TRP Expression Research Articles

Hot topic: Mapping of the human intranasal mucosal thermal sensitivity: A clinical study on thermal threshold and trigeminal receptors.

The olfactory and trigeminal system are closely interlinked. Existing literature has primarily focused on characterizing trigeminal stimulation through mechanical and chemical stimulation, neglecting thermal stimulation thus far. The present study aimed to characterize the intranasal sensitivity to heat and the expression of trigeminal receptors (transient receptor potential channels, TRP). A total of 20 healthy participants (aged 21-27 years, 11 women) were screened for olfactory function and trigeminal sensitivity using several tests. Under endoscopic control, a thermal stimulator was placed in 7 intranasal locations: anterior septum, lateral vestibulum, interior nose tip, lower turbinate, middle septum, middle turbinate, and olfactory cleft to determine the thermal threshold. Nasal swabs were obtained in 3 different locations (anterior septum, middle turbinate, olfactory cleft) to analyze the expression of trigeminal receptors TRP: TRPV1, TRPV3, TRPA1, TRPM8. The thermal threshold differed between locations (p = 0.018), with a trend for a higher threshold at the anterior septum (p = 0.092). There were no differences in quantitative receptor expression (p = 0.46) at the different sites. The highest overall receptor RNA expression was detected for TRPV1 over all sites (p<0.001). The expression of TRPV3 was highest at the anterior septum compared to the middle turbinate or the olfactory cleft. The thermal sensitivity correlated with olfactory sensitivity and results from tests were related to trigeminal function like intensity ratings of ammonium, a questionnaire regarding trigeminal function, nasal patency, and CO2 thresholds. However, no correlation was found between receptor expression and psychophysical measures of trigeminal function. This study provided the first insights about intranasal thermal sensitivity and suggested the presence of topographical differences in thermal thresholds. There was no correlation between thermal sensitivity and trigeminal mRNA receptor expression. However, thermal sensitivity was found to be associated with psychophysical measures of trigeminal and olfactory function.

Dendrobium nobile Polysaccharide Attenuates Blue Light-Induced Injury in Retinal Cells and In Vivo in Drosophila.

Blue light is the higher-energy region of the visible spectrum. Excessive exposure to blue light is known to induce oxidative stress and is harmful to the eyes. The stems of Dendrobium nobile Lindl. (Orchidaceae), named Jinchaishihu, have long been used in traditional Chinese medicine (TCM) for nourishing yin, clearing heat, and brightening the eyes. The polysaccharide is one of the major components in D. nobile. However, the effect on ocular cells remains unclear. This study aimed to investigate whether the polysaccharide from D. nobile can protect the eyes from blue light-induced injury. A crude (DN-P) and a partially purified polysaccharide (DN-PP) from D. nobile were evaluated for their protective effects on blue light-induced damage in ARPE-19 and 661W cells. The in vivo study investigated the electroretinographic response and the expression of phototransduction-related genes in the retinas of a Drosophila model. The results showed that DN-P and DN-PP could improve blue light-induced damage in ARPE-19 and 661W cells, including cell viability, antioxidant activity, reactive oxygen species (ROS)/superoxide production, and reverse opsin 3 protein expression in a concentration-dependent manner. The in vivo study indicated that DN-P could alleviate eye damage and reverse the expression of phototransduction-related genes, including ninaE, norpA, Gαq, Gβ76C, Gγ30A, TRP, and TRPL, in a dose-dependent manner in blue light-exposed Drosophila. In conclusion, this is the first report demonstrating that D. nobile polysaccharide pretreatment can protect retinal cells and retinal photoreceptors from blue light-induced damage. These results provide supporting evidence for the beneficial potential of D. nobile in preventing blue light-induced eye damage and improving eyesight.

