Transient Receptor Potential Vanilloid 6 Channel Research Articles

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.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

A TRPV6 expression atlas for the mouse

The transient receptor potential vanilloid 6 (TRPV6) channel is highly Ca2+-selective and has been implicated in mediating transcellular Ca2+ transport and thus maintaining the Ca2+ balance in the body. To characterize its physiological function(s), a detailed expression profile of the TRPV6 channel throughout the body is essential. Capitalizing on a recently established murine Trpv6-reporter strain, we identified primary TRPV6 channel-expressing cells in an organism-wide manner. In a complementary experimental approach, we characterized TRPV6 expression in different tissues of wild-type mice by TRPV6 immunoprecipitation (IP) followed by mass spectrometry analysis and correlated these data with the reporter gene expression. Taken together, we present a TRPV6 expression atlas throughout the entire body of juvenile and adult mice, providing a novel resource to investigate the role of TRPV6 channels in vivo.

Autoinhibition of TRPV6 Channel by Intramolecular Interactions

Transient receptor potential vanilloid 6 (TRPV6), a calcium-selective channel possessing six transmembrane domains (S1-S6) and intracellular N-/C-termini, plays crucial roles in calcium absorption in epithelia and bone and is involved in human diseases including vitamin-D deficiency, osteoporosis and cancer. The TRPV6 function and regulation remain poorly understood. Here by electrophysiology and Xenopus oocyte expression we found that Arg510 in the S4-S5 linker and Trp633 in the TRP helix are functionally critical. By co-immunoprecipitation and in vitro binding we found that the Arg510:Trp633 pair mediates binding between the linker (L) and C-terminal TRP helix (C) and that disruption of the linker/TRP helix (L/C) interaction substantially increases the channel activity. We also found that Trp361 in the N-terminal pre-S1 helix (N) and Ile637 mediate the pre-S1/TRP helix (N/C) binding which is functionally critical as well. Calcium imaging in human embryonic kidney cells further supported functional importance of Trp361 and Trp633. Besides, disruption of either interaction by blocking peptides activated TRPV6. The L/C interaction was required for the N/C interaction but not reversely. Together, we showed that the TRPV6 intramolecular S4-S5 linker to TRP helix and pre-S1 helix to TRP helix interactions, mediated by Arg510:Trp633 and Trp361:Ile637 bonding, respectively, are autoinhibitory and are required for maintaining TRPV6 at basal states. This study reveals a regulatory mechanism of TRPV6 activation-autoinhibition, which would help elucidating the corresponding mechanisms in other TRP channels. Supported by AIGSS (to RC), NSERC and KFoC (to XZC).

Downregulation of TRPV6 channel activity by cholesterol depletion in Jurkat T cell line.

Transient receptor potential vanilloid 6 (TRPV6) channels are key players in calcium metabolism of healthy and cancerous cells. Nevertheless, the mechanisms controlling abundance of these channels in plasma membrane of the cells to regulate Ca2+ transport is still poorly understood. In this study, we provide the first evidence that TRPV6 calcium channels and Ca 2+ influx in Jurkat T cell line are modulated by cholesterol, a main lipid component of the plasma membrane. Using patch-clamp technique, we found that activity of TRPV6 channels decreased by cholesterol sequestration with methyl-β-cyclodextrin (MβCD). Continuous measurement of intracellular Ca2+ revealed a reduction of Ca2+ influx into Jurkat cells following cholesterol depletion. Immunofluorescence and immunoelectron microscopy analyses of MβCD-treated cells detected the lower surface expression of the TRPV6 proteins in comparison with control cells. In general, our data showed that cholesterol regulates TRPV6 channel activity and TRPV6-mediated Ca2+ influx in cells, apparently affecting the localization and density of the calcium channels in the plasma membrane of Jurkat T cells.

Maternal Transient Receptor Potential Vanilloid 6 (Trpv6) Is Involved In Offspring Bone Development.

