Canonical TRP channels and mechanotransduction: from physiology to disease states

Mechano-gated ion channels play a key physiological role in cardiac, arterial, and skeletal myocytes. For instance, opening of the non-selective stretch-activated cation channels in smooth muscle cells is involved in the pressure-dependent myogenic constriction of resistance arteries. These channels are also implicated in major pathologies, including cardiac hypertrophy or Duchenne muscular dystrophy. Seminal work in prokaryotes and invertebrates highlighted the role of transient receptor potential (TRP) channels in mechanosensory transduction. In mammals, recent findings have shown that the canonical TRPC1 and TRPC6 channels are key players in muscle mechanotransduction. In the present review, we will focus on the functional properties of TRPC1 and TRPC6 channels, on their mechano-gating, regulation by interacting cytoskeletal and scaffolding proteins, physiological role and implication in associated diseases.     

Touch sensitivity in Caenorhabditis elegans

The nematode Caenorhabditis elegans was the first organism for which touch insensitive mutants were obtained. The study of the genes defective in these mutants has led to the identification of components of a mechanosensory complex needed for specific cells to sense gentle touch to the body. Multiple approaches using genetics, cell biology, biochemistry, and electrophysiology have characterized a channel complex, containing two DEG/ENaC pore-forming subunits and several other proteins, that transduces the touch response. Other mechanical responses, sensed by other cells using a variety of other components, are less well understood in C. elegans. Many of these other senses may use TRP channels, although DEG/ENaC channels have also been implicated.     

Multiple roles of calmodulin and other Ca2+-binding proteins in the functional regulation of TRP channels

Transient receptor potential channels (TRP) have emerged as cellular sensors of various internal and external cues. Generally, the activation of TRP canonical (TRPC) channels is triggered by the stimulation of phospholipase C; however, multiple factors are involved in the regulation of these channels. Among them, Ca²⁺-mediated feedback channel modulations are often mediated by calmodulin (CaM) and other Ca²⁺-binding proteins. In vitro binding studies have revealed multiple CaM-binding sites on TRPC proteins. Among them, a common CaM/inositol 1,4,5-trisphosphate receptor-binding site is found at the carboxyl terminus of every TRPC isoform. Additional non-conserved CaM-binding sites are present at the amino and carboxyl termini of several TRPC proteins. Likewise, multiple CaM-binding sites were found in other TRP proteins. These, together with the presence in close vicinity of the interaction sites for the related neuronal Ca²⁺-binding proteins, such as CaBP1, suggest a multitude of diverse intracellular Ca²⁺-dependent regulations of TRP channels. Functional studies have begun to reveal the unique roles of CaM and CaBP1 binding to several TRP channels. This review will focus on the CaM- and CaBP1-mediated regulations of TRPC channels. Related studies on TRPM and TRPV channels will also be highlighted.     

The use of yeast to understand TRP-channel mechanosensitivity

Mechanosensitive (MS) ion channels likely underlie myriad force-sensing processes, from basic osmotic regulation to specified sensations of animal hearing and touch. Albeit important, the molecular identities of many eukaryotic MS channels remain elusive, let alone their working mechanisms. This is in stark contrast to our advanced knowledge on voltage- or ligand-sensitive channels. Several members of transient receptor potential (TRP) ion channel family have been implicated to function in mechanosensation and are recognized as promising candidate MS channels. The yeast TRP homolog, TRPY1, is clearly a first-line force transducer. It can be activated by hypertonic shock in vivo and by membrane stretch force in excised patches under patch clamp, making it a useful model for understanding TRP channel mechanosensitivity in general. TRPY1 offers two additional research advantages: (1) It has a large (∼300 pS) unitary conductance and therefore a favorable S/N ratio. (2) Budding yeast allows convenient and efficient genetic and molecular manipulations. In this review, we focus on the current research of TRPY1 and discuss its prospect. We also describe the use of yeast as a system to express and characterize animal TRP channels.     

