Transient Receptor Potential Channel Subfamily Research Articles

Transient Receptor Potential Channels in Microglia: Roles in Physiology and Disease.

Microglia modulate the nervous system cellular environment and induce neuroprotective and neurotoxic effects. Various molecules are involved in these processes, including families of ion channels expressed in microglial cells, such as transient receptor potential (TRP) channels. TRP channels comprise a family of non-selective cation channels that can be activated by mechanical, thermal, and chemical stimuli, and which contribute to the regulation of intracellular calcium concentrations. TRP channels have been shown to be involved in cellular processes such as osmotic regulation, cytokine production, proliferation, activation, cell death, and oxidative stress responses. Given the significance of these processes in microglial activity, studies of TRP channels in microglia have focused on determining their roles in both neuroprotective and neurotoxic processes. TRP channel activity has been proposed to play an important function in neurodegenerative diseases, ischemia, inflammatory responses, and neuropathic pain. Modulation of TRP channel activity may thus be considered as a potential therapeutic strategy for the treatment of various diseases associated with alterations of the central nervous system (CNS). In this review, we describe the expression of different subfamilies of TRP channels in microglia, focusing on their physiological and pathophysiological roles, and consider their potential use as therapeutic targets in CNS diseases.

Evidence for Novel Pharmacological Sensitivities of Transient Receptor Potential (TRP) Channels in Schistosoma mansoni.

Schistosomiasis, caused by parasitic flatworms of the genus Schistosoma, is a neglected tropical disease affecting hundreds of millions globally. Praziquantel (PZQ), the only drug currently available for treatment and control, is largely ineffective against juvenile worms, and reports of PZQ resistance lend added urgency to the need for development of new therapeutics. Ion channels, which underlie electrical excitability in cells, are validated targets for many current anthelmintics. Transient receptor potential (TRP) channels are a large family of non-selective cation channels. TRP channels play key roles in sensory transduction and other critical functions, yet the properties of these channels have remained essentially unexplored in parasitic helminths. TRP channels fall into several (7–8) subfamilies, including TRPA and TRPV. Though schistosomes contain genes predicted to encode representatives of most of the TRP channel subfamilies, they do not appear to have genes for any TRPV channels. Nonetheless, we find that the TRPV1-selective activators capsaicin and resiniferatoxin (RTX) induce dramatic hyperactivity in adult worms; capsaicin also increases motility in schistosomula. SB 366719, a highly-selective TRPV1 antagonist, blocks the capsaicin-induced hyperactivity in adults. Mammalian TRPA1 is not activated by capsaicin, yet knockdown of the single predicted TRPA1-like gene (SmTRPA) in S. mansoni effectively abolishes capsaicin-induced responses in adult worms, suggesting that SmTRPA is required for capsaicin sensitivity in these parasites. Based on these results, we hypothesize that some schistosome TRP channels have novel pharmacological sensitivities that can be targeted to disrupt normal parasite neuromuscular function. These results also have implications for understanding the phylogeny of metazoan TRP channels and may help identify novel targets for new or repurposed therapeutics.

A Binary Genetic Approach to Characterize TRPM5 Cells in Mice.

Transient receptor potential channel subfamily M member 5 (TRPM5) is an important downstream signaling component in a subset of taste receptor cells making it a potential target for taste modulation. Interestingly, TRPM5 has been detected in extra-oral tissues; however, the function of extra-gustatory TRPM5-expressing cells is less well understood. To facilitate visualization and manipulation of TRPM5-expressing cells in mice, we generated a Cre knock-in TRPM5 allele by homologous recombination. We then used the novel TRPM5-IRES-Cre mouse strain to report TRPM5 expression by activating a τGFP transgene. To confirm faithful coexpression of τGFP and TRPM5 we generated and validated a new anti-TRPM5 antiserum enabling us to analyze acute TRPM5 protein expression. τGFP cells were found in taste bud cells of the vallate, foliate, and fungiform papillae as well as in the palate. We also detected TRPM5 expression in several other tissues such as in the septal organ of Masera. Interestingly, in the olfactory epithelium of adult mice acute TRPM5 expression was detected in only one (short microvillar cells) of two cell populations previously reported to express TRPM5. The TRPM5-IC mouse strain described here represents a novel genetic tool and will facilitate the study and tissue-specific manipulation of TRPM5-expressing cells in vivo.