Shen Yuan extract exerts a hypnotic effect via the tryptophan/5-hydroxytryptamine/melatonin pathway in mice

Ethnopharmacological relevanceSleep plays a critical role in several physiologic processes, and sleep disorders increase the risk of depression, dementia, stroke, cancer, and other diseases. Stress is one of the main causes of sleep disorders. Ginseng Radix et Rhizoma and Polygalae Radix have been reported to have effects of calming the mind and intensifying intelligence in Chinese Pharmacopoeia. Traditional Chinese medicine prescriptions composed of Ginseng Radix et Rhizoma and Polygalae Radix (Shen Yuan, SY) are commonly used to treat insomnia, depression, and other psychiatric disorders in clinical practice. Unfortunately, the underlying mechanisms of the SY extract's effect on sleep are still unknown. Aim of the studyThis study aimed to investigate the hypnotic effect of the SY extract in normal mice and mice with chronic restraint stress (CRS)-induced sleep disorders and elucidate the underlying mechanisms. Materials and methodsThe SY extract (0.5 and 1.0 g/kg) was intragastrically administered to normal mice for 1, 14, and 28 days and to CRS-treated mice for 28 days. The open field test (OFT) and pentobarbital sodium-induced sleep test (PST) were used to evaluate the hypnotic effect of the SY extract. Liquid chromatography-tandem mass spectrometry and enzyme-linked immunosorbent assay were utilized to detect the levels of neurotransmitters and hormones. Molecular changes at the mRNA and protein levels were determined using real-time quantitative polymerase chain reaction and Western blot analysis to identify the mechanisms by which SY improves sleep disorders. ResultsThe SY extract decreased sleep latency and increased sleep duration in normal mice. Similarly, the sleep duration of mice subjected to CRS was increased by administering SY. The SY extract increased the levels of tryptophan (Trp) and 5-hydroxytryptamine (5-HT) and the expression of tryptophan hydroxylase 2 (TPH2) in the cortex of normal mice. The SY extract increased the Trp level, transcription and expression of estrogen receptor beta and TPH2 in the cortex in mice with sleep disorders by decreasing the serum corticosterone level, which promoted the synthesis of 5-HT. Additionally, the SY extract enhanced the expression of arylalkylamine N-acetyltransferase, which increased the melatonin level and upregulated the expressions of melatonin receptor-2 (MT2) and Cryptochrome 1 (Cry1) in the hypothalamus of mice with sleep disorders. ConclusionsThe SY extract exerted a hypnotic effect via the Trp/5-HT/melatonin pathway, which augmented the synthesis of 5-HT and melatonin and further increased the expressions of MT2 and Cry1.

Brain and Pharyngeal Responses Associated with Pharmacological Treatments for Oropharyngeal Dysphagia in Older Patients.

Impaired pharyngo-laryngeal sensory function is a critical mechanism for oropharyngeal dysphagia (OD). Discovery of the TRP family in sensory nerves opens a window for new active treatments for OD. To summarize our experience of the action mechanism and therapeutic effects of pharyngeal sensory stimulation by TRPV1, TRPA1 and TRPM8 agonists in older patients with OD. Summary of our studies on location and expression of TRP in the human oropharynx and larynx, and clinical trials with acute and after 2weeks of treatment with TRP agonists in older patients with OD. (1) TRP receptors are widely expressed in the human oropharynx and larynx: TRPV1 was localized in epithelial cells and TRPV1, TRPA1 and TRPM8 in sensory fibers mainly below the basal lamina. (2) Older people present a decline in pharyngeal sensory function, more severe in patients with OD associated with delayed swallow response, impaired airway protection and reduced spontaneous swallowing frequency. (3) Acute stimulation with TRP agonists improved the biomechanics and neurophysiology of swallowing in older patients with OD TRPV1 = TRPA1 > TRPM8. (4) After 2weeks of treatment, TRPV1 agonists induced cortical changes that correlated with improvements in swallowing biomechanics. TRP agonists are well tolerated and do not induce any major adverse events. TRP receptors are widely expressed in the human oropharynx and larynx with specific patterns. Acute oropharyngeal sensory stimulation with TRP agonists improved neurophysiology, biomechanics of swallow response, and safety of swallowing. Subacute stimulation promotes brain plasticity further improving swallow function in older people with OD.

