OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage.

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

Ca2+ transport via TRPV6 is regulated by rapid internalization of the channel.

Amongst the superfamily of transient receptor potential (TRP) channels, TRPV5 and TRPV6 are specialized members that mediate Ca2+-selective transport across epithelial membranes. Intriguingly, fluorescent fusion proteins of TRPV5 or TRPV6 are hardly discernible within the plasma membrane of living cells. Instead, TRPV6 is mostly found in vesicular membrane compartments, indicating either a rapid degradation or cycling of channel-bearing vesicles between endomembrane compartments and the plasma membrane. In TRPV6-expressing cells, brefeldin A, a toxin that blocks the transit between the endoplasmic reticulum and the Golgi apparatus, caused a drop in [Ca2+]i with a half time in the range of 0.5-1 h. Upon wash-out of the toxin, the [Ca2+]i rose to a steady-state level within 2-3 h. Consistently, the synchronized forward trafficking of TRPV6VL-eGFP after brefeldin A wash-out led to a visible accumulation of the protein within the plasma membrane, as shown by confocal and total internal reflection microscopy. Analysis of the internalization route and differentiation of vesicle populations provided evidence for a clathrin-dependent internalization pathway. Most TRPV6VL-bearing vesicles co-stained with Rab5a, a marker protein for early endosomes. Fewer vesicles were co-localized with Rab7a (late endosomes) or with Rab11 (recycling endosomes). From these data, we propose that the lack of plasma membrane visibility of the channel results from a rapid internalization, which in addition to transcriptional regulation, adds a layer of functional channel regulation to modulate transepithelial Ca2+ transport.

Mutations That Affect the Surface Expression of TRPV6 Are Associated with the Upregulation of Serine Proteases in the Placenta of an Infant.

Recently, we reported a case of an infant with neonatal severe under-mineralizing skeletal dysplasia caused by mutations within both alleles of the TRPV6 gene. One mutation results in an in frame stop codon (R510stop) that leads to a truncated, nonfunctional TRPV6 channel, and the second in a point mutation (G660R) that, surprisingly, does not affect the Ca2+ permeability of TRPV6. We mimicked the subunit composition of the unaffected heterozygous parent and child by coexpressing the TRPV6 G660R and R510stop mutants and combinations with wild type TRPV6. We show that both the G660R and R510stop mutant subunits are expressed and result in decreased calcium uptake, which is the result of the reduced abundancy of functional TRPV6 channels within the plasma membrane. We compared the proteomic profiles of a healthy placenta with that of the diseased infant and detected, exclusively in the latter two proteases, HTRA1 and cathepsin G. Our results implicate that the combination of the two mutant TRPV6 subunits, which are expressed in the placenta of the diseased child, is responsible for the decreased calcium uptake, which could explain the skeletal dysplasia. In addition, placental calcium deficiency also appears to be associated with an increase in the expression of proteases.

Activity of the yeast vacuolar TRP channel TRPY1 is inhibited by Ca2+-calmodulin binding.

Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca2+ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca2+ concentration. However, the mechanism(s) of Ca2+-dependent regulation of TRPY1 and possible contribution(s) of Ca2+-binding proteins are yet not well understood. Our results demonstrate a Ca2+-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1-6 yeast strain, carrying a non-Ca2+-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1-6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca2+ influx and Ca2+ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca2+-CaM required the cytoplasmic amino acid stretch E33-Y92. In summary, our results show that TRPY1 is under inhibitory control of Ca2+-CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca2+-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca2+-CaM.

Comments on the evolution of TRPV6.

