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 Ca²⁺ 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.     

Carvacrol is a novel inhibitor of Drosophila TRPL and mammalian TRPM7 channels

Transient receptor potential (TRP) channels are essential components of biological sensors that detect changes in the environment in response to a myriad of stimuli. A major difficulty in the study of TRP channels is the lack of pharmacological agents that modulate most members of the TRP superfamily. Notable exceptions are the thermoTRPs, which respond to either cold or hot temperatures and are modulated by a relatively large number of chemical agents. In the present study we demonstrate by patch clamp whole cell recordings from Schneider 2 and Drosophila photoreceptor cells that carvacrol, a known activator of the thermoTRPs, TRPV3 and TRPA1 is an inhibitor of the Drosophila TRPL channels, which belongs to the TRPC subfamily. We also show that additional activators of TRPV3, thymol, eugenol, cinnamaldehyde and menthol are all inhibitors of the TRPL channel. Furthermore, carvacrol also inhibits the mammalian TRPM7 heterologously expressed in HEK cells and ectopically expressed in a primary culture of CA3–CA1 hippocampal brain neurons. This study, thus, identifies a novel inhibitor of TRPC and TRPM channels. Our finding that the activity of the non-thermoTRPs, TRPL and TRPM7 channels is modulated by the same compound as thermoTRPs, suggests that common mechanisms of channel modulation characterize TRP channels.     

Identification and functional characterization of ion channels in CD34<Superscript>+</Superscript> hematopoietic stem cells from human peripheral blood

Hematopoietic stem cells (HSCs) are used therapeutically for hematological diseases and may also serve as a source for nonhematopoietic tissue engineering in the future. In other cell types, ion channels have been investigated as potential targets for the regulation of proliferation and differentiation. However, the ion channels of HSCs remain elusive. Here, we functionally characterized the ion channels of CD34⁺ cells from human peripheral blood. Using fluorescence-activated cell sorting, we confirmed that the CD34⁺ cells also express CD45 and CD133. In the CD34⁺/CD45⁺/CD133^high HSCs, RT-PCR of 58 ion channel mRNAs revealed the coexpression of Kv1.3, Kv7.1, Nav1.7, TASK2, TALK2, TWIK2, TRPC4, TRPC6, TRPM2, TRPM7, and TRPV2. Whole-cell patch clamp recordings identified voltage-gated K⁺ currents (putatively Kv1.3), pH-sensitive TASK2-like back-ground K⁺ currents, ADP-ribose-activated TRPM2 currents, temperature-sensitive TRPV2-like currents, and diacylglycerol-analogue-activated TRPC6-like currents. Our results lend new insight into the physiological role of ion channels in HSCs, the specific implications of which require further investigation.     

TRP channels in thermosensation

The ability to sense external temperature is assumed by somatosensory neurons, in which temperature information is converted to neural activity by afferent input to the central nervous system. Somatosensory neurons consist of various populations with specialized gene expression, including thermosensitive transient receptor potential ion channels (thermo-TRPs). Thermo-TRPs are responsible for thermal transduction at the peripheral ends of somatosensory neurons and over a wide range of temperatures. In this review, we focus on several thermo-TRPs expressed in sensory neurons: TRPV1, TRPV4, TRPM2, TRPM3, TRPM8, TRPC5, and TRPA1. TRPV3, TRPV4, and TRPC5 expressed in non-neuronal cells that are also involved in somatosensation are also discussed, whereas TRPM2 and TRPM8 are involved in thermosensation in the brain.     

2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3

The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation.     

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.     

Effect of acyclic monoterpene alcohols and their derivatives on TRP channels

A series of thirty-six geraniol, nerol, citronellol, geranylamine, and nerylamine derivatives was synthesized and tested on TRPA1, TRPM8, and TRPV1 channels. Most of them acted as strong modulators of TRPA1 channels with EC₅₀ and/or IC₅₀ values <1 μM. None was able to significantly activate TRPM8 channels, while thirteen of them behaved as ‘true’ TRPM8 antagonists. Little or no effect was generally observed on TRPV1 channels. Some of the compounds examined, that is, compounds 1d,g,n, 2c,d,h,i,o, 3b,e exhibited an appreciable selectivity for TRPA1 subtype.     

