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TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gβγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gβγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gβγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gβ1 that are individually necessary for TRPM3 inhibition by Gβγ. The 10-amino-acid Gβγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gβγ proteins exert on TRPM3 channels. ispartof: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA vol:117 issue:46 pages:29090-29100 ispartof: location:United States status: published

Tuft cells are a rare population of chemosensory cells at the mucosal surface epithelia of hollow organs. Their name-giving morphological feature is an apical tuft of stiff microvilli. Accordingly, the actin-binding protein, villin, was identified as one of the first tuft cell markers in immunohistochemical analysis. Unfortunately, villin expression is not restricted to tuft cells, but is also prominent e.g. in enterocytes, which limits the use of this gene as a marker and as an experimental tool to genetically target tuft cells. Here, we report that the villin-related protein, advillin, is a specific tuft cell marker in the gastro-intestinal and biliary tract epithelia. In situ hybridization and immunohistochemistry revealed that advillin expression, unlike villin, was restricted to solitary cholinergic tuft cells in the mucosal linings of the small and large intestine, and in the gall bladder. In the glandular stomach, villin and advillin mRNA were present in all epithelial cells, while detectable protein levels were confined to solitary tuft cells. Advillin expression was no longer detectable in the mucosa of the intestinal and biliary tract from Pou2f3 deficient mice that lack tuft cells. Finally, crossing Avil-Cre transgenic mice with a double-fluorescent reporter mouse line resulted in specific targeting of gastro-intestinal and biliary tuft cells. Our analysis introduces advillin as a selective marker and tool in histological and functional analysis of the alimentary tract tuft cell system. Electronic supplementary material The online version of this article (doi:10.1007/s10735-020-09893-6) contains supplementary material, which is available to authorized users.

The gallbladder stores bile between meals and empties into the duodenum upon demand and is thereby exposed to the intestinal microbiome. This exposure raises the need for antimicrobial factors, among them, mucins produced by cholangiocytes, the dominant epithelial cell type in the gallbladder. The role of the much less frequent biliary tuft cells is still unknown. We here show that propionate, a major metabolite of intestinal bacteria, activates tuft cells via the short-chain free fatty acid receptor 2 and downstream signaling involving the cation channel transient receptor potential cation channel subfamily M member 5. This results in corelease of acetylcholine and cysteinyl leukotrienes from tuft cells and evokes synergistic paracrine effects upon the epithelium and the gallbladder smooth muscle, respectively. Acetylcholine triggers mucin release from cholangiocytes, an epithelial defense mechanism, through the muscarinic acetylcholine receptor M3. Cysteinyl leukotrienes cause gallbladder contraction through their cognate receptor CysLTR1, prompting emptying and closing. Our results establish gallbladder tuft cells as sensors of the microbial metabolite propionate, initiating dichotomous innate defense mechanisms through simultaneous release of acetylcholine and cysteinyl leukotrienes.

Steroids have numerous physiological functions associated with cellular signaling or modulation of the lipid membrane structure and dynamics, and as such, they have found broad pharmacological applications. Steroid–membrane interactions are relevant to multiple steps of steroid biosynthesis and action, as steroids are known to interact with neurotransmitter or membrane steroid receptors, and steroids must cross lipid membranes to exert their physiological functions. Therefore, rationalizing steroid function requires understanding of steroid–membrane interactions. We combined molecular dynamics simulations and isothermal titration calorimetry to characterize the conformations and the energetics of partitioning, in addition to the kinetics of flip–flop transitions and membrane exit, of 26 representative steroid compounds in a model lipid membrane. The steroid classes covered in this study include birth control and anabolic drugs, sex and corticosteroid hormones, neuroactive steroids, as well as steroids modulating the lipid membrane structure. We found that the conformational ensembles adopted by different steroids vary greatly, as quantified by their distributions of tilt angles and insertion depths into the membrane, ranging from well-defined steroid conformations with orientations either parallel or normal to the membrane, to wide conformational distributions. Surprisingly, despite their chemical diversity, the membrane/water partition coefficient is similar among most steroids, except for structural steroids such as cholesterol, leading to similar rates for exiting the membrane. By contrast, the rates of steroid flip–flop vary by at least 9 orders of magnitude, revealing that flip–flop is the rate-limiting step during cellular uptake of polar steroids. This study lays the ground for a quantitative understanding of steroid–membrane interactions, and it will hence be of use for studies of steroid biosynthesis and function as well as for the development and usage of steroids in a pharmacological context., Interactions of 26 steroid compounds with lipid membranes were derived using molecular dynamics simulations and isothermal titration calorimetry.

PTEN prevents tumor genesis by antagonizing the PI3 kinase/Akt pathway through D3 site phosphatase activity toward PI(3,4)P2 and PI(3,4,5)P3. The structural determinants of this important specificity remain unknown. Interestingly, PTEN shares remarkable homology to voltage-sensitive phosphatases (VSPs) that dephosphorylate D5 and D3 sites of PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3. Since the catalytic center of PTEN and VSPs differ markedly only in TI/gating loop and active site motif, we wondered whether these differences explained the variation of their substrate specificity. Therefore, we introduced mutations into PTEN to mimic corresponding sequences of VSPs and studied phosphatase activity in living cells utilizing engineered, voltage switchable PTENCiV, a Ci-VSP/PTEN chimera that retains D3 site activity of the native enzyme. Substrate specificity of this enzyme was analyzed with whole-cell patch clamp in combination with total internal reflection fluorescence microscopy and genetically encoded phosphoinositide sensors. In PTENCiV, mutating TI167/168 in the TI loop into the corresponding ET pair of VSPs induced VSP-like D5 phosphatase activity toward PI(3,4,5)P3, but not toward PI(4,5)P2. Combining TI/ET mutations with an A126G exchange in the active site removed major sequence variations between PTEN and VSPs and resulted in D5 activity toward PI(4,5)P2 and PI(3,4,5)P3 of PTENCiV. This PTEN mutant thus fully reproduced the substrate specificity of native VSPs. Importantly, the same combination of mutations also induced D5 activity toward PI(3,4,5)P3 in native PTEN demonstrating that the same residues determine the substrate specificity of the tumor suppressor in living cells. Reciprocal mutations in VSPs did not alter their substrate specificity, but reduced phosphatase activity. In summary, A126 in the active site and TI167/168 in the TI loop are essential determinants of PTEN’s substrate specificity, whereas additional features might contribute to the enzymatic activity of VSPs. Electronic supplementary material The online version of this article (10.1007/s00018-018-2867-z) contains supplementary material, which is available to authorized users.