It is well known that not all biological findings derived from animals can be directly applied to humans. The TRPV6 protein may serve as an example which highlights these inter-species differences as an example of parallel evolutionary pathways. TRPV6 (and TRPV5) belong to a family of ion channels from the transient receptor potential group but are selectively permeable for Ca2+, in contrast to other members of the family. Sequences with recognizable similarity to TRPV6 can already be found in archaebacteria. These ancient sequences show clear similarity to the ion-conducting pore of TRPV6. Over the course of evolution, the duplication of the TRPV6 gene gave rise to TRPV5. Duplications of the complete genome as well as subsequent loss of genetic material have led to a variety of different TRPV5/6 combinations. In addition, there is an N-terminal extension of the protein in placental animals. This extension causes translation of TRPV6 to be initiated from an ACG codon. Inactivation of one TRPV6 allele can be correlated with alcohol-independent pancreatitis in humans while inactivation of both alleles leads to skeletal dysplasia of newborn babies. The latter effect is not observed in mice, implying that the effects due to perturbations in TRPV6 levels are much more pronounced in humans.

Human TRPV6-pathies caused by gene mutations.

The TRP-family of ion channels consists of 27 members in humans. Most TRP channels are non- selective cation channels with the exception of TRPV5 and TRPV6 which exhibit a high permeability for Ca2+ ions. A functional channel is formed by 4 identical subunits [1]. A growing number of mutations are present in human TRPV6 genes which alter channel function and can lead to elevated blood levels of the parathyroid hormone accompanied by transient hyperparathyroidism. Recent publications suggest that TRPV6 mutations could also trigger non-alcoholic chronic pancreatitis. This review summarises the consequences of these mutations within the TRPV6 gene.

Transient Receptor Potential Vanilloid 6 (TRPV6) Proteins Control the Extracellular Matrix Structure of the Placental Labyrinth.

Calcium-selective transient receptor potential Vanilloid 6 (TRPV6) channels are expressed in fetal labyrinth trophoblasts as part of the feto-maternal barrier, necessary for sufficient calcium supply, embryo growth, and bone development during pregnancy. Recently, we have shown a less- compact labyrinth morphology of Trpv6-deficient placentae, and reduced Ca2+ uptake of primary trophoblasts upon functional deletion of TRPV6. Trpv6-/- trophoblasts show a distinct calcium-dependent phenotype. Deep proteomic profiling of wt and Trpv6-/- primary trophoblasts using label-free quantitative mass spectrometry leads to the identification of 2778 proteins. Among those, a group of proteases, including high-temperature requirement A serine peptidase 1 (HTRA1) and different granzymes are more abundantly expressed in Trpv6-/- trophoblast lysates, whereas the extracellular matrix protein fibronectin and the fibronectin-domain-containing protein 3A (FND3A) were markedly reduced. Trpv6-/-placenta lysates contain a higher intrinsic proteolytic activity increasing fibronectin degradation. Our results show that the extracellular matrix formation of the placental labyrinth depends on TRPV6; its deletion in trophoblasts correlates with the increased expression of proteases controlling the extracellular matrix in the labyrinth during pregnancy.

Why endogenous TRPV6 currents are not detectable-what can we learn from bats?

TRPV6 is a calcium selective TRP channel and is expressed in many species. TRPV6 transcripts are abundantly expressed in few tissues but strangely channel properties are only accessible to electrophysiological recordings after overexpression whereas in native tissue functional channel currents seem not to be detectable. Another exceptional property of human and mouse TRPV6 proteins is that the initiation of translation starts from a non-canonical ACG triplet which is translated as methionine. This triplet is located 120 bp upstream of the first in-frame AUG codon of the human/mouse TRPV6 mRNA. In contrast, the TRPV6 gene of bats is initiated from an AUG triplet at the corresponding position of the human ACG. On the basis of these structural nucleotide differences between human and bats we studied the role of the absolute N-Terminus for the regulation of translation by developing chimera and mutants of human/bat TRPV6 channels. The human sequence which is located downstream of the initiation codon slows down ribosomal scanning in 3' direction. We suggest that the mechanism involves most likely the deceleration of ribosome scanning by stem-loop formation and the use of the common initiator tRNA, tRNAiMet, which is placed onto the inappropriate ACG codon resulting in low protein synthesis. The reduced translation efficiency is important to protect TRPV6 expressing cells from toxic calcium overload. The regulation of the TRPV6 translation in bats may be an adaptation to low calcium amounts present in the natural nutrition. In addition, we show that also the GFP protein can be controlled using the translational mechanism of human TRPV6.