Embryonic growth and bone development depend on placental Ca2+ transport across the feto-maternal barrier to supply minerals to the fetus. The individual factors and cellular mechanisms that regulate placental Ca2+ transfer, however, are only beginning to emerge. We find that the Ca2+ -selective transient receptor potential vanilloid 6 (TRPV6) channel is expressed in trophoblasts of the fetal labyrinth, in the yolk sac, and in the maternal part of the placenta. Lack of functional TRPV6 channels in the mother leads to a reduced Ca2+ content in both placenta and embryo. Ca2+ uptake in trophoblasts is impaired in the absence of Trpv6. Trpv6-deficient embryos are smaller, have a lower body weight, and shorter and less calcified femurs. The altered cortical bone microarchitecture persists in adulthood. We show that TRPV6's Ca2+ -conducting property causes this embryonic and bone phenotype. Our results show that TRPV6 is necessary for the Ca2+ uptake in trophoblasts and that TRPV6 deficiency in the placenta leads to reduced embryo growth, minor bone calcification, and impaired bone development. © 2019 American Society for Bone and Mineral Research.

The Mechanism of Regulation of TRPV6 Channels by PI(4,5)P2

Transient Receptor Potential Vanilloid 6 (TRPV6) is a Ca2+ selective ion channel playing important roles in intestinal Ca2+ absorption, male fertility and cancer development. TRPV6 is a member of the highly diverse TRP ion channel family. The only known common functional feature among TRP channels is their dependence on, and modulation by phosphoinositides, mostly phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Most TRP channels require factors other than PI(4,5)P2 to open, which are often in complex interaction with PI(4,5)P2 regulation, which hinders understanding of the molecular mechanism of how PI(4,5)P2 opens these channels. TRPV6 is constitutively active, thus devoid of these complexities, and therefore is an ideal model to gain molecular insight into regulation by PI(4,5)P2. We computationally docked PI(4,5)P2 to both TRPV6, and the heat and capsaicin sensitive TRPV1. We find that PI(4,5)P2 binds to TRPV1 mainly in the proximal C-terminus and in the S4-S5 linker, consistent with earlier predictions, and experimental data. In TRPV6, however, the closest interacting residues were in the proximal N-terminus, close to the first transmembrane region and, similar to TRPV1, in the S4-S5 linker. Introduction of a positive charge in the S4-S5 linker that is missing from TRPV6, but present in TRPV1, markedly increased the affinity of TRPV6 to PI(4,5)P2. Neutralizing a proximal N-terminal positively charged residue predicted to interact with PI(4,5)P2 markedly reduced current TRPV6 amplitudes, but neutralizing neighboring positively charged residues had no effect. Our data will provide mechanistic insight into PI(4,5)P2 regulation of TRPV6.

Abstract B83: Targeting TRPV6 oncochannel for the treatment of pancreatic cancer: A Phase I trial experience

Abstract Background: SOR-C13 is a first-in-human selective peptide inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6) oncochannel. TRPV6 is highly elevated in carcinomas including prostate, breast, lung and ovary and is correlated with poor outcomes. TRPV6-mediated Ca2+ entry is responsible for maintaining a high proliferation rate, increasing cell survival and apoptosis resistance. Since SOR-C13 blocks TRPV6-mediated Ca2+ influx it was evaluated as a single agent in patients with metastatic carcinomas. Methods: This was a Phase I, multi-center, open-label, dose escalation study to assess safety and tolerability of SOR-C13 in subjects with advanced carcinomas commonly known to express the TRPV6 channel (NCT01578564). Subjects received intravenous infusion of SOR-C13 over 90 minutes for 3 d on/4 d off, 3 d on/11 d off (21 d/cycle) until tumor progression, prohibitive toxicity or patient withdrawal. Tumors were assessed according to RECIST 1.1. Results: A total of 23 patients were enrolled to 4 dose levels and 119 cycles of therapy were administered. No study drug-related SAEs occurred. Twelve of 22 evaluable subjects (54.5%) had stable disease after 2 cycles. Promising clinical benefit was observed in two patients with advanced metastatic ductal pancreatic cancer. Subject 304 showed stable disease (SD) at dose level of 2.75 mg/kg for 12 weeks, and was removed from the study for persistent grade 2 hepatic transaminase elevation for more than 21 days. Subject 312, treated at dose level of 6.2 mg/kg, showed 7% decrease in tumor size after 2 cycles and a 27% decrease after 4 cycles. Tumor marker CA 19-9 responses were consistent with most changes in tumor size over the treatment cycles with suggestions of a dose related response and correlation with tumor size. The response of this biomarker was particularly evident in the patient initially treated at 6.2 mg/kg where it decreased by 29% after cycle two and by 55% after cycle 4. This patient was removed from the study after a total of 27 weeks 2 days. Both subjects failed at least three prior regimens of anticancer therapy: Subject 304 (i) IMRT/Tomotherapy plus chemoradiation with Folfirinox, (ii) Carboplatin/gemcitabine/Tarceva (iii) Abraxane/Gemcitabine, (iv) IMRT/Tomotherapy; Subject 312 (i) Folfirinox; (ii) gemcitabine and Abraxane; and, (iii) Xeloda. Conclusions: SOR-C13 was safe and well tolerated in subjects with advanced epithelial malignancies. The clinical benefit observed in patients with metastatic pancreatic cancer, particularly 27% reduction of the sum of tumor diameters after 4 cycles warrants further investigation of this novel treatment in Phase II studies. Citation Format: Siqing Fu, Stephen Welch, Toney T. Ilenchuk, Dominique Dugourd, Tyler Lutes, Chris Rice, Jack M. Stewart.{Authors}. Targeting TRPV6 oncochannel for the treatment of pancreatic cancer: A Phase I trial experience. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B83.

