Conserved Gating Elements in TRPC4 and TRPC5 Channels

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca²⁺-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4–S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4_G503S and TRPC5_G504S mutants causes cell death, which could be prevented by buffering the Ca²⁺ of the culture medium. Current-voltage relationships of the TRPC4_G503S and TRPC5_G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4_G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4–S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.     

Plasma membrane stretch activates transient receptor potential vanilloid and ankyrin channels in Merkel cells from hamster buccal mucosa

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons. Many transient receptor potential (TRP) channels have been identified in mammals; however, the activation properties of these channels in oral mucosal MCs remain to be clarified. We investigated the biophysical and pharmacological properties of TRP vanilloid (TRPV)-1, TRPV2, TRPV4, TRP ankyrin (TRPA)-1, and TRP melastatin (TRPM)-8 channels, which are sensitive to osmotic and mechanical stimuli by measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) using fura-2. We also analyzed their localization patterns through immunofluorescence. MCs showed immunoreaction for TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 channels. In the presence of extracellular Ca²⁺, the hypotonic test solution evoked Ca²⁺ influx. The [Ca²⁺]_i increases were inhibited by TRPV1, TRPV2, TRPV4, or TRPA1 channel antagonists, but not by the TRPM8 channel antagonist. Application of TRPV1, TRPV2, TRPV4, TRPA1, or TRPM8 channel selective agonists elicited transient increases in [Ca²⁺]_i only in the presence of extracellular Ca²⁺. The results indicate that membrane stretching in MCs activates TRPV1, TRPV2, TRPV4, and TRPA1 channels, that it may be involved in synaptic transmission to sensory neurons, and that MCs could contribute to the mechanosensory transduction sequence.     

Expression of Transient Receptor Potential Vanilloid Channels TRPV5 and TRPV6 in Human Blood Lymphocytes and Jurkat Leukemia T Cells

Regulation of Ca²⁺ entry is a key process for lymphocyte activation, cytokine synthesis and proliferation. Several members of the transient receptor potential (TRP) channel family can contribute to changes in [Ca²⁺]_in; however, the properties and expression levels of these channels in human lymphocytes continue to be elusive. Here, we established and compared the expression of the most Ca²⁺-selective members of the TRPs, Ca²⁺ channels transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6), in human blood lymphocytes (HBLs) and leukemia Jurkat T cells. We found that TRPV6 and TRPV5 mRNAs are expressed in both Jurkat cells and quiescent HBLs; however, the levels of mRNAs were significantly higher in malignant cells than in quiescent lymphocytes. Western blot analysis showed TRPV5/V6 proteins in Jurkat T cells and TRPV5 protein in quiescent HBLs. However, the expression of TRPV6 protein was switched off in quiescent HBLs and turned on after mitogen stimulation of the cells with phytohemagglutinin. Inwardly directed monovalent currents that displayed characteristics of TRPV5/V6 currents were recorded in both Jurkat cells and normal HBLs. In outside–out patch-clamp studies, currents were reduced by ruthenium red, a nonspecific inhibitor of TRPV5/V6 channels. In addition, ruthenium red downregulated cell-cycle progression in both activated HBLs and Jurkat cells. Thus, we identified TRPV5 and TRPV6 calcium channels, which can be considered new candidates for Ca²⁺ entry into human lymphocytes. The correlation between expression of TRPV6 channels and the proliferative status of lymphocytes suggests that TRPV6 may be involved in the physiological and/or pathological proliferation of lymphocytes.     

Rapid effects of 17β-estradiol on TRPV5 epithelial Ca channels in rat renal cells

The renal distal tubules and collecting ducts play a key role in the control of electrolyte and fluid homeostasis. The discovery of highly calcium selective channels, Transient Receptor Potential Vanilloid 5 (TRPV5) of the TRP superfamily, has clarified the nature of the calcium entry channels. It has been proposed that this channel mediates the critical Ca²⁺ entry step in transcellular Ca²⁺ re-absorption in the kidney. The regulation of transmembrane Ca²⁺ flux through TRPV5 is of particular importance for whole body calcium homeostasis.In this study, we provide evidence that the TRPV5 channel is present in rat cortical collecting duct (RCCD₂) cells at mRNA and protein levels. We demonstrate that 17β-estradiol (E₂) is involved in the regulation of Ca²⁺ influx in these cells via the epithelial Ca²⁺ channels TRPV5. By combining whole-cell patch-clamp and Ca²⁺-imaging techniques, we have characterized the electrophysiological properties of the TRPV5 channel and showed that treatment with 20-50 nM E₂ rapidly (<5 min) induced a transient increase in inward whole-cell currents and intracellular Ca²⁺ via TRPV5 channels. This rise was significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV5.These data demonstrate for the first time, a novel rapid modulation of endogenously expressed TRPV5 channels by E₂ in kidney cells. Furthermore, the results suggest calcitropic effects of E₂. The results are discussed in relation to present concepts of non-genomic actions of E₂ in Ca²⁺ homeostasis.     