TRPM3 proteins assemble to Ca2+-permeable cation channels in the plasma membrane, which act as nociceptors of noxious heat and mediators of insulin and cytokine release. Here we show that TRPM3 channel activity is strongly dependent on intracellular Ca2+. Conceivably, this effect is attributed to the Ca2+ binding protein calmodulin, which binds to TRPM3 in a Ca2+-dependent manner. We identified five calmodulin binding sites within the amino terminus of TRPM3, which displayed different binding affinities in dependence of Ca2+. Mutations of lysine residues in calmodulin binding site 2 strongly reduced calmodulin binding and TRPM3 activity indicating the importance of this domain for TRPM3-mediated Ca2+ signaling. Our data show that TRPM3 channels are regulated by intracellular Ca2+ and provide the basis for a mechanistic understanding of the regulation of TRPM3 by calmodulin.

Background and Purpose Signalling through phospholipase C (PLC) controls many cellular processes. Much information on the relevance of this important pathway has been derived from pharmacological inhibition of the enzymatic activity of PLC. We found that the most frequently employed PLC inhibitor, U73122, activates endogenous ionic currents in widely used cell lines. Given the extensive use of U73122 in research, we set out to identify these U73122-sensitive ion channels. Experimental Approach We performed detailed biophysical analysis of the U73122-induced currents in frequently used cell lines. Key Results At concentrations required to inhibit PLC, U73122 modulated the activity of transient receptor potential melastatin (TRPM) channels through covalent modification. U73122 was shown to be a potent agonist of ubiquitously expressed TRPM4 channels and activated endogenous TRPM4 channels in CHO cells independently of PLC and of the downstream second messengers PI(4,5)P2 and Ca2+. U73122 also potentiated Ca2+-dependent TRPM4 currents in human Jurkat T-cells, endogenous TRPM4 in HEK293T cells and recombinant human TRPM4. In contrast to TRPM4, TRPM3 channels were inhibited whereas the closely related TRPM5 channels were insensitive to U73122, showing that U73122 exhibits high specificity within the TRPM channel family. Conclusions and Implications Given the widespread expression of TRPM4 and TRPM3 channels, these actions of U73122 must be considered when interpreting its effects on cell function. U73122 may also be useful for identifying and characterizing TRPM channels in native tissue, thus facilitating the analysis of their physiology.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

An increase in light intensity induces a depolarization in retinal ON-bipolar cells via a reduced glutamate release from presynaptic photoreceptor cells. The underlying transduction cascade in the dendritic tips of ON-bipolar cells involves mGluR6 glutamate receptors signaling to TRPM1 proteins that are an indispensable part of the transduction channel. Several other proteins are recognized to participate in the transduction machinery. Deficiency in many of these leads to congenital stationary night blindness, because rod bipolar cells, a subgroup of ON-bipolar cells, constitute the main route for sensory information under scotopic conditions. Here, we review the current knowledge about TRPM1 ion channels and how their activity is regulated within the postsynaptic compartment of ON-bipolar cells. The functional properties of TRPM1 channels in the dendritic compartment are not well understood as they differ substantially from those of recombinant TRPM1 channels. Critical evaluation of possible explanations of these discrepancies indicates that some key components of this transduction pathway might still not be known. The continued exploration of this pathway will yield further clinically useful insights.

TRPM3 proteins assemble to Ca

Opioids, agonists of µ-opioid receptors (µORs), are the strongest pain killers clinically available. Their action includes a strong central component, which also causes important adverse effects. However, µORs are also found on the peripheral endings of nociceptors and their activation there produces meaningful analgesia. The cellular mechanisms downstream of peripheral µORs are not well understood. Here, we show in neurons of murine dorsal root ganglia that pro-nociceptive TRPM3 channels, present in the peripheral parts of nociceptors, are strongly inhibited by µOR activation, much more than other TRP channels in the same compartment, like TRPV1 and TRPA1. Inhibition of TRPM3 channels occurs via a short signaling cascade involving Gβγ proteins, which form a complex with TRPM3. Accordingly, activation of peripheral µORs in vivo strongly attenuates TRPM3-dependent pain. Our data establish TRPM3 inhibition as important consequence of peripheral µOR activation indicating that pharmacologically antagonizing TRPM3 may be a useful analgesic strategy., eLife digest There are very few treatments available for people suffering from strong or long-lasting pain. Currently, substances called opioids – which include the well-known drug morphine – are the strongest painkillers. However, these drugs also cause harmful side effects, which makes them less useful. Like all drugs, opioids mediate their effects by interacting with molecules in the body. In the case of opioids, these interacting molecules belong to a group of receptor proteins called G-protein coupled receptors (or GPCRs for short). These opioid receptors are widely distributed in the nerve cells and brain regions that detect and transmit pain signals. It was poorly understood how activation of opioid receptors reduces the activity of pain-sensing nerve cells, however several lines of evidence had suggested that a protein called TRPM3 might be involved. TRPM3 is a channel protein that allows sodium and calcium ions to enter into nerve cells by forming pores in cell membranes, and mice that lack this protein are less sensitive to certain kinds of pain. Dembla, Behrendt et al. now show that activating opioid receptors on nerve cells from mice, with morphine and a similar substance, rapidly reduces the flow of calcium ions through TRPM3 channels. Further experiments confirmed that activating opioid receptors in a mouse’s paw also reduced the pain caused when TRPM3 proteins are activated. GPCRs interact with a group of small proteins called G-proteins that, when activated by the receptor, split into two subunits. Based on studies with human kidney cells, Dembla, Behrendt et al. found the so-called G-beta-gamma subunit then carries the signal from the opioid receptor to TRPM3. Two independent studies by Quallo et al. and Badheka, Yudin et al. also report similar findings. These new findings show that drugs already used in the treatment of pain can indirectly alter how TRPM3 works in a dramatic way. These results might help scientists to find drugs that work in a more direct way to dial down the activity of TRPM3 and to combat pain with fewer side effects. Though first it will be important to confirm these new findings in human nerve cells.