Abstract CT142: A phase I open-label, dose escalation study of a novel peptide (SOR-C13) antagonistic to the TRPV6 ion channel in patients with advanced solid tumor cancers

Abstract Background: SOR-C13 is a first-in-man selective peptide inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6) calcium oncochannel. TRPV6 is highly expressed in carcinomas including prostate, breast, lung and ovary and expression is correlated with poor outcomes. TRPV6-mediated Ca2+ entry is responsible for maintaining a high proliferation rate, increasing cell survival and apoptosis resistance. Since SOR-C13 blocks TRPV6-mediated Ca2+ influx it was evaluated as a single agent in patients with late-stage carcinoma. Methods: This was a phase I, multi-center, open-label, dose escalation study to assess safety and tolerability of SOR-C13 in subjects with advanced carcinomas commonly known to express the TRPV6 channel (NCT01578564). Subjects received two 21-day cycles of treatment consisting of SOR-C13 for 3 d on/4 d off, 3 d on/11 d off (21 d/cycle). Tested doses were 1.375, 2.75, 4.13 and 6.2 mg/kg given as i.v. infusion initially over 20 m moving to 90 m. In the event of clinically meaningful response (tumor response or stable disease), additional cycles were offered to subjects. Subjects were assessed for safety, tolerability and preliminary efficacy. Results: Twenty-three subjects with advanced refractory solid tumors were in the study. No study drug-related serious adverse events occurred during the study. The most frequently reported treatment-emergent adverse events (TEAEs) were fatigue (30%), hypoalbuminemia (30%), anemia (30%), urinary tract infection (30%), blood calcium decreased (22%), decreased appetite (22%), nausea (22%), constipation (17%), aspartate aminotransferase increased (17%), cough (17%), blood alkaline phosphatase increased (13%), diarrhea (13%), hypercalcemia (13%), hyperkalemia (13%), hypocalcemia (13%). Anemia (Grade 1 to 3) was the only TEAE reflecting clinically significant hematological abnormalities. In all but one case, these events were assessed as unrelated or unlikely related to the study drug. The only safety concern identified definitely related to SOR-C13 infusion was reductions of serum calcium that were short-lived (resolved during the post-infusion observation time (4 h or within <24 h) and asymptomatic at all doses. A range of safe dosing was established between 4.13 and 6.2 mg/kg. Anti-tumor activity was observed across a wide variety of tumor types. Twelve of 22 evaluable subjects (54.5%) had stable disease after 2 cycles. The duration of response ranged from 4 to 7.5 cycles with one subject still in stable disease after 16 cycles (11 months). Two patients with advanced pancreatic cancer showed tumor reduction of 7% (4.13 mg/kg) and 27% (6.2 mg/kg) after two cycles with attendant decreases in CA 19-9. Conclusions: SOR-C13 was safe and well tolerated in subjects with advanced solid tumors of epithelial origin and demonstrates potential activity in patients with advanced solid tumor cancer. Citation Format: Siqing Fu, Hal H. Hirte, Stephen Welch, Toney T. Ilenchuk, Dominique Dugourd, Tyler Lutes, Christopher Rice, Jack M. Stewart. A phase I open-label, dose escalation study of a novel peptide (SOR-C13) antagonistic to the TRPV6 ion channel in patients with advanced solid tumor cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT142.