Diabetes associated cell stress and dysfunction: role of mitochondrial and non-mitochondrial ROS production and activity

The transient receptor potential (TRP) ion channel family was the last major ion channel family to be discovered. The prototypical member (dTRP) was identified by a forward genetic approach in Drosophila , where it represents the transduction channel in the photoreceptors, activated downstream of a Gq-coupled PLC. In the meantime 29 vertebrate TRP isoforms are recognized, distributed amongst seven subfamilies (TRPC, TRPV, TRPM, TRPML, TRPP, TRPA, TRPN). They subserve a wide range of functions throughout the body, most notably, though by no means exclusively, in sensory transduction and in vascular smooth muscle. However, their precise physiological roles and mechanism of activation and regulation are still only gradually being revealed. Most TRP channels are subject to multiple modes of regulation, but a common theme amongst the TRPC/V/M subfamilies is their regulation by lipid messengers. Genetic evidence supports an excitatory role of diacylglycerol (DAG) for the dTRP's, although curiously only DAG metabolites (PUFAs) have been found to activate the Drosophila channels. TRPC2,3,6 and 7 are widely accepted as DAG-activated channels, although TRPC3 can also be regulated via a store-operated mechanism. More recently PIP₂ has been shown to be required for activity of TRPV5, TRPM4,5,7 and 8, whilst it may inhibit TRPV1 and the dTRPs. Although compelling evidence for a direct interaction of DAG with the TRPC channels is lacking, mutagenesis studies have identified putative PIP₂-interacting domains in the C-termini of several TRPV and TRPM channels.     

Caenorhabditis elegans TRPA-1 functions in mechanosensation

Members of the transient receptor potential (TRP) ion channel family mediate diverse sensory transduction processes in both vertebrates and invertebrates. In particular, members of the TRPA subfamily have distinct thermosensory roles in Drosophila, and mammalian TRPA1 is postulated to have a function in noxious cold sensation and mechanosensation. Here we show that mutations in trpa-1, the C. elegans ortholog of mouse Trpa1, confer specific defects in mechanosensory behaviors related to nose-touch responses and foraging. trpa-1 is expressed and functions in sensory neurons required for these mechanosensory behaviors, and contributes to neural responses of these cells to touch, particularly after repeated mechanical stimulation. Furthermore, mechanical pressure can activate C. elegans TRPA-1 heterologously expressed in mammalian cells. Collectively, these data demonstrate that trpa-1 encodes an ion channel that can be activated in response to mechanical pressure and is required for mechanosensory neuron function, suggesting a possible role in mechanosensory transduction or modulation.     

TRPC5 is a regulator of hippocampal neurite length and growth cone morphology

Growth cone motility is regulated by both fast voltage-dependent Ca2+ channels and by unknown receptor-operated Ca2+ entry mechanisms. Transient receptor potential (TRP) homomeric TRPC5 ion channels are receptor-operated, Ca2+-permeable channels predominantly expressed in the brain. Here we show that TRPC5 is expressed in growth cones of young rat hippocampal neurons. Our results indicate that TRPC5 channel subunits interact with the growth cone-enriched protein stathmin 2, are packaged into vesicles and are carried to newly forming growth cones and synapses. Once in the growth cone, TRPC5 channels regulate neurite extension and growth-cone morphology. Dominant-negative TRPC5 expression allowed significantly longer neurites and filopodia to form. We conclude that TRPC5 channels are important components of the mechanism controlling neurite extension and growth cone morphology.     

Function and regulation of TRP family channels in C. elegans

Seventeen transient receptor potential (TRP) family proteins are encoded by the C. elegans genome, and they cover all of the seven TRP subfamilies, including TRPC, TRPV, TRPM, TRPN, TRPA, TRPP, and TRPML. Classical forward and reverse genetic screens have isolated mutant alleles in every C. elegans trp gene, and their characterizations have revealed novel functions and regulatory mechanisms of TRP channels. For example, the TRPC channels TRP-1 and TRP-2 control nicotine-dependent behavior, while TRP-3, a sperm TRPC channel, is regulated by sperm activation and required for sperm–egg interactions during fertilization. Similar to their vertebrate counterparts, C. elegans TRPs function in sensory physiology. For instance, the TRPV channels OSM-9 and OCR-2 act in chemosensation, osmosensation, and touch sensation, the TRPA member TRPA-1 regulates touch sensation, while the TRPN channel TRP-4 mediates proprioception. Some C. elegans TRPM, TRPP, and TRPML members exhibit cellular functions similar to their vertebrate homologues and have provided insights into human diseases, including polycystic kidney disease, hypomagnesemia, and mucolipidosis type IV. The availability of a complete set of trp gene mutants in conjunction with its facile genetics makes C. elegans a powerful model for studying the function and regulation of TRP family channels in vivo.     