Background and Purpose Transient receptor potential melastatin 3 (TRPM3) proteins form non-selective but calcium-permeable membrane channels, rapidly activated by extracellular application of the steroid pregnenolone sulphate and the dihydropyridine nifedipine. Our aim was to characterize the steroid binding site by analysing the structural chemical requirements for TRPM3 activation. Experimental Approach Whole-cell patch-clamp recordings and measurements of intracellular calcium concentrations were performed on HEK293 cells transfected with TRPM3 (or untransfected controls) during superfusion with pharmacological substances. Key Results Pregnenolone sulphate and nifedipine activated TRPM3 channels supra-additively over a wide concentration range. Other dihydropyridines inhibited TRPM3 channels. The natural enantiomer of pregnenolone sulphate was more efficient in activating TRPM3 channels than its synthetic mirror image. However, both enantiomers exerted very similar inhibitory effects on proton-activated outwardly rectifying anion channels. Epiallopregnanolone sulphate activated TRPM3 almost equally as well as pregnenolone sulphate. Exchanging the sulphate for other chemical moieties showed that a negative charge at this position is required for activating TRPM3 channels. Conclusions and Implications Our data demonstrate that nifedipine and pregnenolone sulphate act at different binding sites when activating TRPM3. The latter activates TRPM3 by binding to a chiral and thus proteinaceous binding site, as inferred from the differential effects of the enantiomers. The double bond between position C5 and C6 of pregnenolone sulphate is not strictly necessary for the activation of TRPM3 channels, but a negative charge at position C3 of the steroid is highly important. These results provide a solid basis for understanding mechanistically the rapid chemical activation of TRPM3 channels.

Background and Purpose The melastatin-related transient receptor potential TRPM3 is a calcium-permeable nonselective cation channel that can be activated by the neurosteroid pregnenolone sulphate (PregS) and heat. TRPM3-deficient mice show an impaired perception of noxious heat. Hence, drugs inhibiting TRPM3 possibly get in focus of analgesic therapy. Experimental Approach Fluorometric methods were used to identify novel TRPM3-blocking compounds and to characterize their potency and selectivity to block TRPM3 but not other sensory TRP channels. Biophysical properties of the block were assessed using electrophysiological methods. Single cell calcium measurements confirmed the block of endogenously expressed TRPM3 channels in rat and mouse dorsal root ganglion (DRG) neurones. Key Results By screening a compound library, we identified three natural compounds as potent blockers of TRPM3. Naringenin and hesperetin belong to the citrus fruit flavanones, and ononetin is a deoxybenzoin. Eriodictyol, a metabolite of naringenin and hesperetin, was still biologically active as a TRPM3 blocker. The compounds exhibited a marked specificity for recombinant TRPM3 and blocked PregS-induced [Ca2+]i signals in freshly isolated DRG neurones. Conclusion and Implications The data indicate that citrus fruit flavonoids are potent and selective blockers of TRPM3. Their potencies ranged from upper nanomolar to lower micromolar concentrations. Since physiological functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

TRPM3 channels form ionotropic steroid receptors in the plasma membrane of pancreatic β and dorsal root ganglion cells and link steroid hormone signaling to insulin release and pain perception, respectively. We identified and compared the function of a number of TRPM3 splice variants present in mouse, rat and human tissues. We found that variants lacking a region of 18 amino acid residues display neither Ca(2+) entry nor ionic currents when expressed alone. Hence, splicing removes a region that is indispensable for channel function, which is called the ICF region. TRPM3 variants devoid of this region (TRPM3ΔICF), are ubiquitously present in different tissues and cell types where their transcripts constitute up to 15% of the TRPM3 isoforms. The ICF region is conserved throughout the TRPM family, and its presence in TRPM8 proteins is also necessary for function. Within the ICF region, 10 amino acid residues form a domain essential for the formation of operative TRPM3 channels. TRPM3ΔICF variants showed reduced interaction with other TRPM3 isoforms, and their occurrence at the cell membrane was diminished. Correspondingly, coexpression of ΔICF proteins with functional TRPM3 subunits not only reduced the number of channels but also impaired TRPM3-mediated Ca(2+) entry. We conclude that TRPM3ΔICF variants are regulatory channel subunits fine-tuning TRPM3 channel activity.

