Imbalance and dysfunction of transient receptor potential channels contribute to the pathogenesis of hypertension

Intracellular Ca²⁺ homeostasis is essential for vascular function and blood pressure regulation. Because of their unique roles in regulating intracellular Ca²⁺ concentration and vascular function, a novel class of non-selective cation channels, called transient receptor potential (TRP) channels, have emerged at the frontier of hypertension research. Based on their role in vasculature function regulation, TRP channels can be divided into two functional subtypes: one that participates in vasoconstriction and one that participates in vasodilatation. A functional imbalance of these two subtypes of TRP channels may disturb intracellular calcium ([Ca²⁺]i) homeostasis, and the consequent vascular dysfunction may contribute to the development of hypertension. The potential of these TRP channels as novel pharmacological targets for the treatment of human hypertension is of great interest.     

Muscling in on TRP channels in vascular smooth muscle cells and cardiomyocytes

The human TRP protein family comprises a family of 27 cation channels with diverse permeation and gating properties. The common theme is that they are very important regulators of intracellular Ca²⁺ signaling in diverse cell types, either by providing a Ca²⁺ influx pathway, or by depolarising the membrane potential, which on one hand triggers the activation of voltage-gated Ca²⁺ channels, and on the other limits the driving force for Ca²⁺ entry. Here we focus on the role of these TRP channels in vascular smooth muscle and cardiac striated muscle. We give an overview of highlights from the recent literature, and highlight the important and diverse roles of TRP channels in the pathophysiology of the cardiovascular system.The discovery of the superfamily of Transient Receptor Potential (TRP) channels has significantly enhanced our knowledge of multiple signal transduction mechanisms in cardiac muscle and vascular smooth muscle cells (VSMC). In recent years, multiple studies have provided evidence for the involvement of these channels, not only in the regulation of contraction, but also in cell proliferation and remodeling in pathological conditions.The mammalian family of TRP cation channels is composed by 28 genes which can be divided into 6 subfamilies groups based on sequence similarity: TRPC (Canonical), TRPM (Melastatin), TRPML (Mucolipins), TRPV (Vanilloid), TRPP (Policystin) and TRPA (Ankyrin-rich protein). Functional TRP channels are believed to form four-unit complexes in the plasma, each of them expressed with six transmembrane domain and intracellular N and C termini.Here we review the current knowledge on the expression of TRP channels in both muscle types, and discuss their functional properties and role in physiological and pathophysiological processes.     

The transient receptor potential family of ion channels

The transient receptor potential (TRP) multigene superfamily encodes integral membrane proteins that function as ion channels. Members of this family are conserved in yeast, invertebrates and vertebrates. The TRP family is subdivided into seven subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin) and TRPN (NOMPC-like); the latter is found only in invertebrates and fish. TRP ion channels are widely expressed in many different tissues and cell types, where they are involved in diverse physiological processes, such as sensation of different stimuli or ion homeostasis. Most TRPs are non-selective cation channels, only few are highly Ca²⁺ selective, some are even permeable for highly hydrated Mg²⁺ ions. This channel family shows a variety of gating mechanisms, with modes of activation ranging from ligand binding, voltage and changes in temperature to covalent modifications of nucleophilic residues. Activated TRP channels cause depolarization of the cellular membrane, which in turn activates voltage-dependent ion channels, resulting in a change of intracellular Ca²⁺ concentration; they serve as gatekeeper for transcellular transport of several cations (such as Ca²⁺ and Mg²⁺), and are required for the function of intracellular organelles (such as endosomes and lysosomes). Because of their function as intracellular Ca²⁺ release channels, they have an important regulatory role in cellular organelles. Mutations in several TRP genes have been implicated in diverse pathological states, including neurodegenerative disorders, skeletal dysplasia, kidney disorders and pain, and ongoing research may help find new therapies for treatments of related diseases.     