TRP channels in kidney disease

Mammalian TRP channel proteins form six-transmembrane cation-permeable channels that may be grouped into six subfamilies on the basis of amino acid sequence homology (TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML). Recent studies of TRP channels indicate that they are involved in numerous fundamental cell functions and are considered to play an important role in the pathophysiology of many diseases. Many TRPs are expressed in kidney along different parts of the nephron and growing evidence suggest that these channels are involved in hereditary, as well as acquired kidney disorders. TRPC6, TRPM6, and TRPP2 have been implicated in hereditary focal segmental glomerulosclerosis (FSGS), hypomagnesemia with secondary hypocalcemia (HSH), and polycystic kidney disease (PKD), respectively. In addition, the highly Ca(2+)-selective channel, TRPV5, contributes to several acquired mineral (dys)regulation, such as diabetes mellitus (DM), acid-base disorders, diuretics, immunosuppressant agents, and vitamin D analogues-associated Ca(2+) imbalance whereas TRPV4 may function as an osmoreceptor in kidney and participate in the regulation of sodium and water balance. This review presents an overview of the current knowledge concerning the distribution of TRP channels in kidney and their possible roles in renal physiology and kidney diseases.     

The discovery of potent blockers of the canonical transient receptor channels,TRPC3 and TRPC6, based on an anilino-thiazole pharmacophore

Lead optimization of piperidine amide HTS hits, based on an anilino-thiazole core, led to the identification of analogs which displayed low nanomolar blocking activity at the canonical transient receptor channels 3 and 6 (TRPC3 & 6) based on FLIPR (carbachol stimulated) and electrophysiology (OAG stimulated) assays. In addition, the anilino-thiazole amides displayed good selectivity over other TRP channels (TRPA1, TRPV1, and TRPV4), as well as against cardiac ion channels (CaV1.2, hERG, and NaV1.5). The high oxidation potential of the aliphatic piperidine and aniline groups, as well as the lability of the thiazole amide group contributed to the high clearance observed for this class of compounds. Conversion of an isoquinoline amide to a naphthyridine amide markedly reduced clearance for the bicyclic piperidines, and improved oral bioavailability for this compound series, however TRPC3 and TRPC6 blocking activity was reduced substantially. Although the most potent anilino-thiazole amides ultimately lacked oral exposure in rodents and were not suitable for chronic dosing, analogs such as 14–19, 22, and 23 are potentially valuable in vitro tool compounds for investigating the role of TRPC3 and TRPC6 in cardiovascular disease.     

TRP channels and analgesia

Since cloning and characterizing the first nociceptive ion channel Transient Receptor Potential (TRP) Vanilloid 1 (TRPV1), other TRP channels involved in nociception have been cloned and characterized, which include TRP Vanilloid 2 (TRPV2), TRP Vanilloid 3 (TRPV3), TRP Vanilloid 4 (TRPV4), TRP Ankyrin 1 (TRPA1) and TRP Melastatin 8 (TRPM8), more recently TRP Canonical 1, 5, 6 (TRPC1, 5, 6), TRP Melastatin 2 (TRPM2) and TRP Melastatin 3 (TRPM3). These channels are predominantly expressed in C and Aδ nociceptors and transmit noxious thermal, mechanical and chemical sensitivities. TRP channels are modulated by pro-inflammatory mediators, neuropeptides and cytokines. Significant advances have been made targeting these receptors either by antagonists or agonists to treat painful conditions. In this review, we will discuss TRP channels as targets for next generation analgesics and the side effects that may ensue as a result of blocking/activating these receptors, because they are also involved in physiological functions such as release of vasoactive neuropeptides and regulation of vascular tone, maintenance of the body temperature, gastrointestinal motility, urinary bladder control, etc.     

Role of TRPM2 and TRPV1 cation channels in cellular responses to radiation-induced DNA damage

Background

Radiation exposure causes DNA damage, and DNA repair systems are essential to rescue damaged cells. Although DNA damage or oxidative stress activates transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) cation channels, it has not been established whether these TRP channels are involved in cellular responses to radiation-induced DNA damage. Here, we investigated the contribution of TRPM2 and TRPV1 channels to γ-irradiation- and UVB-induced DNA damage responses in human lung cancer A549 cells.

Methods

A549 cells were irradiated with γ-rays (2.0 Gy) or UVB (5–10 mJ/cm²). γH2AX foci, ATM activation, 53BP1 accumulation and EGFR expression were evaluated by immunofluorescence staining. Extracellular ATP concentration was measured by luciferin–luciferase assay. Knockdown of TRPM2 and TRPV1 expression was done by siRNA transfection.

Results

γ-Irradiation-induced γH2AX focus formation, ATM activation, 53BP1 accumulation and EGFR nuclear translocation, which are all associated with DNA repair, were suppressed by knockdown of TRPM2 and TRPV1 channels in A549 cells. Release of ATP, which mediates DNA damage response-associated activation of P2Y receptors, was suppressed by pre-treatment with catalase or knockdown of TRPM2 channel, but not TRPV1 channel. Similarly, UVB-induced γH2AX focus formation was suppressed in TRPM2- and TRPV1-knockdown cells, while UVB-induced ATP release was blocked in TRPM2- but not TRPV1-knockdown cells.

Conclusion

Our results suggest that the activation of TRPM2 channel, which mediates ATP release, and TRPV1 channel plays significant roles in the cellular responses to DNA damage induced by γ-irradiation and UVB irradiation.