Increased TRPM6 expression in atrial fibrillation patients contribute to atrial fibrosis

BackgroundTransient receptor potential (TRP) family plays important roles in cardiovascular system. We investigated the relationship between transient receptor potential channel subfamily M6 (TRPM6) and atrial fibrosis in rheumatic heart disease patients with atrial fibrillation (AF). MethodsThe right atrial tissue samples were obtained from 64 patients with rheumatic heart diseases who underwent heart valve replacement surgery, and composed of 34 sinus rhythm (SR) patients and 30 AF patients. Hematoxylin and Eosin (HE) staining was used to observe cross-sectional area (CSA) of myocardial cell. Masson staining and measurement of connective tissue growth factor (CTGF), transforming growth factor beta 1 (TGF-β 1), and collagen type I/III (Collagen I/III) were performed to determine atrial fibrosis. The mRNA and protein levels of TRPM6 were detected by real-time quantitative polymerase chain reaction (RT-PCR) and western blotting, respectively. ResultsMarked increases were observed in CSA of myocardial cell and myocardial collagen volume fraction in AF group compared with the SR group (all P<0.05). The mRNA levels of myocardial fibrosis markers (CTGF, TGF-beta 1, Collagen I/III) in AF group increased significantly compared to the SR group (all P<0.05). TRPM6 mRNA and protein levels in AF group were elevated markedly in comparison with SR group (P<0.01). ConclusionThese findings revealed that increased TRPM6 mRNA and protein levels may contribute to atrial fibrosis, and suggested that TRPM6 might be involved in AF development by promoting fibrogenesis.

Transient Receptor Potential Cation Channel Subfamily Vanilloid Member 3 is not Involved in Plasma Membrane Stretch-induced Intracellular Calcium Signaling in Merkel Cells.

Merkel cells (MCs), which form part of the MC-neurite complex, making contact with sensory afferents to drive mechanosensory transduction mechanisms, express transient receptor potential (TRP) cation channel subfamily vanilloid (V) members 1, 2, and 4, as well as ankyrin subfamily member 1. While these proteins are involved in sensing plasma membrane stretch, less is known about the functional properties of TRPV subfamily member 3 (TRPV3) during membrane stretch in MCs. The aim of this study was to determine whether TRPV3 channels were involved in mechanosensory activity by measuring intracellular free Ca(2+) concentrations ([Ca(2+)]i) in MCs isolated from hamster buccal mucosa. Application of a hypotonic extracellular solution to quinacrine-positive MCs elicited a transient increase in [Ca(2+)]i. When TRPV3 channel antagonist 2,2-diphenyltetrahydrofuran was added to the hypotonic extracellular solution, however, no effect was observed on hypotonic stimulation-induced increase in [Ca(2+)]i. These results suggest that TRPV3 channels are not involved in the mechanosensory mechanism during membrane stretch in MCs.

Multimodal use of calcitonin gene-related peptide and substance P in itch and acute pain uncovered by the elimination of vesicular glutamate transporter 2 from transient receptor potential cation channel subfamily V member 1 neurons.

Primary afferents are known to use glutamate as their principal fast neurotransmitter. However, it has become increasingly clear that peptides have an influential role in both mediating and modulating sensory transmission. Here we describe the transmission accounting for different acute pain states and itch transmitted via the transient receptor potential cation channel subfamily V member 1 (TRPV1) population by either ablating Trpv1-Cre-expressing neurons or inducing vesicular glutamate transporter 2 (VGLUT2) deficiency in Trpv1-Cre-expressing neurons. Furthermore, by pharmacological inhibition of substance P or calcitonin gene-related peptide (CGRP) signaling in Vglut2-deficient mice, we evaluated the contribution of substance P or CGRP to these sensory modulations, with or without the presence of VGLUT2-mediated glutamatergic transmission in Trpv1-Cre neurons. This examination, together with c-Fos analyses, showed that glutamate via VGLUT2 in the Trpv1-Cre population together with substance P mediate acute cold pain, whereas glutamate together with CGRP mediate noxious heat. Moreover, we demonstrate that glutamate together with both substance P and CGRP mediate tissue-injury associated pain. We further show that itch, regulated by the VGLUT2-mediated transmission via the Trpv1-Cre population, depends on CGRP and gastrin-releasing peptide receptor (GRPR) transmission because pharmacological blockade of the CGRP or GRPR pathway, or genetic ablation of Grpr, led to a drastically attenuated itch. Our study reveals how different neurotransmitters combined can cooperate with each other to transmit or regulate various acute sensations, including itch.