Interplay between Calmodulin and Phosphatidylinositol 4,5-Bisphosphate in Ca2+-induced Inactivation of Transient Receptor Potential Vanilloid 6 Channels

The epithelial Ca²⁺ channel transient receptor potential vanilloid 6 (TRPV6) undergoes Ca²⁺-induced inactivation that protects the cell from toxic Ca²⁺ overload and may also limit intestinal Ca²⁺ transport. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca²⁺, calmodulin (CaM), and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) in excised inside-out patches. The activity of TRPV6 strictly depended on the presence of PI(4,5)P₂, and Ca²⁺-CaM inhibited the channel at physiologically relevant concentrations. Ca²⁺ alone also inhibited TRPV6 at high concentrations (IC₅₀ = ∼20 μm). A double mutation in the distal C-terminal 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 CaM to the C terminus of the channel. Overall, our data show a complex interplay between CaM and PI(4,5)P₂ and show that Ca²⁺, CaM, and the depletion of PI(4,5)P₂ all contribute to inactivation of TRPV6.     

Phosphorylation on the Ser 824 residue of TRPV4 prefers to bind with F-actin than with microtubules to expand the cell surface area

Previously, we demonstrated that the transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, is one of the serum glucocorticoid-induced protein kinase1 (SGK1) authentic substrate proteins, and that the Ser 824 residue of TRPV4 is phosphorylated by SGK1 [1]. In this study, we demonstrated that phosphorylation on the Ser 824 residue of TRPV4 is required for its interaction with F-actin, using TRPV4 mutants (S824D; a phospho-mimicking TRPV4 mutant and S824A; a non-phosphorylatable TRPV4 mutant) and its proper subcellular localization. Additionally, we noted that the phosphorylation of the Ser824 residue promotes its single channel activity, Ca^2 + influx, protein stability, and cell surface area (expansion of plasma membrane).     

The Xenopus TRPV6 homolog encodes a Mg2+‐permeant channel that is inhibited by interaction with TRPC1

The TRP gene family encodes primarily cation non‐selective, Ca²⁺ permeant channels that are involved in a dizzying array of sensory mechanisms. Two channels in this large family TRPV5 and TRPV6 are highly Ca²⁺ selective and are expressed in epithelia where they are important in Ca²⁺ uptake. TRPV5/6 are constitutively active, yet the mechanisms regulating their activation in native tissue remains elusive. Here we functionally characterize the Xenopus TRPV6 homolog. xTRPV6 is expressed in the oocyte and encodes a channel that is permeant to divalents including Ca²⁺, and displays a high permeability to Mg²⁺. The oocyte does not exhibit functional TRPV6‐like current at rest, showing that the endogenous channel is somehow maintained in an inactive state. We show that endogenous as well as overexpressed xTRPV6 interacts with xTRPC1 and that this interaction inhibits xTRPV6 currents. As such TRPC1 is likely to regulate the activity of TRPV6 under physiological conditions. J. Cell. Physiol. 228: 2386–2398, 2013. © 2013 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Activation of the Ca2+-sensing receptor stimulates the activity of the epithelial Ca2+ channel TRPV5

The extracellular Ca²⁺-sensing receptor (CaR) is a key-player in plasma Ca²⁺ homeostasis. It is essentially expressed in the parathyroid glands and along the kidney nephron. The distal convoluted tubules (DCT) and connecting tubules (CNT) in the kidney are involved in active Ca²⁺ reabsorption, but the function of the CaR has remained unclear in these segments. Here, the Ca²⁺-selective Transient Receptor Potential Vanilloid-subtype 5 channel (TRPV5) determines active Ca²⁺ reabsorption by forming the apical entry gate. In this study we show that the CaR and TRPV5 co-localize at the luminal membrane of DCT/CNT. Furthermore, by patch-clamp and Fura-2-ratiometric measurements we demonstrate that activation of the CaR leads to elevated TRPV5-mediated currents and increases intracellular Ca²⁺ concentrations in cells co-expressing TRPV5 and CaR. Activation of CaR initiated a signaling cascade that activated phorbol-12-myristate-13-acetate (PMA)-insensitive protein kinase C (PKC) isoforms. Importantly, mutation of two putative PKC phosphorylation sites, S299 and S654, in TRPV5 prevented the stimulatory effect of CaR activation on channel activity, as did a dominant negative CaR construct, CaR^R185Q. Interestingly, the activity of TRPV6, TRPV5′ closest homologue, was not affected by the activated CaR. We conclude that activation of the CaR stimulates TRPV5-mediated Ca²⁺ influx via a PMA-insensitive PKC isoform pathway.     