The super-cooling compound icilin stimulates c-Fos and Egr-1 expression and activity involving TRPM8 channel activation, Ca2+ ion influx and activation of the ternary complex factor Elk-1.

The TRPM8 cation channel can be activated by the cooling compound icilin. Recently, we showed that stimulation of TRPM8 channels induces a signaling cascade leading to the activation of the transcription factor AP-1. Additionally, expression of the AP-1 constituent c-Fos has been shown to be induced following TRPM8 stimulation. c-Fos is frequently used as a marker for neuronal activity. Here, we have analyzed the mechanism connecting TRPM8 stimulation and c-Fos expression. Furthermore, we analyzed the expression of the neuronal activity-responsive transcription factor Egr-1 following TRPM8 activation. The results show that icilin-induced stimulation of TRPM8 channels increased c-Fos promoter activity and induced c-Fos expression. Moreover, icilin stimulation increased Egr-1 promoter activity and induced the expression of Egr-1. Pharmacological inhibition of TRPM8 blocked the icilin-induced expression of Egr-1 and c-Fos. An influx of Ca2+ ions into the cells via TRPM8 was necessary to stimulate Egr-1 and c-Fos expression following icilin treatment. Genetic experiments revealed that serum response elements within the Egr-1 and c-Fos promoters are crucial to couple TRPM8 stimulation with enhanced transcription of both the Egr-1 and c-Fos genes. These data were corroborated by experiments showing that TRPM8 stimulation increased the transcriptional activation potential of Elk-1, a SRE binding protein. c-Fos is important for neuronal excitability and survival. Egr-1 plays an important role in synaptic plasticity, consolidation and reconsolidation of long-term memory. Elk-1 may preserve neurons against toxic insults but may also induce depressive behaviour. The fact that TRPM8 stimulation activates the transcription factors c-Fos, Egr-1, and Elk-1 connects TRPM8 signaling with maintaining important brain functions.

Pharmacological inhibition of TRPM8-induced gene transcription.

Transient receptor potential melastatin-8 (TRPM8) channels are activated by cold temperature, menthol and icilin, leading to cold sensation. TRPM8 activation is connected with various diseases, indicating that a specific pharmacological antagonist, allowing nongenetic channel suppression, would be a valuable tool for therapy and basic research. Here, we assessed the biological activity and specificity of various TRPM8 inhibitors following stimulation of TRPM8 channels with either icilin or menthol. Recently, we showed that icilin strikingly upregulates the transcriptional activity of AP-1. By measuring AP-1 activity, we assessed which compound interrupted the TRPM8-induced intracellular signaling cascade from the plasma membrane to the nucleus. We tested the specificity of various TRPM8 inhibitors by analyzing AP-1 activation following stimulation of TRPM3 and TRPV1 channels, L-type voltage-gated Ca2+ channels, and Gαq-coupled receptors, either in the presence or absence of a particular TRPM8 inhibitor. The results show that the TRPM8 inhibitors BCTC, RQ-00203078, TC-1 2014, 2-APB, and clotrimazole blocked TRPM8-mediated activation of AP-1. However, only the compound RQ-00203078 showed TRPM8-specificity, while the other compounds function as broad-spectrum Ca2+ channel inhibitors. In addition, we show that progesterone interfered with TRPM8-induced activation of AP-1, as previously shown for TRPM3 and TRPC6 channels. TRPM8-induced transcriptional activation of AP-1 was additionally blocked by the compound PD98059, indicating that extracellular signal-regulated protein kinase-1/2 is essential to couple TRPM8 stimulation with transcriptional activation of AP-1. Moreover, an influx of Ca2+-ions is essential to induce the intracellular signaling cascade leading to the activation of AP-1.

Dihydrotestosterone activates AP-1 in LNCaP prostate cancer cells.

