TRP channels in lymphocytes

TRP proteins form ion channels that are activated following receptor stimulation. Several members of the TRP family are likely to be expressed in lymphocytes. However, in many studies, messenger RNA (mRNA) but not protein expression was analyzed and cell lines but not primary human or murine lymphocytes were used. Among the expressed TRP mRNAs are TRPC1, TRPC3, TRPM2, TRPM4, TRPM7, TRPV1, and TRPV2. Regulation of Ca2+ entry is a key process for lymphocyte activation, and TRP channels may both increase Ca2+ influx (such as TRPC3) or decrease Ca2+ influx through membrane depolarization (such as TRPM4). In the future, linking endogenous Ca2+/cation channels in lymphocytes with TRP proteins should lead to a better molecular understanding of lymphocyte activation.     

Transient receptor potential (TRP) channels, vascular tone and autoregulation of cerebral blood flow

Members of the transient receptor potential (TRP) channel superfamily are present in vascular smooth muscle cells and play important roles in the regulation of vascular contractility. The TRPC3 and TRPC6 channels are activated by stimulation of several excitatory receptors in vascular smooth muscle cells. Activation of these channels leads to myocyte depolarization, which stimulates Ca2+ entry via voltage-dependent Ca2+ channels (VDCC), leading to vasoconstriction. The TRPV4 channels in arterial myocytes are activated by epoxyeicosatrienoic acids, and activation of the channels enhances Ca2+ spark and transient Ca2+-sensitive K+ channel activity, thereby hyperpolarizing and relaxing vascular smooth muscle cells. The TRPC6 and TRPM4 channels are activated by mechanical stimulation of cerebral artery myocytes. Subsequent depolarization and activation of VDCC Ca2+ entry is directly linked to the development of myogenic tone in vitro and to autoregulation of cerebral blood flow in vivo. These findings imply a fundamental importance of TRP channels in the regulation of vascular smooth muscle tone and suggest that TRP channels could be important targets for drug therapy under conditions in which vascular contractility is disturbed (e.g. hypertension, stroke, vasospasm).     

TRP channels in normal and dystrophic skeletal muscle

TRP proteins constitute non-selective cation-permeable ion channels, most of which are permeable to Ca2?. In skeletal muscle, several isoforms of the TRPC (Canonical), TRPV (Vanilloid) and TRPM (Melastatin) subfamilies are expressed. In particular, TRPC1, C3 and C6, TRPV2 and V4, TRPM4 and TRPM7 have been consistently found in cultured myoblasts or in adult muscles. These channels seem to directly or indirectly respond to membrane stretch or to Ca2? stores depletion; some isoforms might also constitute unregulated Ca2? leak channels. Their function is largely unknown. TRPC1 and C3 have been involved in muscle development, in particular in myoblasts migration and differentiation. TRPC1 and V4 might allow a basal influx of Ca2? at rest. Their lack has consequences on muscle fatigue. TRPV2 seems to be stretch-sensitive. It localizes mainly in intracellular pools at rest, and translocates to the plasma membrane upon IGF-1 stimulation. TRP channels seem to be involved in the pathophysiology of muscle disorders. In particular in Duchenne muscular dystrophy, the lack of the cytoskeletal protein dystrophin induces a disregulation of several ion channels leading to an abnormal influx of Ca2?. We discuss here, the possible involvement of TRP channels in this abnormal influx of Ca2?.     