Molecular Determinants of Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Binding to Transient Receptor Potential V1 (TRPV1) Channels

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P₂ is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P₂ binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P₂ interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P₂ behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P₂ binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P₂ in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P₂ binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.     

Canonical Transient Receptor Potential (TRPC) 1 Acts as a Negative Regulator for Vanilloid TRPV6-mediated Ca2+ Influx

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca²⁺ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca²⁺ and Na⁺ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca²⁺ influx in HEK293 cells.     

The epithelial calcium channels, TRPV5 & TRPV6: from identification towards regulation

The epithelial calcium channels, TRPV5 and TRPV6, have been extensively studied in epithelial tissues controlling the Ca²⁺ homeostasis and exhibit a range of distinctive properties that distinguish them from other TRP channels. This review focuses on the tissue distribution, the functional properties, the architecture and the regulation of the expression and activity of the TRPV5 and TRPV6 channel.     

Molecular determinants for the chemical activation of the warmth-sensitive TRPV3 channel by the natural monoterpenoid carvacrol

Transient receptor potential vanilloid 3 (TRPV3), robustly expressed in the skin, is a nonselective calcium-permeable cation channel activated by warm temperature, voltage, and certain chemicals. Natural monoterpenoid carvacrol from plant oregano is a known skin sensitizer or allergen that specifically activates TRPV3 channel. However, how carvacrol activates TRPV3 mechanistically remains to be understood. Here, we describe the molecular determinants for chemical activation of TRPV3 by the agonist carvacrol. Patch clamp recordings reveal that carvacrol activates TRPV3 in a concentration-dependent manner, with an EC₅₀ of 0.2 mM, by increasing the probability of single-channel open conformation. Molecular docking of carvacrol into cryo-EM structure of TRPV3 combined with site-directed mutagenesis further identified a unique binding pocket formed by the channel S2-S3 linker important for mediating this interaction. Within the binding pocket consisting of four residues (Ile505, Leu508, Arg509, and Asp512), we report that Leu508 is the most critical residue for the activation of TRPV3 by carvacrol, but not 2-APB, a widely used nonspecific agonist and TRP channel modulator. Our findings demonstrate a direct binding of carvacrol to TRPV3 by targeting the channel S2-S3 linker that serves as a critical domain for chemical-mediated activation of TRPV3. We also propose that carvacrol can function as a molecular tool in the design of novel specific TRPV3 modulators for the further understanding of TRPV3 channel pharmacology.     

Alteration of channel characteristics by exchange of pore-forming regions between two structurally related Ca2+ Channels

Several types of structurally homologous high voltage-gated Ca²⁺ channels (L-, P-and N-type) have been identified via biochemical, pharmacological and electrophysiological techniques. Among these channels, the cardiac L-type and the brain BI-2 Ca²⁺ channel display significantly different biophysical properties. The BI-2 channel exhibits more rapid voltage-dependent current activation and inactivation and smaller single-channel conductance compared to the L-type Ca²⁺ channel. To examine the molecular basis for the functional differences between the two structurally related Ca²⁺ channels, we measured macroscopic and single-channel currents from oocytes injected with wild-type and various chimeric channel ₁ subunit cRNAs. The results show that a chimeric channel in which the segment between S5-SS2 in repeat IV of the cardiac L-type Ca²⁺ channel, was replaced by the corresponding region of the BI-2 channel, exhibited macroscopic current activation and inactivation time-courses and single-channel conductance, characteristic of the BI-2 Ca²⁺ channel. The voltage-dependence of steady-state inactivation was not affected by the replacement. Chimeras, in which the SS2-S6 segment in repeat III or IV of the cardiac channel was replaced by the corresponding BI-2 sequence, exhibited altered macroscopic current kinetics without changes in single-channel conductance. These results suggest that part of the S5-SS2 segment plays a critical role in determining voltage-dependent current activation and inactivation and single-channel conductance and that the SS2-S6 segment may control voltage-dependent kinetics of the Ca²⁺ channel.     

