel opening) led to increased Ca(2+) leakage via Sec6 complex, thereby phenocopying the effect of BiP depletion. Thus, BiP facilitates Sec61 channel closure (i.e. limits ER Ca(2+) leakage) via the Sec61 channel with the help of ERj3 and ERj6. Interestingly, deletion of ERj6 causes pancreatic ß-cell failure and diabetes in mice and humans. We suggest that co-chaperone-controlled gating of the Sec61 channel by BiP is particularly important for cells, which are highly active in protein secretion, and that breakdown of this regulatory mechanism can cause apoptosis and disease.

Filamin interacts with epithelial sodium channel and inhibits its channel function.

Epithelial sodium channel (ENaC) in the kidneys is critical for Na(+) balance, extracellular volume, and blood pressure. Altered ENaC function is associated with respiratory disorders, pseudohypoaldosteronism type 1, and Liddle syndrome. ENaC is known to interact with components of the cytoskeleton, but the functional roles remain largely unclear. Here, we examined the interaction between ENaC and filamins, important actin filament components. We first discovered by yeast two-hybrid screening that the C termini of ENaC α and ß subunits bind filamin A, B, and C, and we then confirmed the binding by in vitro biochemical assays. We demonstrated by co-immunoprecipitation that ENaC, either overexpressed in HEK, HeLa, and melanoma A7 cells or natively expressed in LLC-PK1 and IMCD cells, is in the same complex with native filamin. Furthermore, the biotinylation and co-immunoprecipitation combined assays showed the ENaC-filamin interaction on the cell surface. Using Xenopus oocyte expression and two-electrode voltage clamp electrophysiology, we found that co-expression of an ENaC-binding domain of filamin substantially reduces ENaC channel function. Western blot and immunohistochemistry experiments revealed that the filamin A C terminus (FLNAC) modestly reduces the expression of the ENaC α subunit in oocytes and A7 cells. After normalizing the current by plasma membrane expression, we found that FLNAC results in ~50% reduction in the ENaC channel activity. The inhibitory effect of FLNAC was confirmed by lipid bilayer electrophysiology experiments using purified ENaC and FLNAC proteins, which showed that FLNAC substantially reduces ENaC single channel open probability. Taken together, our study demonstrated that filamin reduces ENaC channel function through direct interaction on the cell surface.

Expression of polycystins and fibrocystin on primary cilia of lung cells.

Mutations in polycystin-1, polycystin-2, or fibrocystin account for autosomal dominant or recessive polycystic kidney disease. Renal cystogenesis is linked to abnormal localization and function of these cystoproteins in renal primary cilia. They are also expressed in extrarenal tissues in which their functions are unclear. Here we found that human type-II alveolar epithelial A549, airway submucosal Calu-3 cells, and rat bronchioles contain primary or multiple cilia in which we detected these cystoproteins. At sub-confluency, polycystin-1 was expressed on plasma membrane, while polycystin-2 was localized to the ER of resting cells. Both polycystins were detected on the spindle and mid-body of mitotic cells, while fibrocystin was on centrosome throughout cell cycle. Polycystins and fibrocystin may participate in regulating mucociliary sensing and transport within pulmonary airways.

Translational up-regulation of polycystic kidney disease protein PKD2 by endoplasmic reticulum stress.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, and it affects over 10 million people worldwide. It is characterized by cyst formation in the kidney, liver and pancreas. Dosage changes in PKD1/PKD2 are important in ADPKD pathogenesis; therefore, their expression and function has to be strictly regulated. However, how they are regulated remain poorly understood. Recent studies have linked PKD2 regulation to endoplasmic reticulum (ER) stress that is implicated in neuronal, cardiac, and renal diseases. One major ER stress downstream is phosphorylation of eukaryotic initiation factor eIF2α by kinase PERK, which attenuates global protein translation and enhances translation of selected proteins. Here, we showed in several mammalian cell lines that PKD2 protein expression is up-regulated by different stresses that all increase phosphorylated eIF2α (P-eIF2α). Increasing P-eIF2α by overexpression or inhibiting the phosphatase activity resulted in increased PKD2. PCR and polysome-binding assays showed that ER stress does not affect the PKD2 mRNA level but increase its binding with ribosomes, indicating that P-eIF2α translationally up-regulates PKD2. By mutation analysis, we found that the upstream open reading frame (uORF) in the 5'-untranslated region of PKD2 mRNA represses PKD2 translation. Thus, ER stress and P-eIF2α translationally up-regulates PKD2 through bypassing the inhibitory uORF.

LncRNAs as nodes for the cross-talk between autophagy and Wnt signaling in pancreatic cancer drug resistance.

Pancreatic cancer is a malignancy with high mortality. In addition to the few symptoms until the disease reaches an advanced stage, the high fatality rate is attributed to its rapid development, drug resistance and lack of appropriate treatment. In the selection and research of therapeutic drugs, gemcitabine is the first-line drug for pancreatic cancer. Solving the problem of gemcitabine resistance in pancreatic cancer will contribute to the progress of pancreatic cancer treatment. Long non coding RNAs (lncRNAs), which are RNA transcripts longer than 200 nucleotides, play vital roles in cellular physiological metabolic activities. Currently, our group and others have found that some lncRNAs are aberrantly expressed in pancreatic cancer cells, which can regulate the process of cancer through autophagy and Wnt/ß-catenin pathways simultaneously and affect the sensitivity of cancer cells to therapeutic drugs. This review presents an overview of the recent evidence concerning the node of lncRNA for the cross-talk between autophagy and Wnt/ß-catenin signaling in pancreatic cancer, together with the practicability of lncRNAs and the core regulatory factors as targets in therapeutic resistance.

The TRPV6 Calcium Channel and Its Relationship with Cancer.

Transient receptor potential vanilloid-6 (TRPV6) is a cation channel belonging to the TRP superfamily, specifically the vanilloid subfamily, and is the sixth member of this subfamily. Its presence in the body is primarily limited to the skin, ovaries, kidney, testes, and digestive tract epithelium. The body maintains calcium homeostasis using the TRPV6 channel, which has a greater calcium selectivity than the other TRP channels. Several pieces of evidence suggest that it is upregulated in the advanced stages of thyroid, ovarian, breast, colon, and prostate cancers. The function of TRPV6 in regulating calcium signaling in cancer will be covered in this review, along with its potential applications as a cancer treatment target.