TRP expression signature in tumor-derived endothelial cells: Functional roles in prostate cancer angiogenesis

TRP expression signature in tumor-derived endothelial cells: Functional roles in prostate cancer angiogenesis

XPORT-Dependent Transport of TRP and Rhodopsin

TRP channels have emerged as key biological sensors in vision, taste, olfaction, hearing, and touch. Despite their importance, virtually nothing is known about the folding and transport of TRP channels during biosynthesis. Here, we identify XPORT (exit protein of rhodopsin and TRP) as a critical chaperone for TRP and its G protein-coupled receptor (GPCR), rhodopsin (Rh1). XPORT is a resident ER and secretory pathway protein that interacts with TRP and Rh1, as well as with Hsp27 and Hsp90. XPORT promotes the targeting of TRP to the membrane in Drosophila S2 cells, a finding that provides a critical first step toward solving a longstanding problem inthe successful heterologous expression of TRP. Mutations in xport result in defective transport of TRP and Rh1, leading to retinal degeneration. Our results identify XPORT as a molecular chaperone and provide a mechanistic link between TRP channels and their GPCRs during biosynthesis and transport.

Ascorbic acid increases the activity and synthesis of tyrosinase in B16F10 cells through activation of p38 mitogen-activated protein kinase

Ascorbic acid, a potential antioxidant, is known to inhibit melanogenesis. However, there are conflicting findings that ascorbic acid has very low stability and acts as a pro-oxidant, eventually increasing proliferation and melanin content in melanoma cells. In the present study, we explored the effects of ascorbic acid on the activity and expression of tyrosinase and melanin pigmentation in the presence and absence of α-melanocyte-stimulating hormone (α-MSH) using B16F10 melanoma cells. The mechanism by which ascorbic acid stimulated the expression of tyrosinase was also investigated. No inhibitory effect on melanin content was observed in ascorbic acid-treated cells, regardless of the presence of α-MSH. Ascorbic acid stimulated the activity and expression of tyrosinase and increased the expression of melanogenic regulatory factors, such as tyrosinase-related protein-1 (TRP-1), dihydroxyphenylalaminechrome tautomerase (TRP-2), and microphthalmia-associated transcription factor (MITF). Ascorbic acid also induced phosphorylation of p38 mitogen-activated protein kinase (MAPK). The inhibition of p38 MAPK pathway by SB203580 led to the suppression of tyrosinase, TRP-1, and TRP-2 expression in cells treated with ascorbic acid. Combined treatment with N-acetyl-L: -cysteine and/or desferrioxamine mesylate attenuated the stimulating effect of ascorbic acid on tyrosinase activation in the cells. Collectively, ascorbic acid stimulates tyrosinase activity and expression in B16F10 cells via activation of p38 MAPK signaling and subsequent up-regulation of MITF, tyrosinase, and TRP expression.

TRP expression pattern and the functional importance of TRPC3 in primary human T-cells

TRP proteins form ion channels which are activated following receptor stimulation. In T-cell lines, expression data of TRP proteins have been published. However, almost no data about TRP expression is available in primary human T-cells. Using RT-PCR and quantitative RT-PCR, we compare the expression of TRP mRNA in 1) human peripheral blood lymphocytes, which are a mix of mostly mono-nuclear blood lymphocytes but contain other leucocytes, 2) a pure human CD4 + T-helper cell population in the resting (= naïve) and activated (= effector) state, and 3) two commonly used CD4 + Jurkat T-cell lines, E6-1 and parental. To mimic physiological cell stimulation, we analyzed TRP expression in primary human cells in a quantitative way over several days following formation of an immunological synapse through stimulation with antibody-coated beads. The TRP expression profile of primary human T-cells was significantly different from Jurkat T-cells. Among the TRP mRNAs of the TRPC, TRPM, and TRPV family, we found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 in primary human CD4 + T-cells of all analyzed blood donors. Among these, TRPC3 and TRPM2 were strongly up-regulated following stimulation, but with different kinetics. We found that TRPC3 modulates Ca 2+-dependent proliferation of primary CD4 + T-cells indicating that TRPC3 may be involved in Ca 2+ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca 2+ entry.