Transient receptor potential (TRP) cation channels are renowned for their ability to sense diverse chemical stimuli. Still, for many members of this large and heterogeneous protein family it is unclear how their activity is regulated and whether they are influenced by endogenous substances. On the other hand, steroidal compounds are increasingly recognized to have rapid effects on membrane surface receptors that often have not been identified at the molecular level. We show here that TRPM3, a divalent-permeable cation channel, is rapidly and reversibly activated by extracellular pregnenolone sulphate, a neuroactive steroid. We show that pregnenolone sulphate activates endogenous TRPM3 channels in insulin-producing beta cells. Application of pregnenolone sulphate led to a rapid calcium influx and enhanced insulin secretion from pancreatic islets. Our results establish that TRPM3 is an essential component of an ionotropic steroid receptor enabling unanticipated crosstalk between steroidal and insulin-signalling endocrine systems.

peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=kchl20 ispartof: Channels vol:10 issue:2 pages:83-85 ispartof: location:United States status: published

TRPM3 is dynamically regulated by plasma membrane PI(4,5)P2 and related PIPs., The transient receptor potential (TRP) channel TRPM3 is a calcium-permeable cation channel activated by heat and by the neurosteroid pregnenolone sulfate (PregS). TRPM3 is highly expressed in sensory neurons, where it plays a key role in heat sensing and inflammatory hyperalgesia, and in pancreatic β cells, where its activation enhances glucose-induced insulin release. However, despite its functional importance, little is known about the cellular mechanisms that regulate TRPM3 activity. Here, we provide evidence for a dynamic regulation of TRPM3 by membrane phosphatidylinositol phosphates (PIPs). Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) and ATP applied to the intracellular side of excised membrane patches promote recovery of TRPM3 from desensitization. The stimulatory effect of cytosolic ATP on TRPM3 reflects activation of phosphatidylinositol kinases (PI-Ks), leading to resynthesis of PIPs in the plasma membrane. Various PIPs directly enhance TRPM3 activity in cell-free inside-out patches, with a potency order PI(3,4,5)P3 > PI(3,5)P2 > PI(4,5)P2 ≈ PI(3,4)P2 >> PI(4)P. Conversely, TRPM3 activity is rapidly and reversibly inhibited by activation of phosphatases that remove the 5-phosphate from PIPs. Finally, we show that recombinant TRPM3, as well as the endogenous TRPM3 in insuloma cells, is rapidly and reversibly inhibited by activation of phospholipase C–coupled muscarinic acetylcholine receptors. Our results reveal basic cellular mechanisms whereby membrane receptors can regulate TRPM3 activity.

International audience; : Transient receptor potential (TRP) channels form a diverse family of cation channels comprising 28 members in mammals. Although some TRP proteins can only be found on intracellular membranes, most of the TRP protein isoforms reach the plasma membrane where they form ion channels and control a wide number of biological processes. There, their involvement in the transport of cations such as calcium and sodium has been well documented. However, a growing number of studies have started to expand our understanding of these proteins by showing that they also transport other biologically relevant metal ions like zinc, magnesium, manganese and cobalt. In addition to this newly recognized property, the activity and expression of TRP channels can be regulated by metal ions like magnesium, gadolinium, lanthanum or cisplatin. The aim of this review is to highlight the complex relationship between metal ions and TRP channels.

Anion channels are present in every mammalian cell and serve many different functions, including cell volume regulation, ion transport across epithelia, regulation of membrane potential and vesicular acidification. Here we characterize a proton-activated, outwardly rectifying current endogenously expressed in HEK293 cells. Binding of three to four protons activated the anion permeable channels at external pH below 5.5 (50% activation at pH 5.1). The proton-activated current is strongly outwardly rectifying, due to an outwardly rectifying single channel conductance and an additional voltage dependent facilitation at depolarized membrane potentials. The anion channel blocker 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS) rapidly and potently inhibited the channel (IC50: 2.9 μm). Flufenamic acid blocked this channel only slowly, while mibefradil and amiloride at high concentrations had no effect. As determined from reversal potential measurements under bi-ionic conditions, the relative permeability sequence of this channel was SCN−> I−> NO3−> Br−> Cl−. None of the previously characterized anion channel matches the properties of the proton-activated, outwardly rectifying channel. Specifically, the proton-activated and the volume-regulated anion channels are two distinct and separable populations of ion channels, each having its own set of biophysical and pharmacological properties. We also demonstrate endogenous proton-activated currents in primary cultured hippocampal astrocytes. The proton-activated current in astrocytes is also carried by anions, strongly outwardly rectifying, voltage dependent and inhibited by DIDS. Proton-activated, outwardly rectifying anion channels therefore may be a broadly expressed part of the anionic channel repertoire of mammalian cells.

Like most other members of the TRP family, the Trpm3 gene encodes proteins that form cation-permeable ion channels on the plasma membrane. However, TRPM3 proteins have several unique features that set them apart from the other members of this diverse family. The Trpm3 gene encodes for a surprisingly large number of isoforms generated mainly by alternative splicing. Only for two of the (at least) eight sites at which sequence diversity is generated the functional consequences have been elucidated, one leading to nonfunctional channels, the other one profoundly affecting the ionic selectivity. In the Trpm3 gene an intronic microRNA (miR-204) is co-transcribed with Trpm3. By regulating the expression of a multitude of genes, miR-204 increases the functional complexity of the Trpm3 locus. Over the past years, important progress has been made in discovering pharmacological tools to manipulate TRPM3 channel activity. These substances have facilitated the identification of endogenously expressed functional TRPM3 channels in nociceptive neurons, pancreatic beta cells, and vascular smooth muscle cells, among others. TRPM3 channels, which themselves are temperature sensitive, thus have been implicated in sensing noxious heat, in modulating insulin release, and in secretion of inflammatory cytokines. However, in many tissues where TRPM3 proteins are known to be expressed, no functional role has been identified for these channels so far. Because of the availability of adequate pharmacological and genetic tools, it is expected that future investigations on TRPM3 channels will unravel important new aspects and functions of these channels.