Activation of the melastatin-related cation channel TRPM3 by D-erythro-sphingosine [corrected

TRPM3, a member of the melastatin-like transient receptor potential channel subfamily (TRPM), is predominantly expressed in human kidney and brain. TRPM3 mediates spontaneous Ca2+ entry and nonselective cation currents in transiently transfected human embryonic kidney 293 cells. Using measurements with the Ca2+-sensitive fluorescent dye fura-2 and the whole-cell patch-clamp technique, we found that D-erythro-sphingosine, a metabolite arising during the de novo synthesis of cellular sphingolipids, activated TRPM3. Other transient receptor potential (TRP) channels tested [classic or canonical TRP (TRPC3, TRPC4, TRPC5), vanilloid-like TRP (TRPV4, TRPV5, TRPV6), and melastatin-like TRP (TRPM2)] did not significantly respond to application of sphingosine. Sphingosine-induced TRPM3 activation was not mediated by inhibition of protein kinase C, depletion of intracellular Ca2+ stores, and intracellular conversion of sphingosine to sphingosine-1-phosphate. Although sphingosine-1-phosphate and ceramides had no effect, two structural analogs of sphingosine, dihydro-D-erythro-sphingosine and N,N-dimethyl-D-erythro-sphingosine, also activated TRPM3. Sphingolipids, including sphingosine, are known to have inhibitory effects on a variety of ion channels. Thus, TRPM3 is the first ion channel activated by sphingolipids.     

Role of Ca2+ signaling in the regulation of endothelial permeability

The vascular endothelial cell forms a semipermeable barrier between blood and interstitium. Inflammatory mediators such as thrombin and histamine induce vascular leakage defined as increased endothelial permeability to plasma proteins and other solutes. Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein-coupled receptors (GPCR) trigger increased endothelial permeability by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) activates key signaling pathways, which mediate cytoskeletal reorganization (through myosin light chain (MLC)-dependent contraction) and disassembly of VE-cadherin at the adherens junctions. The Ca(2+)-dependent protein kinase C (PKC) isoform, PKC-alpha, plays a critical role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions. The increase in [Ca(2+)](i) induced by a variety of agonists is achieved by the generation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3R), release of stored intracellular Ca(2+), and Ca(2+) entry through plasma membrane channels. Recent findings demonstrate that IP3-sensitive Ca(2+) store depletion activates plasma membrane cation channels (i.e., store-operated cation channels (SOC) or Ca(2+) release activated channels) to cause Ca(2+) influx in endothelial cells. This mode of Ca(2+) influx is also known as capacitative Ca(2+) entry (CCE). Store-operated Ca(2+) influx signals increase in permeability and nitric oxide (NO) production and provokes changes in gene expression in endothelial cells. Recent studies have established that the Drosophila transient receptor potential (TRP) gene family of channels expressed in endothelial cells can function as SOC. Deletion of one of the TRP homologues, TRPC4, in mouse caused impairment in store-operated Ca(2+) current and Ca(2+) store release activated Ca(2+) influx in aortic and lung endothelial cells (LEC). In TRPC4 knockout (TRPC4(-/-)) mice, acetylcholine-induced endothelium-dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4(-/-) mice LEC exhibited lack of actin stress fiber formation and cell retraction in response to thrombin activation of proteinase-activated receptor-1 (PAR-1) in endothelial cells. The increase in lung microvascular permeability in response to thrombin receptor activation was inhibited in TRPC4(-/-) mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling the increase in endothelial permeability.     

TRP channels in platelet function

Ca2+ entry forms an essential component of platelet activation; however, the mechanisms associated with this process are not understood. Ca2+ entry upon receptor activation occurs as a consequence of intracellular store depletion (referred to as store-operated Ca2+ entry or SOCE), a direct action of second messengers on cation entry channels or the direct occupancy of a ligand-gated P2(Xi) receptor. The molecular identity of the SOCE channel has yet to be established. Transient receptor potential (TRP) proteins are candidate cation entry channels and are classified into a number of closely related subfamilies including TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin) and TRPML (mucolipins). From the TRPC family, platelets have been shown to express TRPC6 and TRPC1, and are likely to express other TRPC and other TRP members. TRPC6 is suggested to be involved with receptor-activated, diacyl-glycerol-mediated cation entry. TRPC1 has been suggested to be involved with SOCE, though many of the suggested mechanisms remain controversial. As no single TRP channel has the properties described for SOCE in platelets, it is likely that it is composed of a heteromeric association of TRP and related subunits, some of which may be present in intracellular compartments in the resting cell.     