General significance

Our results provide a new insight into the function of TRP channels from the viewpoint of radiation biology.     

TRP channels in cancer

The progression of cells from a normal differentiated state in which rates of proliferation and apoptosis are balanced to a tumorigenic and metastatic state involves the accumulation of mutations in multiple key signalling proteins and the evolution and clonal selection of more aggressive cell phenotypes. These events are associated with changes in the expression of numerous other proteins. This process of tumorigenesis involves the altered expression of one or more TRP proteins, depending on the nature of the cancer. The most clearly described changes are those involving TRPM8, TRPV6 and TRPM1. Expression of TRPM8 is substantially increased in androgen-dependent prostate cancer cells, but is decreased in androgen independent and metastatic prostate cancer. TRPM8 expression is regulated, in part, by androgens, most likely through androgen response elements in the TRPM8 promoter region. TRPM8 channels are involved in the regulation of cell proliferation and apoptosis. Expression of TRPV6 is also increased in prostate cancer and in a number of other cancers. In contrast to TRPM8, expression of TRPV6 is not directly regulated by androgens. TRPM1 is highly expressed in early stage melanomas but its expression declines with increases in the degree of aggressiveness of the melanoma. The expression of TRPV1, TRPC1, TRPC6, TRPM4, and TRPM5 is also increased in some cancers. The level of expression of TRPM8 and TRPV6 in prostate cancer, and of TRPM1 in melanomas, potentially provides a good prognostic marker for predicting the course of the cancer in individuals. The Drosophila melanogaster, TRPL, and the TRPV1 and TRPM8 proteins, have been used to try to develop strategies to selectively kill cancer cells by activating Ca²⁺ and Na⁺ entry, producing a sustained increase in the cytoplasmic concentration of these ions, and subsequent cell death by apoptosis and necrosis. TRPV1 is expressed in neurones involved in sensing cancer pain, and is a potential target for pharmacological inhibition of cancer pain in bone metastases, pancreatic cancer and most likely in other cancers. Further studies are required to assess which other TRP proteins are associated with the development and progression of cancer, what roles TRP proteins play in this process, and to develop further knowledge of TRP proteins as targets for pharmaceutical intervention and targeting in cancer.     

Orai and TRP channels in skeletal muscle cells

Modern data concerning expression, localization, biophysical properties, involvement in calcium regulation, and physiological functions of TRP and Orai channels in skeletal muscle cells are analyzed. In skeletal muscles TRPC1/2/3/4/5/6/7, TRPV2/4, TRPM2/7 and Orai1/3 channels are expressed. Activities of TRPC1/3 and TRPV4 facilitate maximal muscle contraction during tetanus. Orai1 channels provide recovery of intracellular calcium stores and are obligatory for proliferation of myoblasts and differentiation of skeletal muscles. TRPC1 knockout results in alterations of the development of skeletal muscles. Enhanced calcium influx via the channels is supposed to be a pathogenic factor of myodystrophy.     

(−)-Menthylamine derivatives as potent and selective antagonists of transient receptor potential melastatin type-8 (TRPM8) channels

A series of twenty-two (?)-menthylamine derivatives was synthesized and tested on TRPM8, TRPV1, and TRPA1 channels. Five of the novel compounds, that is, 1d, 1f, 2b, 2c, and 2e behaved as potent TRPM8 antagonists with IC₅₀ values versus icilin and (?)-menthol between 20 nM and 0.7 μM, and were between 4- and ～150-fold selective versus TRPV1 and TRPA1 activation. Compound 1d also induced caspase 3/7 release in TRPM8-expressing LNCaP prostate carcinoma cells, but not in non-TRPM8 expressing DU-145 cells. Five other derivatives, that is, 1a, 1g, 1h, 2f, and 2h were slightly less potent than previous compounds but still relatively selective versus TRPV1 and TRPA1.     

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 Ca²⁺-dependent proliferation of primary CD4⁺ T-cells indicating that TRPC3 may be involved in Ca²⁺ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca²⁺ entry.     

Phosphoinositide regulation of non-canonical transient receptor potential channels

Transient receptor potential (TRP) channels are involved in a wide range of physiological processes, and characterized by diverse activation mechanisms. Phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PIP₂, or PtdIns(4,5)P₂] recently emerged as regulators of many TRP channels. Several TRP channels require PIP₂ for activity, and depletion of the lipid inhibits them. For some TRP channels, however, phosphoinositide regulation seems more complex, both activating and inhibitory effects have been reported. This review will discuss phosphoinositide regulation of members of the TRPM (Melastatin), TRPV (Vanilloid), TRPA (Ankyrin) and TRPP (Polycystin) families. Lipid regulation of TRPC (Canonical) channels is discussed elsewhere in this volume.     