High Glucose–Induced Oxidative Stress Increases Transient Receptor Potential Channel Expression in Human Monocytes

OBJECTIVETransient receptor potential (TRP) channel–induced cation influx activates human monocytes, which play an important role in the pathogenesis of atherosclerosis. In the present study, we investigated the effects of high glucose–induced oxidative stress on TRP channel expression in human monocytes.RESEARCH DESIGN AND METHODSHuman monocytes were exposed to control conditions (5.6 mmol/l d-glucose), high glucose (30 mmol/l d-glucose or l-glucose), 100 μmol/l peroxynitrite, or high glucose in the presence of the superoxide dismutase mimetic tempol (100 μmol/l). TRP mRNA and TRP protein expression was measured using quantitative real-time RT-PCR and quantitative in-cell Western assay, respectively. Calcium influx and intracellular reactive oxygen species were measured using fluorescent dyes.RESULTSAdministration of high d-glucose significantly increased reactive oxygen species. High d-glucose or peroxynitrite significantly increased the expression of TRP canonical type 1 (TRPC1), TRPC3, TRPC5, TRPC6, TRP melastatin type 6 (TRPM6), and TRPM7 mRNA and TRPC3 and TRPC6 proteins. High d-glucose plus tempol or high l-glucose did not affect TRP expression. Increased oxidative stress by lipopolysaccharide or tumor necrosis factor-α increased TRP mRNA expression, whereas the reduction of superoxide radicals using diphenylene iodonium significantly reduced TRP mRNA expression. Increased TRPC3 and TRPC6 protein expression was accompanied by increased 1-oleoyl-2-acetyl-sn-glycerol–induced calcium influx, which was blocked by the TRPC inhibitor 2-aminoethoxydiphenylborane. TRPC6 mRNA was significantly higher in monocytes from 18 patients with type 2 diabetes compared with 28 control subjects (P < 0.05).CONCLUSIONSHigh d-glucose–induced oxidative stress increases TRP expression and calcium influx in human monocytes, pointing to a novel pathway for increased activation of monocytes and hence atherosclerosis in patients with diabetes.

Tumor Necrosis Factor-Alpha (TNF-α) Enhances Functional Thermal and Chemical Responses of TRP Cation Channels in Human Synoviocytes

BackgroundWe have shown functional expression of several TRP channels on human synovial cells, proposing significance in known calcium dependent proliferative and secretory responses in joint inflammation. The present study further characterizes synoviocyte TRP expression and activation responses to thermal and osmotic stimuli after pre-treatment with proinflammatory mediator tumor necrosis factor alpha (TNF-α, EC50 1.3221 × 10-10g/L).ResultsFluorescent imaging of Fura-2 loaded human SW982 synoviocytes reveals immediate and delayed cytosolic calcium oscillations elicited by (1) TRPV1 agonists capsaicin and resiniferatoxin (20 – 40% of cells), (2) moderate and noxious temperature change, and (3) osmotic stress TRPV4 activation (11.5% of cells). TNF-alpha pre-treatment (1 ng/ml, 8 – 16 hr) significantly increases (doubles) capsaicin responsive cell numbers and [Ca2+]i spike frequency, as well as enhances average amplitude of temperature induced [Ca2+]i responses. With TNF-alpha pre-treatment for 8, 12, and 16 hr, activation with 36 or 45 degree bath solution induces bimodal [Ca2+]i increase (temperature controlled chamber). Initial temperature induced rapid transient spikes and subsequent slower rise reflect TRPV1 and TRPV4 channel activation, respectively. Only after prolonged TNF-alpha exposure (12 and 16 hr) is recruitment of synoviocytes observed with sensitized TRPV4 responses to hypoosmolarity (3–4 fold increase). TNF-alpha increases TRPV1 (8 hr peak) and TRPV4 (12 hr peak) immunostaining, mRNA and protein expression, with a TRPV1 shift to membrane fractions.ConclusionTNF-α provides differentially enhanced synoviocyte TRPV1 and TRPV4 expression and [Ca2+]i response dependent on the TRP stimulus and time after exposure. Augmented relevance of TRPV1 and TRPV4 as inflammatory conditions persist would provide calcium mediated cell signaling required for pathophysiological responses of synoviocytes in inflammatory pain states.