The development of automated patch clamp assays for canonical transient receptor potential channels TRPC3, 6, and 7.

The canonical transient receptor potential channel subfamily (TRPC3, TRPC6, and TRPC7) contains Ca(2+) permeable non-selective cation channels that are widely expressed in a variety of tissues. There is increasing evidence implicating TRPC channels, particularly TRPC3 and 6, in physiological and pathophysiological processes, eliciting interest in these channels as novel drug targets. Electrophysiology remains a benchmark technique for measuring ion channel function and accurately determining the pharmacological effects of compounds. In this report we describe the development of TRPC inhibitor assays on 2 automated planar patch clamp platforms-the IonWorks(®) Quattro™ and QPatch(®) systems. To enable activation of TRPC channels by carbachol, Chinese Hamster Ovary-K1 cells stably expressing the muscarinic M3 receptor were transduced with human TRPC3, TRPC6, or TRPC7 using BacMam viruses. TRPC3, 6, and 7 currents could be recorded on both platforms. However, the design of each platform limits which assay parameters can be recorded. Due to its continuous recording capabilities, the QPatch can capture both the activation and decay of the response. However, the transient nature of TRPC channels, the inability to reactivate and the large variation in peak currents limits the ability to develop assays for compound screening. The IonWorks Quattro, due to its discontinuous sampling, did not fully capture the peak of TRPC currents. However, due to the ability of the IonWorks Quattro to record from 64 cells per well, the variation from well to well was sufficiently reduced allowing for the development of medium-throughput screening assays.

Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in patients with severe asthma

The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of TRPV1 on airway epithelium has yet to be elucidated. In this study we examined the molecular, functional, and immunohistochemical expression of TRPV1 in asthmatic and healthy airways. Bronchial biopsy specimens and bronchial brushings were obtained from healthy volunteers (n = 18), patients with mild-to-moderate asthma (n = 24), and patients with refractory asthma (n = 22). Cultured primary bronchial epithelial cells from patients with mild asthma (n = 4), nonasthmatic coughers (n = 4), and healthy subjects (n = 4) were studied to investigate the functional role of TRPV1. Quantitative immunohistochemistry revealed significantly more TRPV1 expression in asthmatic patients compared with healthy subjects, with the greatest expression in patients with refractory asthma (P = .001). PCR and Western blotting analysis confirmed gene and protein expression of TRPV1 in cultured primary bronchial epithelial cells. Patch-clamp electrophysiology directly confirmed functional TRPV1 expression in all 3 groups. In functional assays the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the antagonist capsazepine. Reduction of external pH from 7.4 to 6.4 activated a capsazepine-sensitive outwardly rectifying membrane current. Functional TRPV1 channels are present in the human airway epithelium and overexpressed in the airways of patients with refractory asthma. These channels might represent a novel therapeutic target for the treatment of uncontrolled asthma.

PharmGKB summary

Thorn, Caroline F.; Ellison, David H.; Turner, Stephen T.; Altman, Russ B.; Klein, Teri E. Author Information

A Novel Ion Channel Formed by Interaction of TRPML3 with TRPV5

TRPML3 and TRPV5 are members of the mucolipin (TRPML) and TRPV subfamilies of transient receptor potential (TRP) cation channels. Based on sequence similarities of the pore forming regions and on structure-function evidence, we hypothesized that the pore forming domains of TRPML and TRPV5/TRPV6 channels have similarities that indicate possible functional interactions between these TRP channel subfamilies. Here we show that TRPML3 and TRPV5 associate to form a novel heteromeric ion channel. This novel conductance is detectable under conditions that do not activate either TRPML3 or TRPV5. It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5. Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations. Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.