Stereo-selective inhibition of transient receptor potential TRPC5 cation channels by neuroactive steroids

BACKGROUND AND PURPOSE

Transient receptor potential canonical 5 (TRPC5) channels are widely expressed, including in the CNS, where they potentiate fear responses. They also contribute to other non-selective cation channels that are stimulated by G-protein-coupled receptor agonists and lipid and redox factors. Steroids are known to modulate fear and anxiety states, and we therefore investigated whether TRPC5 exhibited sensitivity to steroids.

EXPERIMENTAL APPROACH

Human TRPC5 channels were conditionally expressed in HEK293 cells and studied using intracellular Ca²⁺ measurement, whole-cell voltage-clamp and excised patch techniques. For comparison, control experiments were performed with cells lacking TRPC5 channels or expressing another TRP channel, TRPM2. Native TRPC channel activity was recorded from vascular smooth muscle cells.

KEY RESULTS

Extracellular application of pregnenolone sulphate, pregnanolone sulphate, pregnanolone, progesterone or dihydrotestosterone inhibited TRPC5 activity within 1–2 min. Dehydroepiandrosterone sulphate or 17β-oestradiol had weak inhibitory effects. Pregnenolone, and allopregnanolone, a progesterone metabolite and stereo-isomer of pregnanolone, all had no effects. Progesterone was the most potent of the steroids, especially against TRPC5 channel activity evoked by sphingosine-1-phosphate. In outside-out patch recordings, bath-applied progesterone and dihydrotestosterone had strong and reversible effects, suggesting relatively direct mechanisms of action. Progesterone inhibited native TRPC5-containing channel activity, evoked by oxidized phospholipid.

CONCLUSIONS AND IMPLICATIONS

Our data suggest that TRPC5 channels are susceptible to relatively direct and rapid stereo-selective steroid modulation, leading to channel inhibition. The study adds to growing appreciation of TRP channels as non-genomic steroid sensors.     

Block of TRPC5 channels by 2-aminoethoxydiphenyl borate: a differential, extracellular and voltage-dependent effect

1 2-aminoethoxydiphenyl borate (2-APB) has been widely used to examine the roles of inositol 1,4,5-trisphosphate receptors (IP3Rs) and store-operated Ca2+ entry and is an emerging modulator of cationic channels encoded by transient receptor potential (TRP) genes. 2 Using Ca2+-indicator dye and patch-clamp recording we first examined the blocking effect of 2-APB on human TRPC5 channels expressed in HEK-293 cells. 3 The concentration-response curve has an IC50 of 20 microM and slope close to 1.0, suggesting one 2-APB molecule binds per channel. The blocking effect is not shared by other Ca2+ channel blockers including methoxyverapamil, nifedipine, N-propargylnitrendipine, or berberine. 4 In whole-cell and excised membrane patch recordings, 2-APB acts from the extracellular but not intracellular face of the membrane. 5 Block of TRPC5 by 2-APB is less at positive voltages, suggesting that it enters the electric field or acts by modulating channel gating. 6 2-APB also blocks TRPC6 and TRPM3 expressed in HEK-293 cells, but not TRPM2. 7 Block of TRP channels by 2-APB may be relevant to cell proliferation because 2-APB has a greater inhibitory effect on proliferation in cells overexpressing TRPC5. 8 Our data indicate a specific and functionally important binding site on TRPC5 that enables block by 2-APB. The site is only available via an extracellular route and the block shows mild voltage-dependence.     