A cross-talk between androgen/androgen receptor signaling and the AP-1 transcription factor has been proposed. In this study, we asked whether activation of AP-1 modifies androgen-responsive gene transcription, and whether androgens effect AP-1-regulated gene transcription. We show that activation of AP-1 via expression of a constitutively active mutant of mitogen-activated/extracellular signal responsive kinase kinase (MEK) kinase-1 did not increase the activity of the androgen-responsive probasin promoter. Likewise, expression of a constitutively active mutant of the transcription factor c-Jun, which is a major constitutent of AP-1, did not increase the activity of the probasin promoter. In contrast, 5α-dihydrotestosterone (DHT) activated both the probasin promoter and the AP-1-regulated collagenase promoter in LNCaP prostate cancer cells. The AP-1 binding site within the collagenase promoter was identified as DHT-responsive element. In line with this, DHT increased the activities of the c-Jun promoter and the tumor necrosis factor alpha promoter, which both contain AP-1 binding sites. The signal transduction pathway coupling DHT stimulation with AP-1 activation required c-Jun, MAP kinases and androgen receptors, but was independent of transient receptor potential melastatin-8 (TRPM8) channels, proposed to function as ionotropic testosterone receptors. Expression of the GTPase activating protein RGS2 attenuated DHT-induced activation of AP-1, indicating that the DHT-induced signaling cascade involves G proteins.

IP3 Receptor-Dependent Cytoplasmic Ca2+ Signals Are Tightly Controlled by Cavß3.

Voltage-gated calcium channels (Cavs) are major Ca2+ entry pathways in excitable cells. Their ß subunits facilitate membrane trafficking of the channel's ion-conducting α1 pore and modulate its gating properties. We report that one ß subunit, ß3, reduces Ca2+ release following stimulation of phospholipase C-coupled receptors and inositol 1,4,5-trisphosphate (IP3) formation. This effect requires the SH3-HOOK domain of Cavß3, includes physical ß3/IP3 receptor interaction, and prevails when agonist-induced IP3 formation is bypassed by photolysis of caged IP3. In agreement with ß3 acting as a brake on Ca2+ release, fibroblast migration is enhanced in vitro, and in vivo, closure of skin wounds is accelerated in the absence of ß3. To mediate specific physiological responses and to prevent Ca2+ toxicity, cytoplasmic Ca2+ signals must be tightly controlled. The described function of ß3, unrelated to its function as a Cav subunit, adds to this tight control.

TRPC1- and TRPC3-dependent Ca2+ signaling in mouse cortical astrocytes affects injury-evoked astrogliosis in vivo.

Following brain injury astrocytes change into a reactive state, proliferate and grow into the site of lesion, a process called astrogliosis, initiated and regulated by changes in cytoplasmic Ca2+ . Transient receptor potential canonical (TRPC) channels may contribute to Ca2+ influx but their presence and possible function in astrocytes is not known. By RT-PCR and RNA sequencing we identified transcripts of Trpc1, Trpc2, Trpc3, and Trpc4 in FACS-sorted glutamate aspartate transporter (GLAST)-positive cultured mouse cortical astrocytes and subcloned full-length Trpc1 and Trpc3 cDNAs from these cells. Ca2+ entry in cortical astrocytes depended on TRPC3 and was increased in the absence of Trpc1. After co-expression of Trpc1 and Trpc3 in HEK-293 cells both proteins co-immunoprecipitate and form functional heteromeric channels, with TRPC1 reducing TRPC3 activity. In vitro, lack of Trpc3 reduced astrocyte proliferation and migration whereas the TRPC3 gain-of-function moonwalker mutation and Trpc1 deficiency increased astrocyte migration. In vivo, astrogliosis and cortex edema following stab wound injury were reduced in Trpc3-/- but increased in Trpc1-/- mice. In summary, our results show a decisive contribution of TRPC3 to astrocyte Ca2+ signaling, which is even augmented in the absence of Trpc1, in particular following brain injury. Targeted therapies to reduce TRPC3 channel activity in astrocytes might therefore be beneficial in traumatic brain injury.

The stoichiometry of the TMEM16A ion channel determined in intact plasma membranes of COS-7 cells using liquid-phase electron microscopy.