Intragastric Dai-Kenchu-To, a Japanese Herbal Medicine, Stimulates Colonic Motility via Transient Receptor Potential Cation Channel Subfamily V Member 1 in Dogs

Japanese herbal medicine, also known as Kampo, is used for various diseases in Japan. One of those medicines, Dai-Kenchu-To (DKT), is considered clinically effective for adhesive bowel obstruction and chronic constipation. Although scientific evidence of DKT to improve adhesive bowel obstruction was shown in several previous reports, mechanism of DKT to improve constipation remains unknown. Our aim was to study the effect of intragastric DKT on colonic motility and defecation, and the involvement of various receptors in DKT-induced colonic contractions. Five beagle dogs were instructed with serosal strain-gauge force transducers to measure circular muscle activity at the proximal, middle, and distal colon. Dogs are suitable for a present study to administer the drugs repeatedly to the same individual and look at its effect on colonic motility. We studied the effects of DKT (2.5 or 5 g) administered into the stomach on colonic motility. Muscarinic receptor antagonist atropine, nicotinic receptor antagonist hexamthonium, or 5-hydroxytryptamine-3 receptor antagonist ondansetron was injected intravenously 10 min before DKT administration. Capsazepine, an antagonist to transient receptor potential cation channel subfamily V member 1 (TRPV1), was administered into the stomach 5 min before DKT administration. Intragastric DKT (2.5 or 5 g) induced colonic contractions within 10 min after administration but did not induce defecation. Pretreatment with atropine, hexamthonium, ondansetron, or capsazepine inhibited DKT-induced colonic contractions. These results indicate that orally administered DKT stimulates colonic motility via TRPV1, muscarinic, nicotinic, and 5-hydroxytryptamine-3 receptors, thereby providing scientific support for the efficacy of oral DKT in chronic constipation.

Function of Transient Receptor Potential Cation Channel Subfamily V Member 4 (TRPV4) as a Mechanical Transducer in Flow-sensitive Segments of Renal Collecting Duct System

The TRPV4 Ca(2+)-permeable channel is sensitive to mechanical stimuli. In the current study we have employed immunocytochemical staining in kidney slices and functional assessments (Ca(2+) imaging) in isolated, split-opened, tubule segments to define TRPV4 sites of expression and flow-dependent function in the collecting duct system. Staining patterns revealed strong expression of TRPV4 along the entire collecting duct system with highest levels at the apical (luminal)/subapical region of the principal cells (PCs), the dominant cell type, with more diffuse staining in intercalated cells (ICs). Using fluorescence Ca(2+) imaging and the selective TRPV4 agonist, GSK1016790A, we demonstrated functional TRPV4 channels in PCs and ICs of split-opened cortical collecting ducts and connecting tubules. The agonist was ineffective in inducing a rise in [Ca(2+)](i) in the absence of extracellular Ca(2+) or in tubules from TRPV4-deficient animals. Most importantly, a 10-fold elevation in luminal (apical) fluid flow induced a rapid and sustained influx of Ca(2+) that was abolished by the TRPV channel inhibitor, ruthenium red, or in tubules isolated from TRPV4 deficient animals. We concluded that TRPV4 is highly expressed along the entire collecting duct system where it appears to function as a sensor/transducer of flow-induce mechanical stresses.

Dexamethasone Resisted Podocyte Injury via Stabilizing TRPC6 Expression and Distribution

TRPC6, a member of the canonical transient receptor potential channel (TRPC) subfamily, is an important cation selective ion channel on podocytes. Podocytes are highly differentiated cells located on the visceral face of glomerular basement membrane and featured by numerous foot processes, on which nephrin, podocin, and TRPC6 locate. Podocytes and the slit diaphragm (SD) between adjacent foot processes form a selective filtration barrier impermeable to proteins. TRPC6 is very critical for normal podocyte function. To investigate the function of TRPC6 in podocytes and its relation to proteinuria in kidney diseases, we over-expressed TRPC6 in podocytes by puromycin aminonucleoside (PAN) and observed the changes of foot processes, TRPC6 protein distribution, and mRNA expression. Accordingly, in this study, we further investigated the role of specific signaling mechanisms underlying the prosurvival effects of dexamethasone (DEX) on podocyte repair. Our results showed that podocytes processes of overexpressing TRPC6 were reduced remarkably. These changes could be rescued by DEX via blocking TRPC6 channel. Additionally, our results also showed an improvement in TRPC6 arrangement in the cells and decrease of mRNA expression and protein distribution. From these results, we therefore proposed that overexpression of TRPC6 in podocytes may be one of the fundamental changes relating to the dysfunction of the SD and proteinuria. DEX may be maintained the structure and function integrity of SD by blocking TRPC6 signal pathway and played an important role in mechanisms of anti-proteinuria.