N-acetyl cysteine reduces oxidative toxicity,apoptosis, and calcium entry through TRPV1 channels in the neutrophils of patients with polycystic ovary syndrome

Abstract

Polycystic ovary syndrome (PCOS) is a common inflammatory and oxidant disease with an uncertain pathogenesis. N-acetyl cysteine (NAC) decreases oxidative stress, intracellular free calcium ion [Ca²⁺]_i, and apoptosis levels in human neutrophil. We aimed to investigate the effects of NAC on apoptosis, oxidative stress, and Ca²⁺ entry through transient receptor potential vanilloid 1 (TRPV1) and TRP melastatin 2 (TRPM2) channels in neutrophils from patients with PCOS. Neutrophils isolated from PCOS group were investigated in three settings: (1) after incubation with TRPV1 channel blocker capsazepine or TRPM2 channel blocker 2-aminoethyl diphenylborinate (2-APB), (2) after supplementation with NAC (for 6 weeks), and (3) with combination (capsazepine + 2-APB + NAC) exposure. The neutrophils in TRPM2 and TRPV1 experiments were stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 μM) and capsaicin (10 μM) as concentration agonists, respectively. Neutrophil lipid peroxidation and capsaicin-induced increase in [Ca²⁺]_i concentrations were reduced by capsazepine and NAC treatments. However, the [Ca²⁺]_i concentration did not change by fMLP stimulation. Neutrophil lipid peroxidation, apoptosis, caspase-3, caspase-9, cytosolic reactive oxygen species production, and mitochondrial membrane depolarization values were decreased by NAC treatment although neutrophil glutathione peroxidase and reduced glutathione levels were increased by the NAC treatment. Serum lipid peroxidation, luteinizing hormone, testosterone, insulin, interleukin-1 beta, and homocysteine levels were decreased by NAC treatment although serum vitamin A, beta-carotene, vitamin E, and total antioxidant status were increased by the NAC treatment. In conclusion, NAC reduced oxidative stress, apoptosis, cytokine levels, and Ca²⁺ entry through TRPV1 channel, which provide supportive evidence that oxidative stress and TRPV1 channel plays a key role in etiology of PCOS.     

Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages

There is considerable evidence indicating that intracellular Ca²⁺ participates as a second messenger in TLR4-dependent signaling. However, how intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) is increased in response to LPS and how they affect cytokine production are poorly understood. Here we examined the role of transient receptor potential (TRP), a major Ca²⁺ permeation pathway in non-excitable cells, in the LPS-induced cytokine production in macrophages. Pharmacologic experiments suggested that TRPV family members, but neither TRPC nor TRPM family members, are involved in the LPS-induced TNFα and IL-6 production in RAW264 macrophages. RT-PCR and immunoblot analyses showed that TRPV2 is the sole member of TRPV family expressed in macrophages. ShRNA against TRPV2 inhibited the LPS-induced TNFα and IL-6 production as well as IκBα degradation. Experiments using BAPTA/AM and EGTA, and Ca²⁺ imaging suggested that the LPS-induced increase in [Ca²⁺]_i involves both the TRPV2-mediated intracellular and extracellular Ca²⁺ mobilizations. BAPTA/AM abolished LPS-induced TNFα and IL-6 production, while EGTA only partially suppressed LPS-induced IL-6 production, but not TNFα production. These data indicate that TRPV2 is involved in the LPS-induced Ca²⁺ mobilization from intracellular Ca²⁺ store and extracellular Ca²⁺. In addition to Ca²⁺ mobilization through the IP₃-receptor, TRPV2-mediated intracellular Ca²⁺ mobilization is involved in NFκB-dependent TNFα and IL-6 expression, while extracellular Ca²⁺ entry is involved in NFκB-independent IL-6 production.     