TMEM16A is a membrane protein forming a calcium-activated chloride channel. A homodimeric stoichiometry of the TMEM16 family of proteins has been reported but an important question is whether the protein resides always in a dimeric configuration in the plasma membrane or whether monomers of the protein are also present in its native state within in the intact plasma membrane. We have determined the stoichiometry of the human (h)TMEM16A within whole COS-7 cells in liquid. For the purpose of detecting TMEM16A subunits, single proteins were tagged by the streptavidin-binding peptide within extracellular loops accessible by streptavidin coated quantum dot (QD) nanoparticles. The labeled proteins were then imaged using correlative light microscopy and environmental scanning electron microscopy (ESEM) using scanning transmission electron microscopy (STEM) detection. The locations of 19,583 individual proteins were determined of which a statistical analysis using the pair correlation function revealed the presence of a dimeric conformation of the protein. The amounts of detected label pairs and single labels were compared between experiments in which the TMEM16A SBP-tag position was varied, and experiments in which tagged and non-tagged TMEM16A proteins were present. It followed that hTMEM16A resides in the plasma membrane as dimer only and is not present as monomer. This strategy may help to elucidate the stoichiometry of other membrane protein species within the context of the intact plasma membrane in future.

Murine Autoimmune Optic Neuritis Is Not Phenotypically Altered by the Retinal Degeneration 8 Mutation.

Purpose: To investigate whether the presence of the retinal degeneration 8 (rd8) mutation in C57BL/6 mice alters the phenotype of autoimmune optic neuritis (AON). Methods: C57BL/6J and C57BL/6N mice were genotyped for the rd8 mutation and fundus analyses and examination of retinal layer morphology were performed in vivo by scanning laser ophthalmoscopy and optical coherence tomography. Visual function was assessed by recording electroretinographs, and visual evoked potentials and retinae and optic nerves were assessed histopathologically. Retinal ganglion cell numbers were determined by retrograde labeling with fluorogold. Mice were then immunized with myelin oligodendrocyte glycoprotein 35-55 to induce AON before assessment of retinal ganglion cell degeneration, inflammatory infiltration of retinae and optic nerves, and demyelination. Furthermore, visual function was assessed by visual evoked potentials. Results: All C57BL/6N mice were homozygous for the mutation (Crb1rd8/rd8) and had pathology typical of the rd8 mutation; however, this was not seen in the C57BL/6J (Crb1wt/wt) mice. Following induction of AON, no differences were seen between the Crb1rd8/rd8 and Crb1wt/wt mice regarding disease parameters nor regarding inner retinal degeneration either in the retina as a whole or in the inferior nasal quadrant. Conclusions: The presence of the rd8 mutation in C57BL/6 mice does not affect the course of AON and should not provide a confounding factor in the interpretation of experimental results obtained in this model. However, it could be dangerous in other models of ocular pathology.

Identification of Sidt2 as a lysosomal cation-conducting protein.

A screen to identify lysosomal-expressed ion channels led to the discovery of the human Sidt2 protein. Sidt2 is expressed within lysosomal organelles but as a result of heterologous overexpression the protein is also detectable within the plasma membrane of human embryonic kidney cells. The overexpressed protein leads to cell depolarization upon sodium addition. Accordingly in whole-cell patch clamp experiments a spontaneous noninactivating monovalent cation current can be detected in Sidt2-overexpressing cells. Strong overexpression of Sidt2 in HEK293 cells is attended by a significant reduction/loss of detectable lysosomes, indicating that the overexpressed protein leads to lysosomal dysfunction, a hallmark of Alzheimer's disease. Sidt2 is located on chromosome 11q23, a locus repeatedly found by chromosomal mapping of Alzheimer's disease-related genes.

A conserved gating element in TRPV6 channels.