Expression of TRPV6 (Transient Receptor Potential Cation Channel Subfamily V, Member 6) in the Human Endometrium During Menstrual Cycle.

Two highly selective calcium channels at the apical sides of cells, members of the transient receptor potential (TRP) superfamily of ion channels (TRPV6 and TRPV5), are the main calcium ion entry channels. Previously, the location of TRPV6 has been described in the intestine in several species, including human. It is located in the apical brush-border membrane of the intestinal enterocyte where regulates calcium entry into the cell. It is most abundant in the proximal small intestine (duodenum and jejunum) where calbindin and the calcium-pumping ATPase are also found. TRPV6 calcium transporter is also found in the human placenta, pancreas and prostate gland and in some species. However, TRPV6 expression and its potential roles remain to be clarified in the endometrium of human during menstrual cycle. In this study, we employed a human endometrial model to examine the expression of TRPV6, and its potential roles in human menstrual cycle. A significant increase (1.5 fold) in TRPV6 transcript and protein was observed in human uterus at proliferation phase compared to other phases. In addition, the spatial localization of TRPV6 in human uterus was determined by immunohistochemistry. TRPV6 protein was localized in the glandular epithelial cells in menstrual phases and highly expressed in the cytoplasm of the luminal. Overall, these results demonstrate that TRPV6 is abundantly expressed in the uterine human, suggesting that this protein may be involved in reproductive function during the cycle in human. (poster)

Functional TRPV4 channels are expressed in mouse skeletal muscle and can modulate resting Ca2+ influx and muscle fatigue

Skeletal muscle contraction is basically controlled by Ca(2+) release and its reuptake into the sarcoplasmic reticulum. However, the long-term maintenance of muscle function requires an additional Ca(2+) influx from extracellular. Several mechanisms seem to contribute to the latter process, such as store-operated Ca(2+) entry, stretch-activated Ca(2+) influx and resting Ca(2+) influx. Candidate channels that may control Ca(2+) influx into muscle fibers are the STIM proteins, Orai, and the members of the transient receptor potential (TRP) family of cation channels. Here we show that TRPV4, an osmo-sensitive cation channel of the vanilloid subfamily of TRP channels is functionally expressed in mouse skeletal muscle. Western blot analysis showed the presence of TRPV4-specific bands at about 85 and 100kDa in all tested muscles. The bands were absent when muscle proteins from TRPV4 deficient mice were analyzed. Using the manganese quench technique, we studied the resting influx of divalent cations into isolated wild-type muscle fibers. The specific TRPV4-channel activator 4α-phorbol-12,13-didecanoate (4α-PDD) stimulated resting influx by about 60% only in wild-type fibers. Electrical stimulation of soleus muscles did not reveal changes in isometric twitch contractions upon application of 4α-PDD, but tetanic contractions (at 120Hz) were slightly increased by about 15%. When soleus muscles were stimulated with a fatigue protocol, muscle fatigue was significantly attenuated in the presence of 4α-PDD. The latter effect was not observed with muscles from TRPV4(-/-) mice. We conclude that TRPV4 is functionally expressed in mouse skeletal muscle and that TRPV4 activation modulates resting Ca(2+) influx and muscle fatigue.