TRP channels in neurogastroenterology: opportunities for therapeutic intervention

The members of the superfamily of transient receptor potential (TRP) cation channels are involved in a plethora of cellular functions. During the last decade, a vast amount of evidence is accumulating that attributes an important role to these cation channels in different regulatory aspects of the alimentary tract. In this review we discuss the expression patterns and roles of TRP channels in the regulation of gastrointestinal motility, enteric nervous system signalling and visceral sensation, and provide our perspectives on pharmacological targeting of TRPs as a strategy to treat various gastrointestinal disorders. We found that the current knowledge about the role of some members of the TRP superfamily in neurogastroenterology is rather limited, whereas the function of other TRP channels, especially of those implicated in smooth muscle cell contractility (TRPC4, TRPC6), visceral sensitivity and hypersensitivity (TRPV1, TRPV4, TRPA1), tends to be well established. Compared with expression data, mechanistic information about TRP channels in intestinal pacemaking (TRPC4, TRPC6, TRPM7), enteric nervous system signalling (TRPCs) and enteroendocrine cells (TRPM5) is lacking. It is clear that several different TRP channels play important roles in the cellular apparatus that controls gastrointestinal function. They are involved in the regulation of gastrointestinal motility and absorption, visceral sensation and visceral hypersensitivity. TRP channels can be considered as interesting targets to tackle digestive diseases, motility disorders and visceral pain. At present, TRPV1 antagonists are under development for the treatment of heartburn and visceral hypersensitivity, but interference with other TRP channels is also tempting. However, their role in gastrointestinal pathophysiology first needs to be further elucidated.     

New target molecules in the drug control of blood pressure and circulation

Ion channels play a pivotal role in blood pressure regulation. Amongst them, much attention has been directed to dihydropyridine (DHP)-sensitive (L-type) voltage-dependent Ca(2+) channels (VDCCs) and iberiotoxin-sensitive Ca(2+)-dependent K(+) channels which are distributed over the whole vascular tree and contribute to vascular tone regulation. Recent advances in vascular electrophysiology have, however, added novel and interesting molecules to this repertoire. In small mesenteric arterioles, the predominant VDCC phenotype is not L-type but DHP-insensitive, high voltage-activated VDCCs that exhibit unique properties distinguishable from those of hitherto-known VDCCs. Surprisingly, mibefradil, a well-known T-type selective blocker potently inhibits these channels, and the use of this blocker has indicated that Ca(2+) entry through these channels may be one of the important determinants of peripheral vascular tone. Another new candidate likely involved in blood pressure control is the mammalian homologue of Drosophila transient receptor potential (TRP) protein, including TRPC4 and TRPC6. Experiments in genetically engineered TRPC4-deficient mice have suggested that expression of TRPC4 is indispensable for agonist-induced Ca(2+) entry in endothelial cells and production of nitric oxide and vasorelaxation. TRPC6 is likely to contribute to sustained Ca(2+) entry into vascular smooth muscle cells activated by stimulation of sympathetic nerves and elevation of intravascular pressure. Antisense oligonucleotide experiments have suggested that this protein is an essential component of alpha1-adrenoceptor activated and mechanosensitive cation channels in some vascular tissues. This review overviews what is known about the role of ionic channels in blood pressure control with main focus on the above-mentioned new molecules as promising targets for drug discovery and development.     

血管平滑肌钾通道及其调节因素

血管张力是决定血管阻力和血流量的重要因素 ,而改变钾通道活性能直接影响血管张力。钾通道开放引起钾外流 ,细胞膜超极化 ,关闭电压依赖性钙通道 ,钙内流减少 ,血管舒张 ;当钾通道受抑制时 ,可使细胞膜去极化 ,从而使电压依赖的钙通道开放 ,细胞外钙内流 ,钙离子使肌球蛋白轻链磷酸化 ,粗细肌丝发生相对运动 ,血管收缩。本文介绍血管平滑肌上 4种钾通道的基因结构、电生理学与药理学特性     