Conversion of the PIP2 Dependent TRPV6 Channel from Low to High Phosphoinositide Affinity by a Charge Reintroduction Mutation

The activity of Transient Receptor Potential Vanilloid 6 (TRPV6) ion channels depends on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], which is a common feature of TRP channels. The apparent affinity for PI(4,5)P2 is a major determinant of inhibition of ion channels by phosphoinositide depletion upon the activation of phospholipase C (PLC). The constitutively active TRPV6 and its closest relative TRPV5 have low affinity for PI(4,5)P2. The heat- and capsaicin-sensitive TRPV1 on the other hand shows high affinity for this lipid. TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that in TRPV1 was proposed to interact with PI(4,5)P2. We found that the G488R mutant of TRPV6, in which this positive charge is reintroduced, showed higher apparent affinity for PI(4,5)P2 in excised inside out patches, and unlike wild-type TRPV6, it was also activated by PI(4)P. In accordance with its higher affinity and lower specificity for PI(4,5)P2, the mutant was resistant to depletion of PI(4,5)P2 by the voltage-sensitive phosphatase ciVSP. The mutant also showed reduced Ca2+-induced inactivation, both in patch clamp and Ca2+ imaging experiments, highlighting the involvement of PI(4,5)P2 depletion by PLC in this process. Computational modeling show that the 488R residue interacts with PI(4,5)P2 in the mutant TRPV6. We identify a molecular determinant of PI(4,5)P2 affinity and our data support the role of the TM4-TM5 loop in PI(4,5)P2 interactions of TRPV channels.

The Roles Calmodulin and PI(4,5)P2 in Ca2+-Induced Inactivation of TRPV6 Channels

The epithelial Ca2+ channel Transient Receptor Potential Vanilloid 6 (TRPV6) undergoes Ca2+-induced inactivation. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca2+, CaM and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in excised patches. The activity of TRPV6 decreased after excision, (run down) and channels could be re-activated with either PI(4,5)P2 or MgATP, which supplies substrate for endogenous lipid kinases to synthesize PI(4,5)P2. Ca2+-CaM inhibited the channel at physiologically relevant concentrations both when endogenous PI(4,5)P2 was used to maintain channel activity, and when exogenous PI(4,5)P2 was applied. Ca2+ alone also inhibited TRPV6 at high concentrations (IC50 ∼20 μM). Biochemical binding experiments showed that CaM binds to TRPV6 via a distal C-terminal binding site. A double mutation in this CaM binding site of TRPV6 (W695A-R699E) essentially eliminated inhibition by CaM in excised patches. In whole-cell patch clamp experiments, this mutation reduced, but did not eliminate Ca2+-induced inactivation. Providing excess PI(4,5)P2 reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P2 did not inhibit binding of the C-terminus of the channel to CaM. Overall, our data show a complex interplay between CaM and PI(4,5)P2, and that Ca2+, CaM and the depletion of PI(4,5)P2 all contribute to inactivation of TRPV6.

Molecular Mechanisms of Calmodulin Regulation of TRPV6

Transient Receptor Potential Vanilloid 6 (TRPV6) is an epithelial Ca2+ channel that is responsible for active Ca2+ absorption in the intestines. We have recently demonstrated that this channel is activated directly by phosphatidylinositol 4, 5-bisphosphate (PIP2) and that cytoplasmic ATP stimulates TRPV6 channel activity indirectly by providing substrates for lipid kinases and the generation of PIP2. TRPV6 undergoes Ca2+-induced inactivation, preventing toxic Ca2+ overload of the cell. This inactivation has been shown to be mediated both by Ca2+-calmodulin (CaM) and depletion of PIP2.