The Presence of a C − 1 /G + 73 Pair in a tRNA Precursor Influences Processing and Expression In Vivo

To understand whether 5′ and 3′ structural elements of the region corresponding to the mature tRNA affect the expression of the tRNA, we examined several bacterial genomes for tRNA genes where the expression might be potentially affected by structural elements located outside of the mature tRNA. In Pseudomonas aeruginosa, our analysis suggested that the tRNA Trp is transcribed together with a putative stem–loop structure followed by a uridine tract immediately downstream of the tRNA region. This structural element, resembling a Rho-independent transcription terminator, might therefore influence the expression and processing of tRNA Trp. Moreover, the secondary structure suggested that the discriminator base in the tRNA Trp precursor can pair with either the C at position − 1, the 3′ terminal residue in the 5′ leader, or the C immediately 5′ of the uridine tract of the putative Rho-independent transcription terminator. Here, we present in vivo data demonstrating the importance of residue − 1 and the positioning of the putative transcription terminator for the expression of correctly 5′ processed P. aeruginosa tRNA Trp in Escherichia coli. Interestingly, we also detected a difference in the appearance of correctly 5′ processed P. aeruginosa tRNA Trp in E. coli compared to the situation in P. aeruginosa.

Transposon Mutations in the 5′ End ofglnD, the Gene for a Nitrogen Regulatory Sensor, That Suppress the Osmosensitive Phenotype Caused byotsBALesions inEscherichia coli

GlnD of Escherichia coli is a bifunctional signal-transducing enzyme (102.4 kDa) which uridylylates the allosteric regulatory protein PII and deuridylylates PII-UMP in response to growth with nitrogen excess or limitation, respectively. GlnD catalyzes these reactions in response to high or low levels of cytoplasmic glutamine, respectively, and indirectly directs the expression of nitrogen-regulated genes, e.g., the glnK-amtB operon. We report that chromosomal mini-Tn10 insertions situated after nucleotide number 997 or 1075 of glnD partially suppressed the osmosensitive phenotype of DeltaotsBA or otsA::Tn10 mutations (defective osmoregulatory trehalose synthesis). Strains carrying these glnD::mini-Tn10 mutations either completely repressed the expression of trp::(glnKp-lacZ) or induced this reporter system to nearly 60% of the wild-type glnD level in response to nitrogen availability, an essentially normal response. This was in contrast to the much-studied glnD99::Tn10 mutation, which carries its insertion in the 3' end of the gene, causes a complete repression of glnKp-lacZ expression under all growth conditions, and also confers leaky glutamine auxotrophy. When expressed from the Pm promoter in plasmid constructs, the present glnD mutations produced proteins with an apparent mass of 39 or 42 kDa. These proteins were deduced to comprise 344 or 370 N-terminal residues, respectively, harboring the known nucleotidyltransferase domain of GlnD, plus a common C-terminal addition of 12 residues encoded by IS10. They lacked three other domains of GlnD. Apparently, the transferase domain by itself enabled the cells to catalyze the uridylylation reaction and direct nitrogen-regulated gene expression. Our data indicate that there exists a link between osmotic stress and the nitrogen response.

Expression and crystallization of toxoflavin lyase (TflA) and TRP

Expression and crystallization of toxoflavin lyase (TflA) and TRP

Transient receptor potential-like channels are essential for calcium signaling and fluid transport in a Drosophila epithelium.