The light response of fly photoreceptor cells is modulated by changes in free Ca2+concentration. Fly phototransduction and most processes regulating it take place in or very close to the rhabdomere. We therefore measured the kinetics and the absolute values of the free Ca2+concentration in the rhabdomere of fly photoreceptor cellsin vivoby making use of the natural optics of the fly's eye. We show that Ca2+flowing into the rhabdomere after light stimulation of dark-adapted cells causes fast Ca2+transients that reach peak values higher than 200 μmin 2+concentration has declined again to ∼20 μm. The duration of the Ca2+transients becomes still shorter, and their size reduced, when the photoreceptor cell is light-adapted. This reduction in duration and size of the Ca2+transients is graded with the intensity of the adapting light. The kinetics and absolute values of the free calcium concentration found to occur in the rhabdomere are suitable to mediate the fast feedback signals known to act on the fly phototransduction cascade.

Light adaptation in insect photoreceptors is caused by an increase in the cytosolic Ca2+ concentration. To better understand this process, we measured the cytosolic Ca2+ concentration in vivo as a function of adapting light intensity in the white-eyed blowfly mutant chalky. We developed a technique to measure the cytosolic Ca2+ concentration under conditions as natural as possible. The calcium indicator dyes Oregon Green 1, 2, or 5N (Molecular Probes, Inc., Eugene, OR) were iontophoretically injected via an intracellular electrode into a photoreceptor cell in the intact eye; the same electrode was also used to measure the membrane potential. The blue-induced green fluorescence of these dyes could be monitored by making use of the optics of the facet lens and the rhabdomere waveguide. The use of the different Ca2+-sensitive dyes that possess different affinities for Ca2+ allowed the quantitative determination of the cytosolic Ca2+ concentration in the steady state. Determining the cytosolic Ca2+ concentration as a function of the adapting light intensity shows that the Ca2+ concentration is regulated in a graded fashion over the whole dynamic range where a photoreceptor cell can respond to light. When a photoreceptor is adapted to bright light, the cytosolic Ca2+ concentration reaches stable values higher than 10 μM. The data are consistent with the hypothesis that the logarithm of the increase in cytosolic Ca2+ concentration is linear with the logarithm of the light intensity. From the estimated values of the cytosolic Ca2+ concentration, we conclude that the Ca2+-buffering capacity is limited. The percentage of the Ca2+ influx that is buffered gradually decreases with increasing Ca2+ concentrations; at cytosolic Ca2+ concentration levels above 10 μM, buffering becomes minimal.

International audience; Zinc (Zn) is a vital nutrient participating in a myriad of biological processes. The mechanisms controlling its transport through the plasma membrane are far from being completely understood. Two families of eukaryotic zinc transporters are known to date: the Zip (SLC39) and ZnT (SLC30) proteins. In addition, some types of plasmalemmal calcium (Ca)-conducting channels are implied in the cellular uptake of zinc. These ion channels are currently described as systems dedicated to the transport of Ca (and, to some extent, sodium (Na) ions). However, a growing body of evidence supports the view that some of them can also function as pathways for Zn transport. For instance, voltage-gated Ca channels and some types of glutamate-gated receptors have long been known to allow the entry of Zn. More recently, members of the TRP superfamily, another type of Ca-conducting channels, have been shown to permit the uptake of Zn into eukaryotic cells. The aim of this review article is to present the current knowledge supporting the notion that Ca-conducting channels take part in the plasmalemmal transport of Zn.

Type 2 diabetes mellitus is associated with alterations in bile acid (BA) signaling. The aim of our study was to test whether pancreatic β-cells contribute to BA-dependent regulation of glucose homeostasis. Experiments were performed with islets from wild-type, farnesoid X receptor (FXR) knockout (KO), and β-cell ATP-dependent K+ (KATP) channel gene SUR1 (ABCC8) KO mice, respectively. Sodium taurochenodeoxycholate (TCDC) increased glucose-induced insulin secretion. This effect was mimicked by the FXR agonist GW4064 and suppressed by the FXR antagonist guggulsterone. TCDC and GW4064 stimulated the electrical activity of β-cells and enhanced cytosolic Ca2+ concentration ([Ca2+]c). These effects were blunted by guggulsterone. Sodium ursodeoxycholate, which has a much lower affinity to FXR than TCDC, had no effect on [Ca2+]c and insulin secretion. FXR activation by TCDC is suggested to inhibit KATP current. The decline in KATP channel activity by TCDC was only observed in β-cells with intact metabolism and was reversed by guggulsterone. TCDC did not alter insulin secretion in islets of SUR1-KO or FXR-KO mice. TCDC did not change islet cell apoptosis. This is the first study showing an acute action of BA on β-cell function. The effect is mediated by FXR by nongenomic elements, suggesting a novel link between FXR activation and KATP channel inhibition.

TRPM4 (transient receptor potential melastatin 4) channels constitute Ca2+-activated non-selective cationic currents in a variety of tissues (e.g. heart and brain) and in cells of the immune system. The channels are gated by elevated cytosolic Ca2+, and channel activity is modulated by signalling molecules such as ATP and phospholipids. Activation of TRPM4 depolarises the membrane potential, which modulates the driving force for ions and the activity of ion channels. TRPM4 channel agonists and Ca2+-independent gating have not been demonstrated yet.In patch clamp experiments, the maleimide U73122, which is widely used as phospholipase C inhibitor, activated recombinant human TRPM4 and endogenous TRPM4 channels in cell lines. In contrast, U73122 inhibited TRPM3 channels and did not affect TRPM5 channel activity. TRPM4 current amplitudes were independent on phospholipid levels and, strikingly, U73122 activated TRPM4 even in absence of intracellular Ca2+. As the structural analogue U73343 was ineffective, these findings suggested covalent modification as the underlying mechanism. In fact, pre-application of N-ethylmaleimide (NEM) to prevent further covalent modification abolished the activation of TRPM4 by U73122.Taken together, we demonstrated activation of otherwise Ca2+-dependent TRPM4 channels by U73122. These findings raise the possibility that endogenous substances act as physiologically relevant TRPM4 agonists.This work was supported by a Research Grant of the University Medical Center Giessen und Marburg (UKGM) to M.G.L and by Deutsche Forschungsgemeinschaft through SFB 593 to J.O. (TPA16) and D.O. (TPA12).