Regulation of TRP ion channels by phosphatidylinositol-4,5-bisphosphate

Phosphatidylinositol-4,5-bisphosphate (PIP2) has emerged as a versatile regulator of TRP ion channels. In many cases, the regulation involves interactions of channel proteins with the lipid itself independent of its hydrolysis products. The functions of the regulation mediated by such interactions are diverse. Some TRP channels absolutely require PIP2 for functioning, while others are inhibited. A change of gating is common to all, endowing the lipid a role for modulation of the sensitivity of the channels to their physiological stimuli. The activation of TRP channels may also influence cellular PIP2 levels via the influx of Ca2+ through these channels. Depletion of PIP2 in the plasma membrane occurs upon activation of TRPV1, TRPM8, and possibly TRPM4/5 in heterologous expression systems, whereas resynthesis of PIP2 requires Ca2+ entry through the TRP/TRPL channels in Drosophila photoreceptors. These developments concerning PIP2 regulation of TRP channels reinforce the significance of the PLC signaling cascade in TRP channel function, and provide further perspectives for understanding the physiological roles of these ubiquitous and often enigmatic channels.     

TRP Channels in Respiratory Pathophysiology: The Role of Oxidative,Chemical Irritant and Temperature Stimuli

There is rapidly growing evidence indicating multiple and important roles of Ca²⁺-permeable cation TRP channels in the airways, both under normal and disease conditions. The aim of this review was to summarize the current knowledge of TRP channels in sensing oxidative, chemical irritant and temperature stimuli by discussing expression and function of several TRP channels in relevant cell types within the respiratory tract, ranging from sensory neurons to airway smooth muscle and epithelial cells. Several of these channels, such as TRPM2, TRPM8, TRPA1 and TRPV1, are discussed in much detail to show that they perform diverse, and often overlapping or contributory, roles in airway hyperreactivity, inflammation, asthma, chronic obstructive pulmonary disease and other respiratory disorders. These include TRPM2 involvement in the disruption of the bronchial epithelial tight junctions during oxidative stress, important roles of TRPA1 and TRPV1 channels in airway inflammation, hyperresponsiveness, chronic cough, and hyperplasia of airway smooth muscles, as well as TRPM8 role in COPD and mucus hypersecretion. Thus, there is increasing evidence that TRP channels not only function as an integral part of the important endogenous protective mechanisms of the respiratory tract capable of detecting and ensuring proper physiological responses to various oxidative, chemical irritant and temperature stimuli, but that altered expression, activation and regulation of these channels may also contribute to the pathogenesis of respiratory diseases.     

Newly emerging Ca2+ entry channel molecules that regulate the vascular tone

Local blood flow is critically determined by the arterial tone in which sustained Ca(2+) influx, activated by a variety of mechanisms, plays a central regulatory role. Recent progress in molecular biological research has disclosed unexpectedly diverse and complex facets of Ca(2+) entry channel molecules involved in this Ca(2+) influx. Candidates include several transient receptor potential (TRP) superfamily members such as TRPC1, TRPC4, TRPC6, TRPV2, TRPV4 and TRPM4, none of which exhibit simple properties attributable to a single particular role. Rather, they appear to be multimodally activated or modulated by receptor stimulation, temperature, mechanical stress or lipid second messengers generated from various sources, and may be involved in both acute vasomotor control and long-term vascular remodelling. This paper provides an overview of existing knowledge of TRP proteins, and their possible relationships with principal factors regulating the arterial tone (i.e., autonomic nerves, various autocrine and paracrine factors, and intravascular pressure).     

TRPC channels: interacting proteins

TRP channels, in particular the TRPC and TRPV subfamilies, have emerged as important constituents of the receptor-activated Ca2+ influx mechanism triggered by hormones, growth factors, and neurotransmitters through activation ofphospholipase C (PLC). Several TRPC channels are also activated by passive depletion of endoplasmic reticulum (ER) Ca2+. Although in several studies the native TRP channels faithfully reproduce the respective recombinant channels, more often the properties of Ca2+ entry and/or the store-operated current are strikingly different from that of the TRP channels expressed in the same cells. The present review aims to discuss this disparity in the context of interaction of TRPC channels with auxiliary proteins that may alter the permeation and regulation of TRPC channels.     