Role of TRPC3 in the formation of receptor-and store-operated calcium channels in carcinoma A431 cells

Activation of phospholipase C (PLC)-linked signaling cascades in nonexcitable cells stimulates Ca²⁺ release from inositol-1,4,5-trisphosphate (IP₃)-sensitive intracellular Ca²⁺ stores and activation of Ca²⁺ entry via plasma membrane Ca²⁺ channels. The attention of investigators is currently focused on the properties and molecular basis of channels involved in Ca²⁺ entry into nonexcitable cells. According to current views, mammalian TRP proteins are involved in receptor-and store-dependent influx of Ca²⁺; however, little is known about the linkage between specific TRP proteins and endogenous channels responsible for Ca²⁺ entry. The aim of the present study was to elucidate the role of TRPC3 in the formation of store-dependent or receptor-operated pathways of Ca²⁺ entry into A431 cells. Registration of Ca²⁺ influx based on fluorescence measurements of intracellular Ca²⁺ concentrations and analysis of integral membrane currents revealed that partial inhibition of TRPC3 expression by small interfering RNA (siRNA) results in suppression of store-dependent Ca²⁺ entry without any effect on receptor-operated Ca²⁺ influx. In-depth studies of single channels revealed that TRPC3 suppression in A431 cells results in the disappearance of one type of store-operated channels and formation of a novel type of store-independent Ca²⁺-permeable channels. This, in turn, testifies to the crucial role of TRPC3 in normal functioning of store-operated Ca²⁺ channels in A431 cells.     

TRP channels in cell survival and cell death in normal and transformed cells

TRP channels form a superfamily of channel proteins exhibiting versatile regulatory characteristics with many channels participating in the regulation of Ca²⁺ homeostasis and influencing the cell fate. Multitude of evidence is emerging that the colocalization of TRP channels with Ca²⁺-sensing elements of specific regulatory pathways leading to either proliferation or apoptosis is what makes these channels participate in cell fate regulation and, in turn, determines the final effect of Ca²⁺ entry via the particular channel. This review focuses on the aspects of TRP channel localization and function that affect the balance between cell survival and death and how various dysregulations of these channels may lead to perturbed balance and onset of cancer.     

TRPs and Ca2+ in cell death and survival

Transient Receptor Potential (TRP) family mediate the influx of monovalent and/or divalent cations into cells in response to environmental stimuli. Pharmacological or genetic manipulations of TRP channels demonstrate that TRP channels influence cell death rates, prolonging or shortening of cell survival. Due to their diverse cellular localization, TRP channels mediated Ca²⁺ influx generates distinct intracellular Ca²⁺ signals that regulate downstream pathways converging to apoptosis or survival. In this review, we summarize the accumulated knowledge focused on how TRP channel regulate cell fate and may affect different pathologies including cardiovascular, neurological, metabolic or neoplastic disorders.     

Mechanism of Ca2+-dependent desensitization in TRP channels

The 30+ members of the family of TRP channels are diverse in their physiological roles, yet the mechanisms that regulate their gating may be conserved. In particular, all TRP channels show an activity-dependent inhibition which is mediated by Ca²⁺. The mechanism by which Ca²⁺ inhibits TRP channels is currently a matter of intense debate, with Ca²⁺-regulated kinases, phosphatases, phospholipases, and calmodulin all proposed to be involved. In this review, we will discuss different mechanisms for Ca²⁺-dependent desensitization in TRP channels. We will conclude with a model that focuses on Ca²⁺-dependent activation of phospholipase C and Ca²⁺ binding to calmodulin and propose that the phospholipase C and calmodulin pathways are structurally and functionally coupled.     

TRPP2 channels regulate apoptosis through the Ca2+ concentration in the endoplasmic reticulum

Ca²⁺ is an important signalling molecule that regulates multiple cellular processes, including apoptosis. Although Ca²⁺ influx through transient receptor potential (TRP) channels in the plasma membrane is known to trigger cell death, the function of intracellular TRP proteins in the regulation of Ca²⁺‐dependent signalling pathways and apoptosis has remained elusive. Here, we show that TRPP2, the ion channel mutated in autosomal dominant polycystic kidney disease (ADPKD), protects cells from apoptosis by lowering the Ca²⁺ concentration in the endoplasmic reticulum (ER). ER‐resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca²⁺ ATPase by increasing the ER Ca²⁺ permeability. This results in diminished cytosolic and mitochondrial Ca²⁺ signals upon stimulation of inositol 1,4,5‐trisphosphate receptors and reduces Ca²⁺ release from the ER in response to apoptotic stimuli. Conversely, knockdown of TRPP2 in renal epithelial cells increases ER Ca²⁺ release and augments sensitivity to apoptosis. Our findings indicate an important function of ER‐resident TRPP2 in the modulation of intracellular Ca²⁺ signalling, and provide a molecular mechanism for the increased apoptosis rates in ADPKD upon loss of TRPP2 channel function.     

Redox regulation of calcium ion channels: chemical and physiological aspects

Reactive oxygen species (ROS) are increasingly recognized as second messengers in many cellular processes. While high concentrations of oxidants damage proteins, lipids and DNA, ultimately resulting in cell death, selective and reversible oxidation of key residues in proteins is a physiological mechanism that can transiently alter their activity and function. Defects in ROS producing enzymes cause disturbed immune response and disease.Changes in the intracellular free Ca²⁺ concentration are key triggers for diverse cellular functions. Ca²⁺ homeostasis thus needs to be precisely tuned by channels, pumps, transporters and cellular buffering systems. Alterations of these key regulatory proteins by reversible or irreversible oxidation alter the physiological outcome following cell stimulation. It is therefore necessary to understand which proteins are regulated and if this regulation is relevant in a physiological- and/or pathophysiological context. Because ROS are inherently difficult to identify and to measure, we first review basic oxygen redox chemistry and methods of ROS detection with special emphasis on electron paramagnetic resonance (EPR) spectroscopy. We then focus on the present knowledge of redox regulation of Ca²⁺ permeable ion channels such as voltage-gated (CaV) Ca²⁺ channels, transient receptor potential (TRP) channels and Orai channels.     

Lack of correlation between the amplitudes of TRP channel-mediated responses to weak and strong stimuli in intracellular Ca imaging experiments

It is often observed in intracellular Ca²⁺ imaging experiments that the amplitudes of the Ca²⁺ signals elicited by newly characterized TRP agonists do not correlate with the amplitudes of the responses evoked subsequently by a specific potent agonist. We investigated this rather controversial phenomenon by first testing whether it is inherent to the comparison of the effects of weak and strong stimuli. Using five well-characterized TRP channel agonists in commonly used heterologous expression systems we found that the correlation between the amplitudes of the Ca²⁺ signals triggered by two sequentially applied stimuli is only high when both stimuli are strong. Using mathematical simulations of intracellular Ca²⁺ dynamics we illustrate that the innate heterogeneity in expression and functional properties of Ca²⁺ extrusion (e.g. plasma membrane Ca²⁺ ATPase) and influx (TRP channels) pathways across a cellular population is a sufficient condition for low correlation between the amplitude of Ca²⁺ signals elicited by weak and strong stimuli. Taken together, our data demonstrate that this phenomenon is an expected outcome of intracellular Ca²⁺ imaging experiments that cannot be taken as evidence for lack of specificity of low-efficacy stimuli, or as an indicator of the need of other cellular components for channel stimulation.     

Intracellular Long-Chain Acyl CoAs Activate TRPV1 Channels

TRPV1 channels are an important class of membrane proteins that play an integral role in the regulation of intracellular cations such as calcium in many different tissue types. The anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂) is a known positive modulator of TRPV1 channels and the negatively charged phosphate groups interact with several basic amino acid residues in the proximal C-terminal TRP domain of the TRPV1 channel. We and other groups have shown that physiological sub-micromolar levels of long-chain acyl CoAs (LC-CoAs), another ubiquitous anionic lipid, can also act as positive modulators of ion channels and exchangers. Therefore, we investigated whether TRPV1 channel activity is similarly regulated by LC-CoAs. Our results show that LC-CoAs are potent activators of the TRPV1 channel and interact with the same PIP₂-binding residues in TRPV1. In contrast to PIP₂, LC-CoA modulation of TRPV1 is independent of Ca²⁺ _i, acting in an acyl side-chain saturation and chain-length dependent manner. Elevation of LC-CoAs in intact Jurkat T-cells leads to significant increases in agonist-induced Ca²⁺ _i levels. Our novel findings indicate that LC-CoAs represent a new fundamental mechanism for regulation of TRPV1 channel activity that may play a role in diverse cell types under physiological and pathophysiological conditions that alter fatty acid transport and metabolism such as obesity and diabetes.     