The history of the Drosophila TRP channel: the birth of a new channel superfamily

Transient receptor potential (TRP) channels are polymodal cellular sensors involved in a wide variety of cellular processes, mainly by changing membrane voltage and increasing cellular Ca(2+). This review outlines in detail the history of the founding member of the TRP family, the Drosophila TRP channel. The field began with a spontaneous mutation in the trp gene that led to a blind mutant during prolonged intense light. It was this mutant that allowed for the discovery of the first TRP channels. A combination of electrophysiological, biochemical, Ca(2+) measurements, and genetic studies in flies and in other invertebrates pointed to TRP as a novel phosphoinositide-regulated and Ca(2+)-permeable channel. The cloning and sequencing of the trp gene provided its molecular identity. These seminal findings led to the isolation of the first mammalian homologues of the Drosophila TRP channels. We now know that TRP channel proteins are conserved through evolution and are found in most organisms, tissues, and cell-types. The TRP channel superfamily is classified into seven related subfamilies: TRPC, TRPM, TRPV, TRPA, TRPP, TRPML, and TRPN. A great deal is known today about participation of TRP channels in many biological processes, including initiation of pain, thermoregulation, salivary fluid secretion, inflammation, cardiovascular regulation, smooth muscle tone, pressure regulation, Ca(2+) and Mg(2+) homeostasis, and lysosomal function. The native Drosophila photoreceptor cells, where the founding member of the TRP channels superfamily was found, is still a useful preparation to study basic features of this remarkable channel.     

Role of TRP ion channels in cancer and tumorigenesis

Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has captured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca²⁺ distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca²⁺ homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.     

Human Digital Meissner Corpuscles Display Immunoreactivity for the Multifunctional Ion Channels Trpc6 and Trpv4

Ion channels are at the basis of the sensory processes including mechanosensing. Some members of the transient receptor potential (TRP) ion channel superfamily have been proposed as mechanosensors, but their putative role in mechanotransduction is controversial. Among them there are TRP canonical 6 (TRPC6) and TRP vanilloid 4 (TRPV4) ion channels, which are known to cooperate in mechanical hyperalgesia. Here, we investigated the occurrence, distribution, and possible colocalization of TRPC6 and TRPV4 in human digital Meissner sensory corpuscles using immunohistochemistry and double immunofluorescence (associate with markers for specific corpuscular constituents). TRPC6 immunoreactivity was restricted to the axon of Meissner corpuscles, whereas TRPV4 was detected in the axon but also in the lamellar cells. Moreover, axonal colocalization of TRPV4 and TRPC6 was found in the digital Meissner corpuscles. Present results demonstrate for the first time the occurrence and colocalization of two ion channels candidates to mechanosensors in human cutaneous mechanoreceptors. The functional significance of these ion channels in that place remains to be clarified, but should be related to different properties of mechanosensitivity. Anat Rec, 300:1022–1031, 2017. © 2016 Wiley Periodicals, Inc.     

