Members Of Transient Receptor Potential Family Research Articles

Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal transduction.

Mechanotransduction by TRP Channels: General Concepts and Specific Role in the Vasculature

Transient receptor potential (TRP) ion channel superfamily is involved in sensing and transmission of a broad variety of external or internal stimuli, including but not limited to mechanical stress. Based on homology analysis, genetic and molecular studies have recently identified TRP channels in different tissues, comprising blood vessels. In invertebrates, many TRP channels including five TRPV channels identified in Caenorhabditis elegans and two in Drosophila have been implicated in mechanosensory behaviors as molecular basis of volume regulation, hearing and touch sensitivity. Consistently, in mammals many TRP family members such as TRPC1, TRPC3, TRPC6, TRPM4, TRPM7, TRPN1, TRPA1, TRPY1, TRPP1, TRPP2, and notably, TRPV1, TPRV2 as well as TRPV4 have been reported to be involved in mechanotransduction. This review summarizes recent and at times controversial findings on the role and regulation of TRP channels in mechanotransduction. Specifically, we highlight the relevance of TRPV channels in vascular regulation and focus on TRPV4 in the vascular system of the lung, which is constantly exposed to a unique combination of circumferential and longitudinal strains. In light of our observation in intact pulmonary microvessels that mechanical stress induced Ca(2+) signaling in endothelial cells is closely related to TRPV4 activity, we postulate that TRPV4 plays a critical role in lung vascular mechanotransduction. The progress in this rapidly expanding field may allow for the identification of new molecular targets and the development of new therapeutic approaches in a number of intractable diseases related to mechanical stress.

Effect of Menthol on Detrusor Smooth-Muscle Contraction and the Micturition Reflex in Rats

To investigate the effects of menthol, an activator of the temperature-sensitive transient receptor potential family member TRPM8, on detrusor smooth-muscle contraction and the micturition reflex in rats. We evaluated the effect of intravesical infusion of 1 or 3 mM menthol by cystometry in conscious female Sprague-Dawley rats. Some of the animals were pretreated with 125 mg/kg capsaicin subcutaneously 4 days earlier. The parameters measured were voided volume (VV), residual volume (RV), volume threshold for inducing micturition (VT), voiding efficiency (VE), micturition pressure (MP), and pressure threshold for inducing micturition (PT). Strips of detrusor muscle were mounted in organ baths to study the effect of menthol on the contractile response to 0.01 mM carbachol. Intravesical infusion of 3 mM menthol reduced VV by 20%, VT by 19%, and PT by 31%. RV, VE, and MP were unaltered. Capsaicin pretreatment had no effect on baseline cystometric parameters, and 3 mM menthol caused similar reductions in VV (24%), VT (22%), and PT (30%), while having no effect on RV, VE, and MP. Menthol at 0.1, 0.3, and 1 mM inhibited carbachol-induced contractions by 10.7%, 36.7%, and 97.3%, respectively. Our results demonstrated that intravesical infusion of menthol facilitated the micturition reflex, and capsaicin pretreatment had no effect on this response. Menthol inhibited carbachol-induced contraction of the detrusor smooth muscle. This suggests that intravesically infused menthol cannot relax detrusor muscle, and acts on capsaicin-resistant afferents (probably through TRPM8 in urothelium or sensory nerve endings) to facilitate the micturition reflex.

Pucker Up: Sour Taste Receptors Revealed

Although the receptors for most taste modalities have been identified, the mechanisms by which sour and salty tastes are detected have remained elusive. Ishimaru et al. show that two members of the transient receptor potential (TRP) family, PKD1L3 and PKD2L1, are expressed in particular regions of taste tissue (in situ mRNA hybridization and reverse transcription polymerase chain reaction analysis) and that their expression does not overlap with that of TRPM5, a downstream effector in some forms of taste perception. Antibodies raised against PKD2L1 showed that the protein was present at the taste pore, the predicted location of a taste receptor. Coexpression of tagged versions of the two proteins in human embryonic kidney (HEK) 293 cells showed that both proteins had to be coexpressed for abundant surface expression, and coimmunoprecipitation experiments indicated that the proteins interacted. Electrophysiological analysis and calcium imaging showed that HEK293 cells expressing the two proteins responded to acids, such as citric acid, HCl, and malic acid, with a rapidly inactivating current and with an increase in intracellular calcium concentration. The response did not appear to be mediated solely by a change in pH, because citric acid was more potent at activating the channel than HCl at the same pH. Y. Ishimaru, H. Inada, M. Kubota, H. Zhuang, M. Tominaga, H. Matsunami, Transient receptor potential family members PKD1L3 and PKD2L1 form a candidate sour taste receptor. Proc. Natl. Acad. Sci. U.S.A. 103 , 12569-12574 (2006). [Abstract] [Full Text]