Intracellular ATP supports TRPV6 activityvialipid kinases and the generation of PtdIns(4,5)P2

Transient receptor potential vanilloid 6 (TRPV6) channels play an important role in Ca(2+) absorption in the intestines. Both phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and cytoplasmic ATP have been proposed to be important for maintaining TRPV6 activity. To evaluate whether PtdIns(4,5)P(2) and ATP affect channel activity directly or indirectly, we have used a dual approach, examining channel activity in excised patches and planar lipid bilayers. In excised inside-out patch-clamp measurements, ATP reactivated the human TRPV6 channels after current rundown only in the presence of Mg(2+). The effect of MgATP was inhibited by 3 structurally different compounds that inhibit type III phosphatidylinositol 4-kinases (PI4Ks). PtdIns(4,5)P(2) also activated TRPV6 in excised patches, while its precursor PtdIns(4)P had only minimal effect. These data demonstrate that MgATP provides substrate for lipid kinases, allowing the resynthesis of PtdIns(4,5)P(2). To determine whether PtdIns(4,5)P(2) is a direct activator of TRPV6, we purified and reconstituted the channel protein in planar lipid bilayers. The reconstituted channel showed high activity in the presence of PtdIns(4,5)P(2), while PtdIns(4)P induced only minimal activity. Our data establish PtdIns(4,5)P(2) as a direct activator of TRPV6 and demonstrate that intracellular ATP regulates the channel indirectly as a substrate for type III PI4Ks.

Direct Activation of TRPV6 Channels by Phosphatidylinositol 4,5-Bisphosphate

Transient Receptor Potential Vanilloid 6 (TRPV6) is an inwardly rectifying Ca2+ selective ion channel that plays a role in Ca2+ absorption in the intestines. The activity of these channels have been shown to depend on the presence of phosphatidylinositol 4,5-bisphosphate (PIP2). Increased in cytoplasmic Ca2+ concentrations inhibit these channels, protecting cells from toxic Ca2+ overload. Both Ca2+ Calmodulin (CaCaM) and depletion of PIP2 have been implicated in Ca2+ induced inactivation. In addition, it has been proposed that cytoplasmic ATP binds directly to the channels, and this binding is important for maintaining TRPV6 channel activity in whole-cell patch clamp experiments. To evaluate whether PIP2, CaCaM and ATP affects channel activity directly or indirectly, we have performed excised inside-out patch clamp measurements with these compounds. Channel activity upon excision showed marked current run-down, characteristic of PIP2 dependent channels. ATP reactivated the channels, but only in the presence of Mg2+. This could mean that MgATP provides substrate for lipid kinases present in the patch membrane, allowing the resynthesis of PIP2. To test this hypothesis, we have used wortmannin and LY294002 at concentrations where they inhibit phosphatidylinositol 4-kinases, and found that they both inhibit the effect of MgATP in excised patches. At lower concentrations, where they specifically inhibit PI3Kinases, they did not inhibit the effects of MgATP. We also found that PIP2 reactivates TRPV6 in excised patches, and this effect is inhibited by CaCaM. To further establish PIP2 as a direct activator of TRPV6, we purified the channel protein and reconstituted it in planar lipid bilayers. We found that the reconstituted protein showed inward rectification, and its activity depended on the presence of PIP2. Our data establish PIP2 as a direct activator of TRPV6 and raise doubt that intracellular ATP regulates the channel through direct binding.

Altered Biochemical Properties of Transient Receptor Potential Vanilloid 6 Calcium Channel by Peptide Tags

Ion channels are commonly expressed in recombinant forms with peptide tags, which facilitates their molecular and electrophysiological studies. However, peptide tags may alter ion channel properties. Here we describe the differential effect of peptide tags on the biochemical properties of transient receptor potential vanilloid 6 (TRPV6) channels. Yellow fluorescent protein (YFP)-tagged wild-type TRPV6 (YFP-TRPV6(WT)) showed much lower levels of aggregate-like bands in Western blots than those of Myc-TRPV6(WT). By contrast, the glycosylation level was higher with Myc-TRPV6(WT) than that with YFP-TRPV6(WT). We additionally demonstrate that peptide tags affect the protein integrity of TRPV6 channels. Myc-TRPV6(WT) was expressed as an intact channel, whereas the pore mutants Myc-TRPV6(D542A) and Myc-TRPV6(D542K) were observed to be partially fragmented. By contrast, all YFP-tagged channels were intact, although the YFP-tagged pore mutants were less glycosylated than YFP-TRPV6(WT). However, regardless of the peptide tag used, TRPV6(D542A) and TRPV6(D542K) electrophysiologically inhibited TRPV6(WT) which indicates that all pore mutants are equivalent electrophysiologically, not biochemically. Thus, our findings suggest that peptide tags can produce unintended biochemical changes of ion channels which highlight the necessity of careful biochemical evaluation to clarify the roles of ion channels.