TRPC6

TRPC6 is a Ca(2+)-permeable non-selective cation channel expressed in brain, smooth muscle containing tissues and kidney, as well as in immune and blood cells. Channel homomers heterologously expressed have a characteristic doubly rectifying current-voltage relationship and are six times more permeable for Ca2+ than for Na+. In smooth muscle tissues, however, Na+ influx and activation of voltage-gated calcium channels by membrane depolarization rather than Ca2+ elevation by TRPC6 channels is the driving force for contraction. TRPC6 channels are directly activated by the second messenger diacylglycerol (DAG) and regulated by specific tyrosine or serine phosphorylation. Extracellular Ca2+ has inhibitory effects, while Ca2+/calmodulin acting from the intracellular side has potentiator effects on channel activity. Given its specific expression, TRPC6 is likely to play a number of physiological roles. Studies with TRPC6(-/-) mice suggest a role for the channel in the regulation of vascular and pulmonary smooth muscle contraction. TRPC6 was identified as an essential component of the slit diaphragm architecture of kidney podocytes. Other functions in immune and blood cells, as well as in brain and in smooth muscle-containing tissues such as stomach, colon and myometrium, remain elusive.     

The TRPM ion channel subfamily: molecular, biophysical and functional features

Significant progress in the molecular and functional characterization of a subfamily of genes that encode melastatin-related transient receptor potential (TRPM) cation channels has been made during the past few years. This subgroup of the TRP superfamily of ion channels contains eight mammalian members and has isoforms in most eukaryotic organisms. The individual members of the TRPM subfamily have specific expression patterns and ion selectivity, and their specific gating and regulatory mechanisms are tailored to integrate multiple signaling pathways. The diverse functional properties of these channels have a profound effect on the regulation of ion homoeostasis by mediating direct influx of Ca2+, controlling Mg2+ entry, and determining the potential of the cell membrane. TRPM channels are involved in several physiological and pathological conditions in electrically excitable and non-excitable cells, which make them exciting targets for drug discovery.     

An introduction on TRP channels

The transient receptor potential (TRP) ion channels are named after the role of the channels in Drosophila phototransduction. Mammalian TRP channel subunit proteins are encoded by at least 28 genes. TRP cation channels display an extraordinary assortment of selectivities and activation mechanisms, some of which represent previously unrecognized modes of regulating ion channels. In addition, the biological roles of TRP channels appear to be equally diverse and range from roles in thermosensation and pain perception to Ca2+ and Mg2+ absorption, endothelial permeability, smooth muscle proliferation and gender-specific behaviour.     

Psychiatric Disorders and TRP Channels: Focus on Psychotropic Drugs

Psychiatric and neurological disorders are mostly associated with the changes in neural calcium ion signaling pathways required for activity-triggered cellular events. One calcium channel family is the TRP cation channel family, which contains seven subfamilies. Results of recent papers have discovered that calcium ion influx through TRP channels is important. We discuss the latest advances in calcium ion influx through TRP channels in the etiology of psychiatric disorders.Activation of TRPC4, TRPC5, and TRPV1 cation channels in the etiology of psychiatric disorders such as anxiety, fear-associated responses, and depression modulate calcium ion influx. Evidence substantiates that anandamide and its analog (methanandamide) induce an anxiolytic-like effect via CB1 receptors and TRPV1 channels. Intracellular calcium influx induced by oxidative stress has an significant role in the etiology of bipolar disorders (BDs), and studies recently reported the important role of TRP channels such as TRPC3, TRPM2, and TRPV1 in converting oxidant or nitrogen radical signaling to cytosolic calcium ion homeostasis in BDs. The TRPV1 channel also plays a function in morphine tolerance and hyperalgesia. Among psychotropic drugs, amitriptyline and capsazepine seem to have protective effects on psychiatric disorders via the TRP channels. Some drugs such as cocaine and methamphetamine also seem to have an important role in alcohol addiction and substance abuse via activation of the TRPV1 channel.Thus, we explore the relationships between the etiology of psychiatric disorders and TRP channel-regulated mechanisms. Investigation of the TRP channels in psychiatric disorders holds the promise of the development of new drug treatments.