Neuroepithelial Bodies as Mechanotransducers in the Intrapulmonary Airway Epithelium: Involvement of TRPC5

In rodent lungs, a major part of the myelinated vagal airway afferents selectively contacts pulmonary neuroepithelial bodies (NEBs). Because most myelinated vagal airway afferents concern physiologically characterized mechanoreceptors, the present study aimed at unraveling the potential involvement of NEB cells in transducing mechanosensory information from the airways to the central nervous system. Physiological studies were performed using confocal Ca(2+) imaging of airway epithelium in murine lung slices. Mechanical stimulation by short-term application of a mild hypoosmotic solution (230 mosmol) resulted in a selective, fast, reversible, and reproducible Ca(2+) rise in NEB cells. Other airway epithelial cells could only be activated using more severe hypoosmotic stimuli (< 200 mosmol). NEB cells selectively expressed the Ca(2+)-permeable osmo- and mechanosensitive transient receptor potential canonical channel 5 (TRPC5) in their apical membranes, whereas immunoreactivity for TRP vanilloid-4 and TRP melastatin-3 was abundant in virtually all other airway epithelial cells. Hypoosmotic activation of NEB cells was prevented by GsMTx-4, an inhibitor of mechanosensitive ion channels, and by SKF96365, an inhibitor of TRPC channels. Short application of gadolinium, reported to activate TRPC5 channels, evoked a transient Ca(2+) rise in NEB cells. Osmomechanical activation of NEB cells gave rise to a typical delayed activation of Clara-like cells due to the release of ATP from NEB cells. Because ATP may activate the NEB-associated P2X(2/3) ATP receptor expressing myelinated vagal afferents, the current observations strongly suggest that pulmonary NEB cells are fully equipped to initiate mechanosensory signal transduction to the central nervous system via a purinergic signaling pathway.     

Genetic models of mechanotransduction: the nematode Caenorhabditis elegans

Mechanotransduction, the conversion of a mechanical stimulus into a biological response, constitutes the basis for a plethora of fundamental biological processes such as the senses of touch, balance, and hearing and contributes critically to development and homeostasis in all organisms. Despite this profound importance in biology, we know remarkably little about how mechanical input forces delivered to a cell are interpreted to an extensive repertoire of output physiological responses. Recent, elegant genetic and electrophysiological studies have shown that specialized macromolecular complexes, encompassing mechanically gated ion channels, play a central role in the transformation of mechanical forces into a cellular signal, which takes place in mechanosensory organs of diverse organisms. These complexes are highly efficient sensors, closely entangled with their surrounding environment. Such association appears essential for proper channel gating and provides proximity of the mechanosensory apparatus to the source of triggering mechanical energy. Genetic and molecular evidence collected in model organisms such as the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the mouse highlight two distinct classes of mechanically gated ion channels: the degenerin (DEG)/epithelial Na(+) channel (ENaC) family and the transient receptor potential (TRP) family of ion channels. In addition to the core channel proteins, several other potentially interacting molecules have in some cases been identified, which are likely parts of the mechanotransducing apparatus. Based on cumulative data, a model of the sensory mechanotransducer has emerged that encompasses our current understanding of the process and fulfills the structural requirements dictated by its dedicated function. It remains to be seen how general this model is and whether it will withstand the impiteous test of time.     

Revisiting TRPC1 and TRPC6 mechanosensitivity

This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-activated mechano-sensitive Ca²⁺ permeable cation channel (MscCa). We have transiently expressed TRPC1 and TRPC6 in African green monkey kidney (COS) or Chinese hamster ovary (CHO) cells and monitored the activity of the stretch-activated channels using a fast pressure clamp system. Although both TRPC1 and TRPC6 are highly expressed at the protein level, the amplitude of the mechano-sensitive current is not significantly altered by overexpression of these subunits. In conclusion, although several TRPC channel members, including TRPC1 and TRPC6, have been recently proposed to form MscCa in vertebrate cells, the functional expression of these TRPC subunits in heterologous systems remains problematic. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Co-first authors: Philip Gottlieb, Joost Folgering, and Rosario Maroto.     