Regulation of CRAC channels by protein interactions and post-translational modification

Store-operated Ca²⁺ entry (SOCE) is a widespread mechanism to elevate the intracellular Ca²⁺ concentrations and stimulate downstream signaling pathways affecting proliferation, secretion, differentiation and death in different cell types. In immune cells, immune receptor stimulation induces intracellular Ca²⁺ store depletion that subsequently activates Ca²⁺-release-activated-Ca²⁺ (CRAC) channels, a prototype of store-operated Ca²⁺ (SOC) channels. CRAC channel opening leads to activation of diverse downstream signaling pathways affecting proliferation, differentiation, cytokine production and cell death. Recent identification of STIM1 as the endoplasmic reticulum Ca²⁺ sensor and Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tools to dissect the mechanism of activation and regulation of CRAC channels. In this review, we discuss the recent advances in understanding the associating partners and posttranslational modifications of Orai1 and STIM1 proteins that regulate diverse aspects of CRAC channel function.     

Plasma membrane stretch activates transient receptor potential vanilloid and ankyrin channels in Merkel cells from hamster buccal mucosa

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons. Many transient receptor potential (TRP) channels have been identified in mammals; however, the activation properties of these channels in oral mucosal MCs remain to be clarified. We investigated the biophysical and pharmacological properties of TRP vanilloid (TRPV)-1, TRPV2, TRPV4, TRP ankyrin (TRPA)-1, and TRP melastatin (TRPM)-8 channels, which are sensitive to osmotic and mechanical stimuli by measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) using fura-2. We also analyzed their localization patterns through immunofluorescence. MCs showed immunoreaction for TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 channels. In the presence of extracellular Ca²⁺, the hypotonic test solution evoked Ca²⁺ influx. The [Ca²⁺]_i increases were inhibited by TRPV1, TRPV2, TRPV4, or TRPA1 channel antagonists, but not by the TRPM8 channel antagonist. Application of TRPV1, TRPV2, TRPV4, TRPA1, or TRPM8 channel selective agonists elicited transient increases in [Ca²⁺]_i only in the presence of extracellular Ca²⁺. The results indicate that membrane stretching in MCs activates TRPV1, TRPV2, TRPV4, and TRPA1 channels, that it may be involved in synaptic transmission to sensory neurons, and that MCs could contribute to the mechanosensory transduction sequence.     

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca²⁺ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca²⁺ imaging, we found that the depletion of ER/SR Ca²⁺ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca²⁺ ([Ca²⁺]_i), followed by sustained increase depending on extracellular Ca²⁺. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na⁺/Ca²⁺ exchanger inhibitors, inhibited [Ca²⁺]_i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl₃) or by an increased extracellular Ca²⁺([Ca²⁺]_o) increased the concentration of intracelluar Ca²⁺, whereas, the sustained elevation of [Ca²⁺]_i was reduced in the presence of SKF96365. Similarly, the duration of [Ca²⁺]_i increase was also shortened in the absence of extracellular Ca²⁺. Western blot analysis showed that GdCl₃ increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl₃. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca²⁺-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.     