Summary Transient receptor potential melastatin-3 (TRPM3) is a broadly expressed Ca 2+ -permeable nonselective cation channel. Previous work has demonstrated robust activation of TRPM3 by the neuroactive steroid pregnenolone sulfate (PS), but its in vivo gating mechanisms and functions remained poorly understood. Here, we provide evidence that TRPM3 functions as a chemo- and thermosensor in the somatosensory system. TRPM3 is molecularly and functionally expressed in a large subset of small-diameter sensory neurons from dorsal root and trigeminal ganglia, and mediates the aversive and nocifensive behavioral responses to PS. Moreover, we demonstrate that TRPM3 is steeply activated by heating and underlies heat sensitivity in a subset of sensory neurons. TRPM3-deficient mice exhibited clear deficits in their avoidance responses to noxious heat and in the development of inflammatory heat hyperalgesia. These experiments reveal an unanticipated role for TRPM3 as a thermosensitive nociceptor channel implicated in the detection of noxious heat.

TRPM1 is the founding member of the melastatin subgroup of transient receptor potential (TRP) proteins, but it has not yet been firmly established that TRPM1 proteins form ion channels. Consequently, the biophysical and pharmacological properties of these proteins are largely unknown. Here we show that heterologous expression of TRPM1 proteins induces ionic conductances that can be activated by extracellular steroid application. However the current amplitudes observed were too small to enable a reliable biophysical characterization. We overcame this limitation by modifying TRPM1 channels in several independent ways that increased the similarity to the closely related TRPM3 channels. The resulting constructs produced considerably larger currents after overexpression. We also demonstrate that unmodified TRPM1 and TRPM3 proteins form functional heteromultimeric channels. With these approaches, we measured the divalent permeability profile and found that channels containing the pore of TRPM1 are inhibited by extracellular zinc ions at physiological concentrations, in contrast to channels containing only the pore of TRPM3. Applying these findings to pancreatic β cells, we found that TRPM1 proteins do not play a major role in steroid-activated currents of these cells. The inhibition of TRPM1 by zinc ions is primarily due to a short stretch of seven amino acids present only in the pore region of TRPM1 but not of TRPM3. Combined, our data demonstrate that TRPM1 proteins are bona fide ion-conducting plasma membrane channels. Their distinct biophysical properties allow a reliable identification of endogenous TRPM1-mediated currents.

Zinc is stored in insulin-containing dense core vesicles of pancreatic beta-cells where it forms crystals together with insulin and calcium ions. Zinc ions are therefore released together with insulin upon exocytosis of these vesicles. Consequently, pancreatic beta-cells need to take up large amounts of zinc from the extracellular space across their plasma membrane. The pathways for zinc uptake are only partially understood. TRPM3 channels are present in pancreatic beta-cells and can be activated by the endogenous steroid pregnenolone sulfate. We demonstrate here that recombinant TRPM3 channels are highly permeable for many divalent cations, in particular also for zinc ions. Importantly, TRPM3 channels endogenously expressed in pancreatic beta-cells are also highly permeable for zinc ions. Using FluoZin3 to image changes of the intracellular zinc concentration, we show that pancreatic beta-cells take up zinc through TRPM3 channels even when extracellular zinc concentrations are low and physiological levels of calcium and magnesium are present. Activation of TRPM3 channels also leads to depolarization of beta-cells and to additional zinc influx through voltage-gated calcium channels. Our data establish that TRPM3 channels constitute a regulated entry pathway for zinc ions in pancreatic beta-cells.

TRPM channels have emerged as key mediators of diverse physiological functions. However, the ionic permeability relevant to physiological function in vivo remains unclear for most members. We report that the single Drosophila TRPM gene (dTRPM) generates a conductance permeable to divalent cations, especially Zn(2+) and in vivo a loss-of-function mutation in dTRPM disrupts intracellular Zn(2+) homeostasis. TRPM deficiency leads to profound reduction in larval growth resulting from a decrease in cell size and associated defects in mitochondrial structure and function. These phenotypes are cell-autonomous and can be recapitulated in wild-type animals by Zn(2+) depletion. Both the cell size and mitochondrial defect can be rescued by extracellular Zn(2+) supplementation. Thus our results implicate TRPM channels in the regulation of cellular Zn(2+) in vivo. We propose that regulation of Zn(2+) homeostasis through dTRPM channels is required to support molecular processes that mediate class I PI3K-regulated cell growth.

TRPM3 [TRP (transient receptor potential) melastatin 3] is one of the least investigated proteins of the TRP family of ion channels. Heterologously expressed TRPM3 channels are constitutively active, have an outwardly rectifying current–voltage relationship and are inhibited by intracellular Mg2+ ions. Besides these rather common features, in which TRPM3 channels resemble the closely related channels TRPM6 and TRPM7, TRPM3 channels have several unique characteristics. The TRPM3 gene encodes a plethora of different proteins owing to alternative splicing and alternative exon usage. One site of alternative splicing affects the ion-conducting pore region and profoundly alters the pore properties of the encoded channels. The channels having the longer pore region efficiently conduct univalent cations, but are only poorly permeated by bivalent cations. Conversely, the channels with the shorter pore region are highly permeable to bivalent cations. Unusually, the short-pore TRPM3 channels are inhibited by extracellular Na+ ions. At physiological sodium concentration, this block is very strong, making it difficult to envision a physiological function for these ion channels. Recently, pharmacological investigations have been initiated in order to identify substances that influence TRPM3 channel activity. With the use of such substances, it might be possible to identify TRPM3 channels in their native environment and to elucidate some of their physiological roles. Hopefully, TRPM3 channels will then no longer appear to be as enigmatic as they do right now.