The Ca2+-selective tetrameric Transient Receptor Potential Vanilloid 6 (TRPV6) channel is an inwardly rectifying ion channel. The constitutive current endures Ca2+-induced inactivation as a result of the activation of phospholipase C followed depletion of phosphatidylinositol 4,5-bisphosphate, and calmodulin binding. Replacing a glycine residue within the cytosolic S4-S5 linker of the human TRPV6 protein, glycine 516, which is conserved in all TRP channel proteins, by a serine residue forces the channels into an open conformation thereby enhancing constitutive Ca2+ entry and preventing inactivation. Introduction of a second mutation (T621A) into TRPV6G516S reduces constitutive activity and partially rescues the TRPV6 function. According to the recently revealed crystal structure of the rat TRPV6 the T621 is adjacent to the distal end of the transmembrane segment 6 (S6) within a short linker between S6 and the helix formed by the TRP domain. These results indicate that the S4-S5 linker and the S6-TRP-domain linker are critical constituents of TRPV6 channel gating and that disturbance of their sequences foster constitutive Ca2+ entry.

TRPV6 channels.

TRPV6 (former synonyms ECAC2, CaT1, CaT-like) displays several specific features which makes it unique among the members of the mammalian Trp gene family (1) TRPV6 (and its closest relative, TRPV5) are the only highly Ca(2+)-selective channels of the entire TRP superfamily (Peng et al. 1999; Wissenbach et al. 2001; Voets et al. 2004). (2) Translation of Trpv6 initiates at a non-AUG codon, at ACG, located upstream of the annotated AUG, which is not used for initiation (Fecher-Trost et al. 2013). The ACG codon is nevertheless decoded by methionine. Not only a very rare event in eukaryotic biology, the full-length TRPV6 protein existing in vivo comprises an amino terminus extended by 40 amino acid residues compared to the annotated truncated TRPV6 protein which has been used in most studies on TRPV6 channel activity so far. (In the following numbering occurs according to this full-length protein, with the numbers of the so far annotated truncated protein in brackets). (3) Only in humans a coupled polymorphism of Trpv6 exists causing three amino acid exchanges and resulting in an ancestral Trpv6 haplotype and a so-called derived Trpv6 haplotype (Wissenbach et al. 2001). The ancestral allele encodes the amino acid residues C197(157), M418(378) and M721(681) and the derived alleles R197(157), V418(378) and T721(681). The ancestral haplotype is found in all species, the derived Trpv6 haplotype has only been identified in humans, and its frequency increases with the distance to the African continent. Apparently the Trpv6 gene has been a strong target for selection in humans, and its derived variant is one of the few examples showing consistently differences to the orthologues genes of other primates (Akey et al. 2004, 2006; Stajich and Hahn 2005; Hughes et al. 2008). (4) The Trpv6 gene expression is significantly upregulated in several human malignancies including the most common cancers, prostate and breast cancer (Wissenbach et al. 2001; Zhuang et al. 2002; Fixemer et al. 2003; Bolanz et al. 2008). (5) Male mice lacking functional TRPV6 channels are hypo-/infertile making TRPV6 one of the very few channels essential for male fertility (Weissgerber et al. 2011, 2012).

Trans-activation response (TAR) RNA-binding protein 2 is a novel modulator of transient receptor potential canonical 4 (TRPC4) protein.

TRPC4 proteins function as Ca(2+) conducting, non-selective cation channels in endothelial, smooth muscle, and neuronal cells. To further characterize the roles of TRPC4 in vivo, detailed information about the molecular composition of native channel complexes and their association with cellular signaling networks is needed. Therefore, a mouse brain cDNA library was searched for novel TRPC4-interacting proteins using a modified yeast two-hybrid assay. This screen identified Trans-activation Response RNA-binding protein 2 (Tarpb2), a protein that recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Tarbp2 was found to bind to the C terminus of TRPC4 and TRPC5 and to modulate agonist-dependent TRPC4-induced Ca(2+) entry. A stretch of basic residues within the Tarbp2 protein is required for these actions. Tarbp2 binding to and modulation of TRPC4 occurs in the presence of endogenously expressed Dicer but is no longer detectable when the Dicer cDNA is overexpressed. Dicer activity in crude cell lysates is increased in the presence of Ca(2+), most probably by Ca(2+)-dependent proteolytic activation of Dicer. Apparently, Tarbp2 binding to TRPC4 promotes changes of cytosolic Ca(2+) and, thereby, leads to a dynamic regulation of Dicer activity, essentially at low endogenous Dicer concentrations.