Modeling the structural and dynamical changes of the epithelial calcium channel TRPV5 caused by the A563T variation based on the structure of TRPV6

TRPV5, transient receptor potential cation channel vanilloid subfamily member 5, is an epithelial Ca²⁺ channel that plays a key role in the active Ca²⁺ reabsorption process in the kidney. A single nucleotide polymorphism (SNP) rs4252499 in the TRPV5 gene results in an A563T variation in the sixth transmembrane (TM) domain of TRPV5. Our previous study indicated that this variation increases the Ca²⁺ transport function of TRPV5. To understand the molecular mechanism, a model of TRPV5 was established based on the newly deposited structure of TRPV6 that has 83.1% amino acid identity with TRPV5 in the modeled region. Computational simulations were performed to study the structural and dynamical differences between the TRPV5 variants with A563 and T563. Consistent with the TRPV1-based simulation, the results indicate that the A563T variation increases the contacts between residues 563 and V540, which is one residue away from the key residue D542 in the Ca²⁺-selective filter. The variation enhanced the stability of the secondary structure of the pore region, decreased the fluctuation of residues around residue 563, and reduced correlated and anti-correlated motion between monomers. Furthermore, the variation increases the pore radius at the selective filter. These findings were confirmed using simulations based on the recently determined structure of rabbit TRPV5. The simulation results provide an explanation for the observation of enhanced Ca²⁺ influx in TRPV5 caused by the A563T variation. The A563T variation is an interesting example of how a residue distant from the Ca²⁺-selective filter influences the Ca²⁺ transport function of the TRPV5 channel.

Communicated by Ramaswamy H. Sarma     

The S4–­­S5 linker – gearbox of TRP channel gating

Transient receptor potential (TRP) channels are cation channels which participate in a wide variety of physiological processes in organisms ranging from fungi to humans. They fulfill roles in body homeostasis, are sensors for noxious chemicals and temperature in the mammalian somatosensory system and are activated by light stimulated phospholipase C activity in Drosophila or by hypertonicity in yeast. The transmembrane topology of TRP channels is similar to that of voltage-gated cation channels. TRP proteins assemble as tetramers with each subunit containing six transmembrane helices (S1–S6) and intracellular N- and C-termini. Here we focus on the emerging functions of the cytosolic S4–S5 linker on TRP channel gating. Most of this knowledge comes from pathogenic mutations within the S4–S5 linker that alter TRP channel activities. This knowledge has stimulated forward genetic approaches to identify additional residues around this region which are essential for channel gating and is supported, in part, by recent structures obtained for TRPV1, TRPV2, TRPV6, TRPA1, and TRPP2.     

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca²⁺ channel TRPV5 constitutes the apical entry gate for Ca²⁺ transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca²⁺ reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using fura-2 analysis. Removal of amino acid His⁷¹² elevated the [Ca²⁺]_i, indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His⁷¹² for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His⁷¹² was confirmed by patch clamp analysis, which demonstrates increased Na⁺ and Ca²⁺ currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca²⁺-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca²⁺]_i. Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His⁷¹² plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.     

Regulation of the Ca2+ Channel TRPV6 by the Kinases SGK1, PKB/Akt, and PIKfyve

The serum- and glucocorticoid-inducible kinase SGK1 and the protein kinase PKB/Akt presumably phosphorylate and, by this means, activate the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3), which has in turn been shown to regulate transporters and channels. SGK1-regulated channels include the Ca²⁺ channel TRPV6, which is expressed in a variety of epithelial and nonepithelial cells including tumor cells. SGK1 and protein kinase B PKB/Akt foster tumor growth. The present study thus explored whether TRPV6 is regulated by PIKfyve. TRPV6 was expressed in Xenopus laevis oocytes with or without additional coexpression of constitutively active ^S422DSGK1, constitutively active ^T308D,S473DPKB, wild-type PIKfyve, and ^S318APIKfyve lacking the SGK1 phosphorylation site. TRPV6 activity was determined from the current (I_Ca) resulting from TRPV6-induced Ca²⁺ entry and subsequent activation of Ca²⁺-sensitive endogenous Cl^? channels. TRPV6 protein abundance in the cell membrane was determined utilizing immunohistochemistry and Western blotting. In TRPV6-expressing oocytes I_H was increased by coexpression of ^S422DSGK1 and by ^T308D,S473DPKB. Coexpression of wild-type PIKfyve further increased I_H in TRPV6 + ^S422DSGK1-expressing oocytes but did not significantly modify I_Ca in oocytes expressing TRPV6 alone. ^S318APIKfyve failed to significantly modify I_Ca in the presence and absence of ^S422DSGK1. ^S422DSGK1 increased the TRPV6 protein abundance in the cell membrane, an effect augmented by additional expression of wild-type PIKfyve. We conclude that PIKfyve participates in the regulation of TRPV6.