Transient receptor potential (TRP) channel function in the reproductive axis

Transient receptor potential (TRP) channels play important functional roles in the signal transduction machinery of hormone-secreting cells and have recently been implicated in reproductive physiology. While expression studies have demonstrated TRP channel expression at all levels of the hypothalamic–pituitary–gonadal (hpg) axis, functional details about TRP channel action at the level of the individual cells controlling reproduction are just beginning to emerge. Canonical TRP (TRPC) channels are prominently expressed in the reproductive center of the neuroendocrine brain, i.e. in kisspeptin and gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin neurons are depolarized by leptin via activation of TRPC channels and kisspeptin depolarizes GnRH neurons through TRPC4 activation. Recent studies have functionally identified TRPC channels also in gonadotrope cells in the anterior pituitary gland, which secrete gonadotropins in response to GnRH and thus regulate gonadal function. TRP channel expression in these cells exhibits remarkable plasticity and depends on the hormonal status of the animal. Subsequent functional analyses have demonstrated that TRPC5 in gonadotropes contributes to depolarization of the plasma membrane upon GnRH stimulation and increases the intracellular Ca²⁺ concentration via its own Ca²⁺ permeability and via the activation of voltage-gated Ca²⁺ channels. However, conditional gene targeting experiments will be needed to unambiguously dissect the physiological role of TRPC channels in the different cell types of the reproductive axis in vivo.     

Functional organization of TRPC-Ca channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1–TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca²⁺ influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP₂) hydrolysis, generation of IP₃ and DAG, and IP₃-induced Ca²⁺ release from the intracellular Ca²⁺ store via inositol trisphosphate receptor (IP₃R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca²⁺ entry mechanisms. The former is regulated by the emptying/refilling of internal Ca²⁺ store(s) while the latter depends on PIP₂ hydrolysis (due to changes in PIP₂ per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca²⁺ entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca²⁺]_i signals which are critical for precise control of downstream cellular functions.     

Magnetic field-induced Ca2+ intake by mesenchymal stem cells is mediated by intracellular Zn2+ and accompanied by a Zn2+ influx

Chronic exposure to magnetic fields (MFs) has a diverse range of effects on biological systems but definitive molecular mechanisms of the interaction remain largely unknown. One of the most frequently reported effects of MF exposure is an elevated concentration of intracellular Ca²⁺ through disputed pathways. Other prominent effects include increased oxidative stress and upregulation of neural markers through EGFR activation in stem cells. Further characterization of cascades triggered by MF exposure is hindered by the phenotype diversity of biological models used in the literature. In an attempt to reveal more mechanistic data in this field, we combined the most commonly used biological model and MF parameters with the most commonly reported effects of MFs.Based on clues from the pathways previously defined as sensitive to MFs (EGFR and Zn²⁺-binding enzymes), the roles of different types of channels (voltage gated Ca²⁺ channels, NMDA receptors, TRP channels) were inquired in the effects of 50 Hz MFs on bone marrow-derived mesenchymal stem cells. We report that, an influx of Zn²⁺ accompanies MF-induced Ca²⁺ intake, which is only attenuated by the broad-range inhibitor of TRP channels and store-operated Ca²⁺ entry (SOCE), 2-Aminoethoxydiphenyl borate (2-APB) among other blockers (memantine, nifedipine, ethosuximide and gabapentin). Interestingly, cation influx completely disappears when intracellular Zn²⁺ is chelated. Our results rule out voltage gated Ca²⁺ channels as a gateway to MF-induced Ca²⁺ intake and suggest Zn²⁺-related channels as a new focus in the field.     

Lack of TRPV2 impairs thermogenesis in mouse brown adipose tissue

Brown adipose tissue (BAT), a major site for mammalian non‐shivering thermogenesis, could be a target for prevention and treatment of human obesity. Transient receptor potential vanilloid 2 (TRPV2), a Ca²⁺‐permeable non‐selective cation channel, plays vital roles in the regulation of various cellular functions. Here, we show that TRPV2 is expressed in brown adipocytes and that mRNA levels of thermogenic genes are reduced in both cultured brown adipocytes and BAT from TRPV2 knockout (TRPV2KO) mice. The induction of thermogenic genes in response to β‐adrenergic receptor stimulation is also decreased in TRPV2KO brown adipocytes and suppressed by reduced intracellular Ca²⁺ concentrations in wild‐type brown adipocytes. In addition, TRPV2KO mice have more white adipose tissue and larger brown adipocytes and show cold intolerance, and lower BAT temperature increases in response to β‐adrenergic receptor stimulation. Furthermore, TRPV2KO mice have increased body weight and fat upon high‐fat‐diet treatment. Based on these findings, we conclude that TRPV2 has a role in BAT thermogenesis and could be a target for human obesity therapy.