TRP channels in cancer

The progression of cells from a normal differentiated state in which rates of proliferation and apoptosis are balanced to a tumorigenic and metastatic state involves the accumulation of mutations in multiple key signalling proteins and the evolution and clonal selection of more aggressive cell phenotypes. These events are associated with changes in the expression of numerous other proteins. This process of tumorigenesis involves the altered expression of one or more TRP proteins, depending on the nature of the cancer. The most clearly described changes are those involving TRPM8, TRPV6 and TRPM1. Expression of TRPM8 is substantially increased in androgen-dependent prostate cancer cells, but is decreased in androgen independent and metastatic prostate cancer. TRPM8 expression is regulated, in part, by androgens, most likely through androgen response elements in the TRPM8 promoter region. TRPM8 channels are involved in the regulation of cell proliferation and apoptosis. Expression of TRPV6 is also increased in prostate cancer and in a number of other cancers. In contrast to TRPM8, expression of TRPV6 is not directly regulated by androgens. TRPM1 is highly expressed in early stage melanomas but its expression declines with increases in the degree of aggressiveness of the melanoma. The expression of TRPV1, TRPC1, TRPC6, TRPM4, and TRPM5 is also increased in some cancers. The level of expression of TRPM8 and TRPV6 in prostate cancer, and of TRPM1 in melanomas, potentially provides a good prognostic marker for predicting the course of the cancer in individuals. The Drosophila melanogaster, TRPL, and the TRPV1 and TRPM8 proteins, have been used to try to develop strategies to selectively kill cancer cells by activating Ca(2+) and Na(+) entry, producing a sustained increase in the cytoplasmic concentration of these ions, and subsequent cell death by apoptosis and necrosis. TRPV1 is expressed in neurones involved in sensing cancer pain, and is a potential target for pharmacological inhibition of cancer pain in bone metastases, pancreatic cancer and most likely in other cancers. Further studies are required to assess which other TRP proteins are associated with the development and progression of cancer, what roles TRP proteins play in this process, and to develop further knowledge of TRP proteins as targets for pharmaceutical intervention and targeting in cancer.     

Role and regulation of TRP channels in neutrophil granulocytes

Members of the transient receptor potential (TRP) family for which mRNA can be demonstrated in neutrophil granulocytes with RT-PCR include TRPC6 (as only "short" TRP), TRPM2, TRPV1, TRPV2, TRPV5 and TRPV6. When these are analyzed in heterologous overexpression experiments, TRPM2 is the only cation channel with characteristic properties that can be used as fingerprint to provide functional evidence for its expression in neutrophil granulocytes. As cells transfected with TRPM2, neutrophil granulocytes display non-selective cation currents and typical channel activity evoked by intracellular ADP-ribose and NAD. Thus, stimulation of TRPM2 is likely to occur after activation of CD38 (producing ADP-ribose) and during the oxidative burst (enhancing the NAD concentration). This novel mode of cation entry regulation may be of particular importance for the response of granulocytes to chemoattractants. TRPV6 is a likely but not exclusive candidate as subunit of the channels mediating store-operated Ca2+ entry (SOCE). Evidence for SOCE in granulocytes has been presented with the fura-2 technique but not with electrophysiological methods although Ca2+-selective store-operated currents can be demonstrated in HL-60 cells, a cell culture model of neutrophil granulocytes.     

Side chain flexibility and coupling between the S4‐S5 linker and the TRP domain in thermo‐sensitive TRP channels: Insights from protein modeling

The transient receptor potential (TRP) superfamily is subdivided into several subfamilies on the basis of sequence similarity, which is highly heterogeneous but shows a molecular architecture that resembles the one present in members of the Kv channel superfamily. Because of this diversity, they produce a large variety of channels with different gating and permeability properties. Elucidation of these particular features necessarily requires comparative studies based on structural and functional data. The present study aims to compilate, analyze, and determine, in a coherent way, the relationship between intrinsic side‐chain flexibility and the allosteric coupling in members of the TRPV, TRPM, and TRPC families. Based on the recently determined structures of TRPV1 and TRPV2, we have generated protein models for single subunits of TRPV5, TRPM8, and TRPC5 channels. With these models, we focused our attention on the apparently crucial role of the GP dipeptide at the center of the S4‐S5 linker and discussed its role in the interaction with the TRP domain, specifically with the highly‐conserved Trp during this coupling. Our analysis suggests an important role of the S4‐S5L flexibility in the thermosensitivity, where heat‐activated channels possess rigid S4‐S5 linkers, whereas cold‐activated channels have flexible ones. Finally, we also present evidence of the key interaction between the conserved Trp residue of the TRP box and of several residues in the S4‐S5L, importantly the central Pro. Proteins 2017; 85:630–646. © 2016 Wiley Periodicals, Inc.