Calcium signaling is an important mediator of neuropeptide-stimulated fluid transport by Drosophila Malpighian (renal) tubules. We demonstrate the first epithelial role, in vivo, for members of the TRP family of calcium channels. RT-PCR revealed expression of trp, trpl, and trpgamma in tubules. Use of antipeptide polyclonal antibodies for TRP, TRPL, and TRPgamma showed expression of all three channels in type 1 (principal) cells in the tubule main segment. Neuropeptide (CAP(2b))-stimulated fluid transport rates were significantly reduced in tubules from the trpl(302) mutant and the trpl;trp double mutant, trpl(302);trp(343). However, a trp null, trp(343), had no impact on stimulated fluid transport. Measurement of cytosolic calcium concentrations ([Ca(2+)](i)) in tubule principal cells using an aequorin transgene in trp and trpl mutants showed a reduction in calcium responses in trpl(302). Western blotting of tubule preparations from trp and trpl mutants revealed a correlation between TRPL levels and CAP(2b)-stimulated fluid transport and calcium signaling. Rescue of trpl(302) with a trpl transgene under heat-shock control resulted in a stimulated fluid transport phenotype that was indistinguishable from wild-type tubules. Furthermore, restoration of normal stimulated rates of fluid transport by rescue of trpl(302) was not compromised by introduction of the trp null, trp(343). Thus, in an epithelial context, TRPL is sufficient for wild-type responses. Finally, a scaffolding component of the TRPL/TRP-signaling complex, INAD, is not expressed in tubules, suggesting that inaD is not essential for TRPL/TRP function in Drosophila tubules.

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes.

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.

TRP4 (CCE1) Protein Is Part of Native Calcium Release-activated Ca2+-like Channels in Adrenal Cells

Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca(2+) entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry that TRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only other TRP transcript found at considerable levels was TRP1, whereas TRP2, TRP3, TRP5(CCE2), and TRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca(2+) (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.

Trp1, a Candidate Protein for the Store-operated Ca2+Influx Mechanism in Salivary Gland Cells

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Olfactory Adaptation Depends on the Trp Ca2+Channel inDrosophila

Olfactory adaptation is shown to occur in Drosophila, at both behavioral and physiological levels. In a behavioral paradigm, the extent of adaptation is shown to depend on the dose and duration of the adapting stimulus. Half-maximal adaptation occurred after 15 sec of exposure to an odor, and recovery occurred with a half-time of 1. 5 min, under a set of test conditions. Cross-adaptation was observed among all odor combinations tested, although to a lesser extent than when the same odor was used as both the adapting and the test stimulus. Mutants of the transient receptor potential (Trp) Ca2+ channel were normal in olfactory response, but defective in olfactory adaptation, when measured either behaviorally or in tests of antennal physiology. These results indicate that olfactory response and adaptation can be distinguished. Trp expression was detected in the developing antenna but, surprisingly, not in the mature antenna. These results, together with temperature-shift analysis of a temperature-sensitive trp mutant, provide evidence of a role of Trp in olfactory system development.

Molecular cloning and functional expression of rat trp encoding store-operated Ca 2+ channel