TRPM3 is a poorly understood member of the large family of transient receptor potential (TRP) ion channels. Here we describe five novel splice variants of TRPM3, TRPM3alpha1-5. These variants are characterized by a previously unknown amino terminus of 61 residues. The differences between the five variants arise through splice events at three different sites. One of these splice sites might be located in the pore region of the channel as indicated by sequence alignment with other, better-characterized TRP channels. We selected two splice variants, TRPM3alpha1 and TRPM3alpha2, that differ only in this presumed pore region and analyzed their biophysical characteristics after heterologous expression in human embryonic kidney 293 cells. TRPM3alpha1 as well as TRPM3alpha2 induced a novel, outwardly rectifying cationic conductance that was tightly regulated by intracellular Mg(2+). However, these two variants are highly different in their ionic selectivity. Whereas TRPM3alpha1-encoded channels are poorly permeable for divalent cations, TRPM3alpha2-encoded channels are well permeated by Ca(2+) and Mg(2+). Additionally, we found that currents through TRPM3alpha2 are blocked by extracellular monovalent cations, whereas currents through TRPM3alpha1 are not. These differences unambiguously show that TRPM3 proteins constitute a pore-forming channel subunit and localize the position of the ion-conducting pore within the TRPM3 protein. Although the ionic selectivity of ion channels has traditionally been regarded as rather constant for a given channel-encoding gene, our results show that alternative splicing can be a mechanism to produce channels with very different selectivity profiles.

In fly photoreceptor cells, two processes dominate the Ca2+ homeostasis: light-induced Ca2+ influx through members of the TRP family of ion channels, and Ca2+ extrusion by Na+/Ca2+ exchange. Ca2+ release from intracellular stores is quantitatively insignificant. Both, the light-activated channels and the Ca2+-extruding exchangers are located in or close to the rhabdomeric microvilli, small protrusions of the plasma membrane. The microvilli also contain the molecular machinery necessary for generating quantum bumps, short electrical responses caused by the absorption of a single photon. Due to this anatomical arrangement, the light-induced Ca2+ influx results in two separate Ca2+ signals that have different functions: a global, homogeneous increase of the Ca2+ concentration in the cell body, and rapid but large amplitude Ca2+ transients in the microvilli. The global rise of the Ca2+ concentration mediates light adaptation, via regulatory actions on the phototransduction cascade, the voltage-gated K+ channels and small pigment granules controlling the light intensity. The local Ca2+ transients in the microvilli are responsible for shaping the quantum bumps into fast, all-or-nothing events. They achieve this by facilitating strongly the phototransduction cascade at early stages ofthe light response and subsequently inhibiting it. Many molecular targets of these feedback mechanisms have been identified and characterized due to the availability of numerous Drosophila mutant showing defects in the phototransduction.

In fly photoreceptor cells, two processes dominate the Ca2+ homeostasis: light-induced Ca2+ influx through members of the TRP family of ion channels, and Ca2+ extrusion by Na+/Ca2+ exchange. Ca2+ release from intracellular stores is quantitatively insignificant. Both, the light-activated channels and the Ca2+-extruding exchangers are located in or close to the rhabdomeric microvilli, small protrusions of the plasma membrane. The microvilli also contain the molecular machinery necessary for generating quantum bumps, short electrical responses caused by the absorption of a single photon. Due to this anatomical arrangement, the light-induced Ca2+ influx results in two separate Calf signals that have different functions: a global, homogeneous increase of the Ca2+ concentration in the cell body, and rapid but large amplitude Ca2+ transients in the microvilli. The global rise of the Ca2+ concentration mediates light adaptation, via regulatory actions on the phototransduction cascade, the voltage-gated K+ channels and small pigment granules controlling the light intensity. The local Ca2+ transients in the microvilli are responsible for shaping the quantum bumps into fast, all-or-nothing events. They achieve this by facilitating strongly the phototransduction cascade at early stages of the light response and subsequently inhibiting it. Many molecular targets of these feedback mechanisms have been identified and characterized due to the availability of numerous Drosophila mutant showing defects in the phototransduction. been identified that are regulated by Calf, either directly or by binding to Ca2+1 calmodulin or by Ca2+-dependent phosphorylation and dephosphorylations (Figure.12). In the overwhelming majority of cases, interfering with these mechanisms produces flies that have abnormally prolonged termination phases of the light response, i.e., show defects in the negative regulation of the phototransduction. The PKC INAC and the unconventional myosin NINAC have been proposed to additionally play a role in light adaptation, i.e., in the regulation of the sensitivity of the phototransduction cascade. Also, two candidates for positive Ca2+-dependent feedback have been identified, the channel-protein TRP and the PLC(3 NORPA. However, the more interesting, albeit much harder, question how these different regulatory events play together to produce their macroscopic effect on the physiology of the photoreceptor cells has not yet been tackled. An advance of our knowledge in this direction will probably demand a more quantitative understanding of the Ca2+-dependent processes and their consequences, as well as a detailed analysis of their time course, probably down to the millisecond range. This will allow to integrate the different pathways that have already been identified into a combined model of how Ca2+ brings about its regulation of fly phototransduction. The search for answers to these questions will no doubt increase not only our knowledge of fly phototransduction, but will also further our general understanding of how complex signaling cascades can be modulated and adapted to the prevailing environmental needs.

During illumination, Ca 2+ enters fly photoreceptor cells through light-activated channels that are located in the rhabdomere, the compartment specialized for phototransduction. From the rhabdomere, Ca 2+ diffuses into the cell body. We visualize this process by rapidly imaging the fluorescence in a cross section of a photoreceptor cell injected with a fluorescent Ca 2+ indicator in vivo . The free Ca 2+ concentration in the rhabdomere shows a very fast and large transient shortly after light onset. The free Ca 2+ concentration in the cell body rises more slowly and displays a much smaller transient. After ≈400 ms of light stimulation, the Ca 2+ concentration in both compartments reaches a steady state, indicating that thereafter an amount of Ca 2+ , equivalent to the amount of Ca 2+ flowing into the cell, is extruded. Quantitative analysis demonstrates that during the steady state, the free Ca 2+ concentration in the rhabdomere and throughout the cell body is the same. This shows that Ca 2+ extrusion takes place very close to the location of Ca 2+ influx, the rhabdomere, because otherwise gradients in the steady-state distribution of Ca 2+ should be measured. The close colocalization of Ca 2+ influx and Ca 2+ extrusion ensures that, after turning off the light, Ca 2+ removal from the rhabdomere is faster than from the cell body. This is functionally significant because it ensures rapid dark adaptation.

Publisher Summary This chapter covers two rather distinct areas of photoreceptor research that have been treated with quantitative models. The first area investigates the visual pigments, specifically the wavelength dependence of their absorption characteristics and the cycling of the visual pigment molecules between the different states. The second area tries to quantitatively understand how changes in membrane permeability translate into changes of membrane currents and potentials, and causes changes of ion concentrations. Most of the reviewed work has been done on fly photoreceptors, specifically Drosophila. The first step in vision is the absorption of a photon by a visual pigment molecule, which then triggers the phototransduction process. The spectral characteristics of insect photoreceptors are emphasized. The consequences of packing visual pigments in the optical waveguides are formally described, and then the consequences for the resulting sensitivity spectra of the photoreceptors are discussed.

The colour-vision system of these crustaceans includes four types of UV photoreceptor. Humans cannot see ultraviolet light, but many arthropods and vertebrates can because they have a single photo-receptor with a peak sensitivity to light at wavelengths of around 350 nanometres (ref. 1). Here we use electrophysiological methods to investigate the vision of the mantis shrimp, Neogonodactylus oerstedii. We find that this marine crustacean has at least four types of photoreceptor for ultraviolet light that are located in cells of the eye known as R8 cells. These photoreceptors are maximally sensitive to light of wavelengths 315, 330, 340 and 380 nm. Together with previous evidence2, this finding indicates that the remarkable colour-vision system in these stomatopod crustaceans may be unique, as befits their habitat of kaleidoscopically colourful tropical coral reefs.

TRPM3 channels are non-selective cation channels that have been implicated in a variety of functions, including the detection of noxious heat and the secretion of insulin. These channels can be activated by the endogenous steroid pregnenolone sulfate and the voltage-gated calcium channel blocker nifedipine, but it is unclear how these compounds interact with the channels.In electrophysiological and calcium imaging experiments we provide evidence that pregnenolone sulfate and nifedipine bind to distinct binding sites. Furthermore, we show that pregnenolone sulfate needs to bind to a chiral, and thus proteinaceous, binding site in order to activate TRPM3 by utilizing the enantiomer of pregnenolone sulfate. Employing further structural analogs of pregnenolone sulfate, we additionally determined that the binding site of pregnenolone sulfate needs to accommodate a large, negatively charged substituent at the C3 position of the steroid backbone.By combining these data we devised a strategy to find candidate amino acid residues of TRPM3 important for channel activation, possibly by being part of the pregnenolone sulfate binding site. We systematically mutated positively charged amino acids accessible from the extracellular side (from which pregnenolone sulfate is capable of activating TRPM3). We then evaluated whether mutated channels that displayed a reduced response to pregnenolone sulfate still responded to nifedipine comparably to wild-type channels. We identified one amino acid with these properties, which is predicted to be located at the transmembrane-extracellular domain interface.These data will lead to the characterization of the steroid binding site responsible for activating TRPM3 channels. Likely, this will help to identify more potent and specific pharmacological tools, with the ultimate goal to manipulate these channels for experimental and therapeutic purposes.

Summary Phototransduction in Drosophila is mediated by a phospholipase C (PLC) cascade culminating in activation of transient receptor potential (TRP) channels [1, 2]. Ca 2+ influx via these channels is required for light adaptation, but although several molecular targets of Ca 2+ -dependent feedback have been identified [3], their contribution to adaptation is unclear. By manipulating cytosolic Ca 2+ via the Na + /Ca 2+ exchange equilibrium, we found that Ca 2+ inhibited the light-induced current (LIC) over a range corresponding to steady-state light-adapted Ca 2+ levels (0.1–10 μM Ca 2+ ) and accurately mimicked light adaptation. However, PLC activity monitored with genetically targeted PIP 2 -sensitive ion channels (Kir2.1) was first inhibited by much higher (≥ ∼50 μM) Ca 2+ levels, which occur only transiently in vivo. Ca 2+ -dependent inhibition of PLC, but not the LIC, was impaired in mutants ( inaC ) of protein kinase C (PKC). The results indicate that light adaptation is primarily mediated downstream of PLC and independently of PKC by Ca 2+ -dependent inhibition of TRP channels. This is interpreted as a strategy to prevent inhibition of PLC by global steady-state light-adapted Ca 2+ levels, whereas rapid inhibition of PLC by local Ca 2+ transients is required to terminate the response and ensures that PIP 2 reserves are not depleted during stimulation.