Neurobiology of TRPC2: from gene to behavior

The mammalian vomeronasal organ (VNO), a part of the accessory olfactory system, plays an essential role in the sensing of pheromonal signals. The VNO has emerged as an excellent model to investigate the functional role of transient receptor potential (TRP) channels in intact neurons and intact physiological systems. TRPC2, a member of the (canonical) TRPC subfamily, is highly localized to the dendritic tip of vomeronasal sensory neurons. Phenotypic analysis of mice exhibiting a targeted deletion in the TRPC2 gene has established that TRPC2 occupies a fundamental role in the transduction machinery underlying the detection of pheromone signals by the VNO. TRPC2-deficient mice exhibit striking behavioral defects in the regulation of sexual and social behaviors. A previously unknown Ca²⁺-permeable, diacylglycerol (DAG)-activated cation channel found at the dendritic tip of vomeronasal neurons is severely defective in TRPC2 mutants, providing the first clear example for the existence of native DAG-gated cation channels in the mammalian nervous system. The experimental strategy employed in the mouse VNO now serves as a powerful model for examining the native functions of other TRP genes.     

Determining the functional role of TRPC channels in primary cells

Although the TRPC members of the mammalian transient receptor potential TRP cation channel family were the first to be described in 1995, the depth of knowledge of TRPC channels has fallen behind that of their counterparts in the TRPV and TRPM subfamilies in the intervening years. The complexities and controversies of TRPC channel composition and regulation have hindered their progress as therapeutic targets in the drug discovery environment to date, however embracing these challenges as opportunities may bring TRPC channels to the forefront of the discovery of novel therapies for many diseases. These challenges and opportunities of exploring TRPC channels as therapeutic targets are highlighted and discussed in this review with respect to respiratory diseases.     

Specification of auditory sensitivity by Drosophila TRP channels

Ears achieve their exquisite sensitivity by means of mechanical feedback: motile mechanosensory cells through their active motion boost the mechanical input from the ear. Examination of the auditory mechanics in Drosophila melanogaster mutants shows that the transient receptor potential (TRP) channel NompC is required to promote this feedback, whereas the TRP vanilloid (TRPV) channels Nan and Iav serve to control the feedback gain. The combined function of these channels specifies the sensitivity of the fly auditory organ.     

The history of TRP channels, a commentary and reflection

The transient receptor potential (TRP) family of cation channels has redefined our understanding of sensory physiology. In one animal or another, all senses depend on TRP channels. These include vision, taste, smell, hearing, and various forms of touch, including the ability to sense changes in temperature. The first trp gene was identified because it was disrupted in a Drosophila mutant with defective vision. However, there was no clue as to its biochemical function until the cloning, and analysis of the deduced amino acid sequence suggested that trp encoded a cation channel. This concept was further supported by subsequent electrophysiological studies, including alteration of its ion selectivity by an amino acid substitution within the pore loop. The study of TRP channels emerged as a field with the identification of mammalian homologs, some of which are direct sensors of environmental temperature. At least one TRP channel is activated downstream of a thermosensory signaling cascade, demonstrating that there exist two modes of activation, direct and indirect, through which TRP channels respond to changes in temperature. Mutations in many TRP channels result in disease, including a variety of sensory impairments.     

Transient receptor potential cation channels in disease

The transient receptor potential (TRP) superfamily consists of a large number of cation channels that are mostly permeable to both monovalent and divalent cations. The 28 mammalian TRP channels can be subdivided into six main subfamilies: the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and the TRPA (ankyrin) groups. TRP channels are expressed in almost every tissue and cell type and play an important role in the regulation of various cell functions. Currently, significant scientific effort is being devoted to understanding the physiology of TRP channels and their relationship to human diseases. At this point, only a few channelopathies in which defects in TRP genes are the direct cause of cellular dysfunction have been identified. In addition, mapping of TRP genes to susceptible chromosome regions (e.g., translocations, breakpoint intervals, increased frequency of polymorphisms) has been considered suggestive of the involvement of these channels in hereditary diseases. Moreover, strong indications of the involvement of TRP channels in several diseases come from correlations between levels of channel expression and disease symptoms. Finally, TRP channels are involved in some systemic diseases due to their role as targets for irritants, inflammation products, and xenobiotic toxins. The analysis of transgenic models allows further extrapolations of TRP channel deficiency to human physiology and disease. In this review, we provide an overview of the impact of TRP channels on the pathogenesis of several diseases and identify several TRPs for which a causal pathogenic role might be anticipated.