This paper provides a brief overview of the diversity of voltage-gated Ca2+ channels and our recent work on neuronal Ca2+ channels with novel pharmacological and biophysical properties that distinguish them from L, N, P or T-type channels. The Ca2+ channel α1 subunit known as α1A or BI [Mori Y., Friedrich T., Kim M.-S., Mikami A., Nakai J., Ruth P., Bosse E., Hofmann F., Flockerzi V., Furuichi T., Mikoshiba K., Imoto K., Tanabe T. and Numa S. (1991) Nature350, 398–402] is generally assumed to encode the P-type Ca2+ channel. However, we find that α1A expressed in Xenopus oocytes differs from P-type channels in its kinetics of inactivation and its degree of sensitivity to block by the peptide toxins ω-Aga-IVA and ω-CTx-MVIIC [Sather W. A., Tanabe T., Zhang J.-F., Mori Y., Adams M. E. and Tsien R. W. (1993) Neuron11, 291–303]. Thus, α1A is capable of generating a Ca2+ channel with characteristics quite distinct from P-type channels. Doe-1, recently cloned from the forebrain of a marine ray, is another α1 subunit which exemplifies a different branch of the Ca2+ channel family tree [Home W. A., Ellinor P. T., Inman I., Zhou M., Tsien R. W. and Schwarz T. L. (1993) Proc. Natn. Acad. Sci. U.S.A. 90, 3787–3791]. When expressed in Xenopus oocytes, doe-1 forms a high voltage-activated (HVA) Ca2+ channel [Ellinor P. T., Zhang J.-F., Randall A. D., Zhou M., Schwarz T. L., Tsien R. W. and Home W. (1993) Nature363, 455–458]. It inactivates more rapidly than any previously expressed calcium channel and is not blocked by dihydropyridine antagonists or ω-Aga-IVA. Doe-1 current is reduced by ω-CTx-GVIA, but the inhibition is readily reversible and requires micromolar toxin, in contrast to this toxin's potent and irreversible block of N-type channels. Doe-1 shows considerable sensitivity to block by Ni2+ or Cd2+. We have identified components of Ca2+ channel current in rat cerebellar granule neurons with kinetic and pharmacological features similar to α1A and doe-1 in oocytes [Randall A. D., Wendland B., Schweizer F., Miljanich G., Adams M. E. and Tsien R. W. (1993) Soc. Neurosci. Abstr.19, 1478]. The doe-1-like component (R-type current) inactivates much more quickly than L, N or P-type channels, and also differs significantly in its pharmacology. The rapid inactivation and slow repriming of R-type current would make its contribution to Ca2+ entry strongly frequency-dependent.

Cloning and Expression of a Novel Mammalian Homolog ofDrosophila Transient Receptor Potential (Trp) Involved in Calcium Entry Secondary to Activation of Receptors Coupled by the Gq Class of G Protein

Hormonal stimulation of Gq-protein coupled receptors triggers Ca2+ mobilization from internal stores. This is followed by a Ca2+ entry through the plasma membrane. Drosophila Trp and Trpl proteins have been implicated in Ca2+ entry and three mammalian homologues of Drosophila Trp/Trpl, hTrp1, hTrp3 and bTrp4 (also bCCE) have been cloned and expressed. Using mouse brain RNA as template, we report here the polymerase chain reaction-based cloning and functional expression of a novel Trp, mTrp6. The cDNA encodes a protein of 930 amino acids, the sequence of which is 36.8, 36.3, 43.1, 38.6, and 74. 1% identical to Drosophila Trp and Trpl, bovine Trp4, and human Trp1 and Trp3, respectively. Transient expression of mTrp6 in COS.M6 cells by transfection of the full-length mTrp6 cDNA increases Ca2+ entry induced by stimulation of co-transfected M5 muscarinic acetylcholine receptor with carbachol (CCh), as seen by dual wavelength fura 2 fluorescence ratio measurements. The mTrp6-mediated increase in Ca2+ entry activity was blocked by SKF-96365 and La3+. Ca2+ entry activity induced by thapsigargin was similar in COS cells transfected with or without the mTrp6 cDNA. The thapsigargin-stimulated Ca2+ entry could not be further stimulated by CCh in control cells but was markedly increased in mTrp6-transfected cells. Records of whole cell transmembrane currents developed in response to voltage ramps from -80 to +40 mV in control HEK cells and HEK cells stably expressing mTrp6 revealed the presence of a muscarinic receptor responsive non-selective cation conductance in Trp6 cells that was absent in control cells. Our data support the hypothesis that mTrp6 encodes an ion channel subunit that mediates Ca2+ entry stimulated by a G-protein coupled receptor, but not Ca2+ entry stimulated by intracellular Ca2+ store depletion.