Transient receptor potential family members PKD1L3 and PKD2L1 form a candidate sour taste receptor

Animals use their gustatory systems to evaluate the nutritious value, toxicity, sodium content, and acidity of food. Although characterization of molecular identities that receive taste chemicals is essential, molecular receptors underlying sour taste sensation remain unclear. Here, we show that two transient receptor potential (TRP) channel members, PKD1L3 and PKD2L1, are coexpressed in a subset of taste receptor cells in specific taste areas. Cells expressing these molecules are distinct from taste cells having receptors for bitter, sweet, or umami tastants. The PKD2L1 proteins are accumulated at the taste pore region, where taste chemicals are detected. PKD1L3 and PKD2L1 proteins can interact with each other, and coexpression of the PKD1L3 and PKD2L1 is necessary for their functional cell surface expression. Finally, PKD1L3 and PKD2L1 are activated by various acids when coexpressed in heterologous cells but not by other classes of tastants. These results suggest that PKD1L3 and PKD2L1 heteromers may function as sour taste receptors.

Capsaicin, transient receptor potential (TRP) protein subfamilies and the particular relationship between capsaicin receptors and small primary sensory neurons

A number of subfamilies of the capsaicin receptor, collectively called TRP, have been reported since the discovery of vanilloid receptor 1 (VR1). The term 'TRP' is derived from 'transient receptor potential', which means the transient and rapid defect of reaction following long stimulation with light in the photoreceptor cells of mutant Drosophila. The common features of TRP family members are the centrally situated six transmembrane domain, in which an ion channel is located, three to four ankirin repeats at the N-terminus and a TRP domain comprising 25 amino acids at the C-terminus. The TRP family members are present in animals, including invertebrates and vertebrates, and in the cells in various tissues in individual animals. During evolution, the original TRP seems to have acquired a wide variety of functions related to sensing the inner or outer environment (e.g. to sensing light (Drosophila), osmolarity, protons, temperature, ligands and mechanical force). In mammals, the TRPV subfamily is exclusively expressed in small- to medium-sized primary sensory neurons that also co-express some chemical markers (i.e. isolectin B4 (IB4), fluoride-resistant acid phosphatase (FRAP), the P2X3 purinoceptor (a receptor provoked by ATP-induced nociception) and Ret, a glial cell line-derived neurotrophic factor receptor). There is a paradox in that regardless of the marked or complete loss of noxious, small sensory neurons (polymodal nociceptors) in mice treated with capsaicin during the neonatal period, as well as in VR1 (TRPV1)-deficient knock-out mice, the responses to noxious heat are normal. Regarding the paradox in mice treated with capsaicin as neonates, our explanation is that although capsaicin probably reduces the number of a subgroup of small neurons (IB4-, VR1+), the remaining IB4+ (VR1-) neurons can sense noxious heat normally. One working hypothesis is that mice lacking TRPV1/2 can sense noxious heat under normal conditions, presumably via another still unknown pathway, and TRPV1 has been suggested to be involved in noxious heat transduction under pathological conditions, such as inflammation and tissue injury. Further studies will be required to clarify these complexities. Mice treated with capsaicin as neonates would provide a model to investigate the above paradoxes, as would TRPV1-knock-out mice, although different mechanisms may be operating in the two models.

Peptide‐Specific Antibody as a Tool to Evaluate TRPM3 Ion Channel Function

Calcium channel blockers can be used to selectively inhibit calcium influx across a membrane, making them potentially useful as therapeutic tools. Existing transient receptor potential (TRP) channel blockers are relatively non-specific, but have previously been used to characterise individual members of the TRP family. The activation of TRP channels can be more specifically inhibited through the use of anti-peptide antibodies targeted to particular regions of the channel structure (Xu & Beech, 2001. Circ. Res. 88:84–87). The polyclonal antibody TM3E3 was designed to specifically bind to a peptide corresponding to the 3rd extracellular loop of TRPM3, a member of the melastatin-like TRP family. The location and sequence of the peptide were determined using hydrophilicity analysis of the channel sequence (Xu et al., 2005. Nat. Biotechnol. 23:1289–1293). Here we use Ca2+-imaging to study the specificity of TM3E3 for human TRPM3 expressed in HEK-293 cells. Extracellular application of the antibody has a functional effect, inhibiting the sphingosine-induced activation of TRPM3. It does not inhibit activation of the closely related channel TRPM2 or the canonical family member TRPC5. TM3E3 will serve as a useful tool for further characterisation of endogenous TRPM3 in a variety of cells and tissues, and more importantly the results strengthen the idea that E3 targeting may be used to generate specific ion channel blockers for a variety of channels, with therapeutic potential. This work is supported by the BBSRC, the Welcome Trust and the BHF. Many thanks to C. Harteneck for providing the TRPM3 clone.

Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca(2+) store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. We report that two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation was mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induced the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and was accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na(+)/H(+) exchanger regulatory factor. Furthermore, this translocation and association was defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs was concomitant with a reduction in Ca(2+) influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function.

TRPM7 and Ischemic CNS Injury

Ischemic brain damage represents a major source of morbidity and mortality in westernized society and poses a significant financial burden on the health care system. To date, few effective therapies have been realized to treat stroke and once promising avenues such as antiexcitotoxic therapy with NMDA receptor antagonists have not proven clinically useful. Thus, we need to identify new targets for research and therapeutic intervention of the neurodegeneration caused by stroke. Transient receptor potential (TRP) channels are an exciting new family of cation channels that respond to intracellular and extracellular stimuli. Indeed, several members can be induced by oxidative stress and oxygen free radicals. We have recently demonstrated that one member, TRPM7, is an essential mediator of anoxic neuronal death that is activated by oxidative stress, in parallel to excitotoxic signal pathways. Thus, future treatment of ischemic brain injury may need to include strategies that inhibit or modulate TRPM7 activity. Further investigation of the physiology and pathophysiology of TRPM7 and other TRP family members is needed to provide both pharmacological targets and a better understanding of ischemic brain disorders.

Expression of transient receptor potential C6 and related transient receptor potential family members in human airway smooth muscle and lung tissue.

Elevation of the intracellular free Ca(2+) concentration regulates many functional responses in airway smooth muscle, including contraction, proliferation, adhesion, and cell survival. This increase in calcium can be achieved by a release from internal stores (sarcoplasmic reticulum) and/or entry across the cell membrane from the extracellular environment. The molecular identity of this calcium influx pathway in human airway smooth muscle (HASM) remains unclear. Functional studies using Fluo 4-loaded HASM suggest the presence of a histamine H(1) receptor-activated Ca(2+) entry pathway with characteristics similar to those seen with transient receptor potential (TRP) family homologs. Using a range of molecular and cell biological approaches we defined the expression pattern of transient receptor potential classics (TRPC) homologs in airway cells and tissue. Here we show that HASM and human bronchial epithelial cells both express TRPC1, -4, and -6, with HASM also expressing TRPC3 at the mRNA level. Identification of TRPC6 protein by western blot and confocal microscopy indicated that the protein is localized in specific cell types, suggesting that it plays an important role in regulating key functions in airway cells. These data demonstrate the expression of a range of TRPC homologs in the airway and the presence of a functional Ca(2+) entry pathway with characteristics typical of TRPC family members. TRPC homologs may provide an important novel target for the treatment of airway disease.

A comparison of the genes coding for canonical TRP channels and their M, V and P relatives

The mammalian transient receptor potential (TRP) protein gene family consists of a diverse group of cation channels that currently contain at least 26 members. The physiologic functions of many remain unknown. They are structurally similar to Drosophila TRP and have a wide tissue distribution. In the present report, we compare the chromosomal locations, the gene, and primary structures of each of these 26 human TRP family members. Based on primary amino acid analyses, these channels comprise four different subfamilies: C- (canonical or classical), V- (or vaniloid receptor related), M- (melastatin related), and P (PKD)-type. The highest homology within each subfamily and between subfamilies exists in the predicted ion channel domains. Belonging to a given subfamily, however, does not determine the activating stimuli. This is exemplified by the V- and M-subfamilies, both of which have members that respond to temperature and osmolarity. TRP genes vary in their intron–exon organization, with the greatest diversity in the P subfamily. Chromosomal organization analyses revealed that two TRP members are found as direct repeats; TRPV3 follows TRPV1 and TRPV6 follows TRPV5. Both of these duplications appear to be recent as TRPV1 and V3 are more similar to each other than to other members of the TRPV subfamily. The same holds true for TRPV5 and V6. The article presents complication of comparisons including exon–intron boundaries, the amino acid sequence alignments, and the chromosomal organization of each of the presently known TRP channels.