Na+-independent, nifedipine-resistant rat afferent arteriolar Ca2+ responses to noradrenaline: possible role of TRPC channels

In rat afferent arterioles we investigated the role of Na(+) entry in noradrenaline (NA)-induced depolarization and voltage-dependent Ca(2+) entry together with the importance of the transient receptor potential channel (TRPC) subfamily for non-voltage-dependent Ca(2+) entry. R (340/380) Fura-2 fluorescence was used as an index for intracellular free Ca(2+) concentration ([Ca(2+)](i)). Immunofluorescence detected the expression of TRPC channels. TRPC 1, 3 and 6 were expressed in afferent arteriolar vascular smooth muscle cells. Under extracellular Na(+)-free (0 Na) conditions, the plateau response to NA was 115% of the baseline R(340/380) (control response 123%). However, as the R(340/380) baseline increased (7%) after 0 Na the plateau reached the same level as during control conditions. Similar responses were obtained after blockade of the Na(+)/Ca(2+) exchanger. The L-type blocker nifedipine reduced the plateau response to NA both under control (from 134% to 116% of baseline) and 0 Na conditions (from 112% to 103% of baseline). In the presence of nifedipine, the putative TRPC channel blockers SKF 96365 (30 μm) and Gd(3+) (100 μm) further reduced the plateau Ca(2+) responses to NA (from 117% to 102% and from 117% to 110% respectively). We found that Na(+) is not crucial for the NA-induced depolarization that mediates Ca(2+) entry via L-type channels. In addition, the results are consistent with the idea that TRPC1/3/6 Ca(2+) -permeable cation channels expressed in afferent arteriolar smooth muscle cells mediate Ca(2+) entry during NA stimulation.

TRPM3 Expression and Function in Vascular Smooth Muscle

The Transient Receptor Potential (TRP) channels form a diverse superfamily of cation channels. Currently there are few well characterised, selective, physiological regulators of TRP channels, which can impede the discovery of native channel functions. TRPM3, a member of the melastatin subfamily of TRP channels, is reported to form constitutively active Ca2+-permeable cation channels activated by sphingolipids, hypotonic shock, and the neurosteroid pregnenolone sulphate. Expression is predominantly in brain and kidney, although expression has recently been reported in pancreatic β-cells. Here we report TRPM3 expression and function in vascular smooth muscle cells. Anti-TRPM3 blocking antibody and short-interfering RNA targeted to TRPM3 were used to confirm the involvement of TRPM3 in the pregnenolone sulphate elicited Ca2+ influx in cultured human vascular smooth muscle cells. Although pregnenolone sulphate is a useful pharmacological and potentially therapeutic agent, the physiological significance in the vasculature is unknown. We therefore screened for novel modulators, and found that cholesterol inhibited both pregnenolone sulphate-induced and constitutive TRPM3 activity. The data suggest TRPM3 is a novel calcium-entry channel of vascular smooth muscle cells.The research was supported by a BBSRC Industrial Co-operative Award in Science and Engineering, the Wellcome Trust and the British Heart Foundation.

Gitelman Syndrome

Gitelman Syndrome

Capsaicin: A review of its pharmacology and clinical applications

Summary Capsaicin is a naturally occurring alkaloid derived from chillies that is responsible for its hot pungent taste. It is an odourless fat soluble compound that is rapidly absorbed through the skin. Capsaicin is thought to produce analgesia by depleting substance P in small fibre nociceptor neurons on which Transient Receptor Potential action channel (subfamily V), type 1 (TRPV1) is predominantly located. It binds to the vanilloid receptor TRPV1, which acts as a molecular integrator of chemical and physical painful stimuli. Topically applied capsaicin is useful in alleviating pain associated with diabetic neuropathy and chronic musculoskeletal pain. It is used to increase the bladder capacity and reduce incontinence in patients with bladder hyperactivity. Capsaicin can reduce post-operative nausea and vomiting when applied at acupressure points. It may be used to treat pruritis associated with renal failure and protect the stomach against non-steroidal anti-inflammatory drug induced gastritis. The development of capsaicin analogues and TRPV1 antagonists may provide more effective and better tolerated therapeutic agents in the future. Synthetic agents that are less pungent and target the TRPV1 receptor may prove to be more efficacious and more tolerable than naturally occurring capsaicin for the treatment of pain and inflammatory disease states.

Yeast gain-of-function mutations reveal structure–function relationships conserved among different subfamilies of transient receptor potential channels

Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across subfamilies. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550I) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of phototaxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure-function principles that can be generalized across different TRP subfamilies. It appears that the conserved aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed.