TRP Channels as Therapeutic Targets: From Basic Science to Clinical Use

TRP Channels as Therapeutic Targets: From Basic Science to Clinical Use is authored by experts across academia and industry, providing readers with a complete picture of the therapeutic potential and challenges associated with using TRP channels as drug targets.

This book offers a unique clinical approach by covering compounds that target TRP channels in pre-clinical and clinical phases, also offering a discussion of TRP channels as biomarkers.

An entire section is devoted to the novel and innovative uses of these channels across a variety of diseases, offering strategies that can be used to overcome the adverse effects of first generation TRPV1 antagonists.

Intended for all researchers and clinicians working toward the development of successful drugs targeting TRP channels, this book is an essential resource chocked full of the latest clinical data and findings.

• Contains comprehensive coverage of TRP channels as therapeutic targets, from emerging clinical indications to completed clinical trials
• Discusses TRP channels as validated targets, ranging from obesity and diabetes through cancer and respiratory disorders, kidney diseases, hypertension, neurodegenerative disorders, and more
• Provides critical analysis of the complications and side effects that have surfaced during clinical trials, offering evidence-based suggestions for overcoming them

• Language: English
• Publisher: Academic Press
• Release date: Apr 9, 2015
• ISBN: 9780124200791

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Chapter 1

An Introduction to Transient Receptor Potential Ion Channels and Their Roles in Disease

Michael J. Caterina¹,²,³,⁴,*    ¹ Department of Neurosurgery, Johns Hopkins School of Medicine, Baltimore, Maryland, USA

² Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland, USA

³ Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, Maryland, USA

⁴ Neurosurgery Pain Research Institute, Johns Hopkins School of Medicine, Baltimore, Maryland, USA

* Corresponding author: [e-mail]

Abstract

The transient receptor potential (TRP) cation channel family consists of seven subfamilies that are widely expressed in mammalian tissues. By mediating flux of calcium, sodium, and other cations across cell membranes, in addition to nonionic signaling mechanisms, these channels contribute to many sensory and nonsensory processes throughout the body. Abnormalities in TRP channel function, whether a consequence of mutations in their sequence, alterations in their expression levels, or changes in their myriad regulators, have been associated with numerous disease states ranging from chronic pain to cardiovascular disease, skeletal abnormalities, and cancer. Such prevalent involvement in disease stems not only from the ubiquity of TRP channels but also from their complex pattern of polymodal gating. The connection between TRP channels and disease creates numerous opportunities for therapeutic intervention at these channels, whether through inhibition, activation, or co-opting of their ability to transport cations to alter the course of pathophysiological processes.

Keywords

Transient receptor potential

Ion channel

Channelopathy

Pain

Calcium

Outline

Discovery and General Properties of TRP Channels   2

TRP Channels in Normal Physiology   4

TRP Channels and Sensory Physiology   4

TRP Channels and Cardiovascular Function   5

TRP Channels and Gastrointestinal Function   5

TRP Channels and Urological Function   5

TRP Channels and the Brain   5

TRP Channels and Immune Function   5

TRP Channels and Development   6

TRP Channels and Disease   6

Therapeutic Strategies Based on TRP Channel Modulation   9

Acknowledgment   10

References   10

Discovery and General Properties of TRP Channels

The diverse repertoire of ion channels expressed in mammalian and nonmammalian species is encoded by a multitude of gene families. Among these, the transient receptor potential (TRP) ion channel family exhibits an especially prevalent and complex link with disease. Fittingly for the theme of this book, the TRP channel name emerged as a consequence of a disease state, although the victims of this disease were not human beings, but rather members of a line of visually impaired fruit flies [1]. Electroretinograms recorded from photoreceptors of these flies revealed that the electrical response to a light pulse (receptor potential), instead of remaining robust throughout a pulse of several seconds, decayed prematurely. Subsequent molecular and physiological studies revealed that the gene mutated in these so-called transient receptor potential (trp) flies encoded an ion channel subunit that, together with a homologous channel subunit, TRPL, forms the functional photoreceptor channel. This channel is not gated directly by light, but rather is activated by a G protein-coupled phospholipase C signaling pathway following the photoisomerization of the light receptor protein, rhodopsin.

Following the identification of the Drosophila TRP channel, numerous homologous proteins were discovered, both in invertebrate species, such as fruit flies and nematodes, and in vertebrate species from fish to mammals [2]. Based on their domain structure and details of their sequences, members of the TRP channel family can be divided into seven subfamilies: TRPA (ankyrin, 1 human member), TRPC (canonical, 6 human members, plus 1 human pseudogene), TRPM (melastatin, 8 human members), TRPML (mucolipin, 3 human members), TRPN (NompC, no human members), TRPP (polycystin, 3 human members), and TRPV (vanilloid, 6 human members). There is also a distantly related family, TRPY, found in yeast. Functional TRP channels consist of homomeric or heteromeric tetramers of subunits from these subfamilies. The domain structure of an example TRP channel subunit, TRPV1, is shown in Figure 1.1a. A common structural feature of all TRP channel subunits is a core of six transmembrane domains (S1-S6), flanked by intracellular amino- and carboxyl-termini. Between S5 and S6 there is a complex pore-loop structure, which breaches the extracellular plane of the plasma membrane and forms the ion selectivity filter. This overall architecture resembles that of the voltage-gated and cyclic nucleotide-gated channel families. The TRPC, TRPM, TRPV, TRPA, and TRPN subfamilies, referred to as Group I TRP channels, resemble one another more closely than they do the TRPP or TRPML subfamilies, which are classified as Group II. Two features found among most Group I TRP channels include a TRP box homology element (absent in the TRPA subfamily), just distal to the sixth transmembrane domain, that participates in channel multimerization and modulation of gating, and a string of 4-16 sequential ankyrin repeat domains in the amino terminus (absent in the TRPM subfamily) that serves as a site of channel regulation (Figure 1.1a). Several TRPM subfamily members also contain kinase or nucleotide binding domains within their carboxyl termini and are therefore referred to as chanzymes. Ion flux through TRP channels occurs via a central pore lined by the pore loop domains of the four channel subunits (Figure 1.1b). All known TRP channels are selective for cations, although their degree of discrimination among cations can vary. For example, although some channels such as TRPV5 and TRPV6 are highly selective for Ca² +, and TRPM4 and TRPM5 are relatively Ca² + impermeant, most TRP channels are nonselective cation channels that can mediate flux of multiple monovalent and divalent cations [3]. Whereas most of these channels function at the plasma membrane, some are also found in organellar membranes. For example, TRPM2, TRPML, and TRPV2 channels can reside and function within the endolysosomal pathway [4]. A higher-resolution understanding of structural features of TRP channels has recently emerged with the solution of the atomic-level structure of one family member, TRPV1, by cryo-electron microscopy (Figure 1.1b and c) [5,6]. A few details and implications of this important advance will be described later in this chapter.

Figure 1.1 Representative TRP channel structure. (a) Domain map of a TRPV1 subunit. Amino terminus is at left. (b) TRPV1 holochannel structure in the apo (closed) state, solved by cryo-electron microscopy. Each subunit is in a different color. At left the channel is viewed from the side, illustrating distinct sites at which several agonists and regulators bind to allosterically control gating. At right, the transmembrane portion of the channel is viewed from the bottom and illustrates the separation between the S1-S4 domain and the S5-pore loop-S6 domain that forms the pore module lining the central pore axis (RTX, resiniferatoxin; DkTx, tarantula double-knot toxin). (c) Comparison of the TRPV1 pore module in the apo form (left) vs. a strongly activated state (right) evoked by a combination of RTX and DkTx. Path available for ion permeation is marked by dotted volume. For clarity, only two opposing subunits are shown. Sites of maximal constriction (G643 in the upper pore and I679 in the lower pore) are indicated. Note widening of both constrictions on activation. Modified, with permission, from Liao et al. [3] and Cao et al. [4].

TRP Channels in Normal Physiology

TRP channels as a family are broadly expressed in mammalian tissues. In fact, every cell in the body likely expresses at least one family member, and often more. Moreover, these channels can be activated by a number of heterogeneous stimuli, including a plethora of endogenous and exogenous chemical ligands, physical stimuli such as temperature and mechanical force, free cytosolic Ca² + ions, depletion of endoplasmic reticulum Ca² + stores, and many others. It should therefore not be surprising that these channels have been linked to numerous physiological functions. The following examples provide a glimpse into the ubiquitous involvement of TRP channels in the fundamental processes of life. As will be emphasized later in this chapter, and throughout this book, the pervasiveness of TRP channels in normal mammalian biology sets the stage for them to serve as contributors to, modulators of, or even primary causes of numerous human diseases.

TRP Channels and Sensory Physiology

Perhaps the best understood physiological functions of TRP channels are in the realm of sensory signal transduction. Just as the Drosophila TRP channel is a key effector in phototransduction, many other TRP channels serve either as primary transducers of environmental stimuli or as amplifiers or modulators of signals transduced by other receptors. The most extensively studied example from mammalian systems is TRPV1, a channel expressed at disproportionately high levels in a subpopulation of primary afferent nociceptors, sensory neurons that trigger the perception of pain [7]. TRPV1 was discovered on the basis of, and derives its name from, its ability to be gated by painful vanilloid compounds such as capsaicin (the main pungent ingredient in chili peppers) and resiniferatoxin (a highly potent irritant produced in the latex of Euphorbia plant species). Functional studies subsequently revealed that TRPV1 could alternatively be activated by other, nonvanilloid stimuli, most notably noxious heat (> 42 °C), protons (< pH 6), and various lipid metabolites. Activation of TRPV1 triggers calcium and sodium ion flux into nociceptor terminals, resulting in depolarization and consequent action potential firing. It also causes local neuronal release of bioactive peptides and consequent neurogenic inflammation. The necessity of TRPV1 for responsiveness to vanilloids and for normal heat-evoked pain has been demonstrated through the study of TRPV1 knockout mice, as well as the development and application of highly selective TRPV1 antagonists [7,8]. Subsequent to the discovery of TRPV1, a number of other TRP channels were identified as mediators of chemosensation, thermosensation, and perhaps most controversially, mechanosensation. Several examples include TRPA1, which is activated by pungent cysteine-reactive compounds such as mustard oil and acrolein, is a key mediator of itch sensation, and also appears to play roles in cold- and mechanically evoked pain [9]; TRPM8, which is activated by menthol and moderately cold temperatures and which is essential for mouse avoidance of such temperatures [10]; TRPC1, which has been implicated in transduction of low-intensity mechanical stimuli [11]; and TRPM3, another heat-gated channel that appears to contribute to heat-evoked pain [12]. In many cases, these channels appear to be functionally important not only at the peripheral terminals of sensory neurons, but also at their spinal and trigeminal central terminals, where they modulate the release of neurotransmitters onto second-order neurons [13,14]. Beyond somatosensory neurons, TRPM5 is a key participant in the detection of taste stimuli [15], whereas TRPC6 and TRPC7 appear to be involved in phototransduction by intrinsically photosensitive retinal ganglion cells [16].

TRP Channels and Cardiovascular Function

Many TRP channels, including TRPC1, TRPC3, TRPC6, TRPM4, TRPV2, and TRPV4, are expressed in endothelial or muscle cells within the heart and blood vessels, where they regulate vascular tone and permeability, as well as cardiac contractility [17]. These functions are accomplished predominantly by increasing free cytoplasmic calcium levels.

TRP Channels and Gastrointestinal Function

Many TRP channels are expressed intrinsically within the gastrointestinal tract [18]. For example, TRPV6 is a key effector of vitamin D-stimulated calcium absorption in the gut, whereas TRPV2 is expressed in enteric neurons, where it appears to regulate gastrointestinal motility. There is also abundant innervation of the gastrointestinal tract by extrinsic sensory afferents that express TRPV1, TRPA1, and other TRP channels. These neuronal channels regulate such processes as mucosal bloodflow and sensitivity to luminal distension. Within the exocrine portion of the gastrointestinal tract, TRPC1 and TRPV4 regulate saliva production and/or pancreatic acinar cell secretion, through the modulation of intracellular calcium levels and the regulation of aquaporin water channels. In the endocrine pancreas, TRPM5 and TRPM2, among others, contribute to the regulation of beta cell insulin release.

TRP Channels and Urological Function

TRP channels contribute to multiple processes within the kidneys and lower urinary tract [19–21]. These include regulation of renal glomerular filtration function by TRPC6, regulation of nephron osmoregulatory function by TRPV4, regulation of calcium and magnesium uptake by TRPV5 and TRPV6, and the contribution of TRPV1, TRPA1, and TRPV4 to the detection of stretch and irritation by bladder afferents and the urinary bladder epithelium.

TRP Channels and the Brain

All the TRP channel subfamilies expressed in humans have been implicated in important functions within the central nervous system [22]. Examples include neurotransmitter release (TRPC3, TRPV1, TRPV2), neurogenesis (TRPC6), astrocyte calcium homeostasis (TRPA1), responses to oxidative stress (TRPM7, TRPM2), osmoregulation (TRPV1, TRPV4), respiratory control (TRPM4), and neuronal lysosomal function (TRPML).

TRP Channels and Immune Function

Among the immune cell processes demonstrated to involve TRP channels are regulation of T-cell membrane excitability (TRPM4) and macrophage and microglial phagocytosis, lysosomal acidification, and cytokine release (TRPM2, TRPM7, TRPV2) [23,24]. Other cell types that contribute to innate immune function, including skin keratinocytes and primary sensory afferents, also employ TRP channels to carry out these functions. For example, TRPA1 and TRPV4 were recently shown to mediate the release of immune modulatory molecules such as TSLP and endothelin-1 from keratinocytes [25,26], whereas TRPV1- and TRPA1-mediated neurogenic release of neuropeptides from sensory afferents can promote tissue swelling and regulate recruitment of immune cells [9,27].

TRP Channels and Development

TRP channels also perform important functions related to reproduction and embryonic development. For example, elimination of TRPM7 is lethal at very early embryonic stages [28]. TRPV3 controls differentiation of the skin and hair, at least in part by regulating keratinocyte production of EGF receptor ligands [29], whereas TRPV2, TRPV4, and TRPM7 all may be involved in the differentiation of human adipocytes [30].

TRP Channels and Disease

Given the involvement of TRP channels in so many physiological processes, it should not be surprising that dysregulation of TRP channels has been linked to numerous pathophysiological conditions. In the ensuing chapters, the reader will encounter a host of situations in which an excess or shortage of TRP channel activity is a contributing factor to a human disease, or in which modulation of TRP channels provides a potential opportunity to circumvent a disease process.

In some cases, the contribution of a particular TRP channel to a specific disease process is direct and paramount. As will be highlighted both in the chapter on hereditary TRP channelopathies (Chapter 2) and on TRP gene polymorphism (Chapter 4), as well as in chapters dedicated to diseases of specific organ systems, point mutations in a number of TRP channels are sufficient to produce clinical syndromes characterized by well-defined inheritance patterns [31,32]. Examples include mutations in TRPV4 leading to either sensorimotor axonopathies such as Type 2C Charcot-Marie-Tooth disease or heritable skeletal abnormalities such as brachyolmia, mutations in TRPA1 resulting in familial episodic pain syndrome, mutations in TRPC6 giving rise to focal segmental glomerulosclerosis, mutations in TRPV3 producing palmoplantar keratoderma and pruritus in Olmsted syndrome, mutations in TRPML3 causing the lysosomal storage disease type IV mucolipidosis, and mutations in TRPP1 and TRPP2 causing polycystic kidney disease. Although many of these are autosomal dominant conditions, this is not always so, and it is sometimes difficult to differentiate whether a given mutation produces disease through loss- or gain-of-function.

There also exist situations in which the link between a given TRP channel and a given disease apparently arises from alterations in the expression of the channel or of factors that regulate the channel’s localization or activity. For example, following tissue inflammation or nerve injury, MAP kinase-dependent changes in the abundance of TRPV1 at the nociceptor terminal, protein kinase C and protein kinase A phosphorylation-mediated increase in TRPV1 sensitivity to its agonists or TRPV1 localization at the plasma membrane, and increases in the abundance of endogenous stimulators and potentiators of this channel, such as protons, endocannabinoids, and serotonin, all conspire to augment TRPV1 signaling [7,33]. A similarly complex regulatory pattern appears to exist for TRPA1 [9]. As a consequence of these events, TRPV1, TRPA1, and other TRP channels have emerged as key contributors to chemical, thermal, and mechanical hypersensitivity in animal models of nerve injury or inflammation. In humans, immunostaining studies have revealed upregulation of TRPV1 in peripheral nerves in a number of pain states [34]. Furthermore, a recent study showed that epigenetic changes in TRPA1 promoter methylation in white blood cells and gene expression in skin biopsies are predictive of human thermal pain sensitivity [35]. Finally, as discussed in two later chapters, the development of selective TRPV1 (Chapter 8) and TRPA1 (Chapter 9) antagonists has set the stage for the direct pharmacological assessment of the involvement of these channels in human disease [36].

As will be detailed throughout this book, many nonsensory pathophysiological conditions also appear to involve hyper- or hypoactivity of TRP channels. For example, abnormalities in TRP channel function in immune or inflammatory processes contribute to conditions such as asthma, dermatitis, gastrointestinal inflammation, and autoimmunity. TRP channels are also involved in cardiovascular diseases such as heart failure and hypertension and in both vascular and nonvascular diseases of the central nervous system. There is a also a growing body of evidence to support the intimate involvement of TRP channels, including TRPM8, TRPC6, TRPV2, TRPV1, and TRPM1, in cancer [37]. Finally, links have emerged between channels such as TRPM2, TRPV1, and TRPV4 and diseases involving tissue and organismal homeostasis, such as diabetes and obesity.

Factors that contribute to the many diverse consequences of TRP channel dysfunction and likely explain the pervasive association of TRP channels with disease:

(1) Both widespread and specific expression patterns. As discussed earlier, every cell in the body is likely to express one or more TRP channels, providing numerous opportunities for their aberrant function to contribute to disease pathogenesis. At the same time, the exceptionally high levels of expression of TRP channels in specific cell types, such as particular sensory neuron populations, creates a scenario in which channel dysfunction can have a disproportionately large effect on that cell’s function or health.

(2) Importance of calcium signaling. The ability of many TRP channels to mediate calcium influx into cells makes them gatekeepers for one of the most potent of biological signals. Cytoplasmic calcium levels are normally tightly controlled and regulate many cellular processes, such as secretion, motility, action potential firing and propagation, neurotransmitter release, and gene expression. At the same time, high levels of calcium can be cytotoxic. This creates a situation in which either gain or loss of TRP channel function can profoundly influence cellular behavior and survival.

(3) Diversity of signaling outputs. Calcium is not the only ion that can flow through TRP channels. As discussed earlier, many TRP channels are nonselective cation channels that can pass sodium, potassium, and magnesium ions, which in turn can influence cell behavior through the control of membrane polarization, cellular osmolarity, or more specific functions of the particular ions involved. In addition, some TRP channels, such as TRPV1, can be induced to mediate the influx of unusually large cations, such as potentially toxic aminoglycosides [38]. The repertoire of TRP channel signaling is expanded even further by the existence of catalytic domains on some subtypes, such as TRPM7, and the ability of TRP channels to functionally interact with other signaling proteins, such as Homer and STIM family members [39,40].

(4) Polymodal regulation and polygating. A remarkable feature of many TRP channels is that a given subtype can be alternatively regulated by multiple diverse stimuli that might be chemical, thermal, or mechanical in nature. Furthermore, multiple stimuli can converge on a given TRP channel to evoke additive, supra-additive, or antagonistic activities.

Mutagenesis studies on many TRP channels, coupled with the recent solution of the TRPV1 atomic structure, have provided some insights into TRP channel polymodality. Examination of TRPV1 structures, solved in the absence vs. presence of exogenous activators [5,6], has revealed that multiple domains previously implicated in responsiveness to various agonists (a vanilloid binding pocket between transmembrane domains 3 and 4, a pore turret domain adjacent to the selectivity filter, the ankyrin repeat region that can bind and be modulated by intracellular ATP and calmodulin) are connected via conserved motifs (a linker prior to the first transmembrane domain, a helix between transmembrane domains 4 and 5, the helical TRP domain distal to transmembrane domain 6, a short pore helix adjacent to the selectivity filter) to not one, but two gates within the channel pore. The first gate is located toward the cytoplasmic end of the pore, at a site where the sixth transmembrane helices of each of the four subunits come in close proximity to restrict ion access to the pore. A similar (though not identical) gate has long been recognized in the voltage-gated channel family. The second, and somewhat surprising, gate in TRPV1 is located within the selectivity filter itself, which widens measurably with strong stimulation. The existence of this second gate explains why stimuli like protons and the tarantula-derived double-knot vanillotoxin activate the channel on binding to an adjacent pore turret region and may provide a hint at the structural basis for the observation that large cation permeability through TRPV1 is augmented by strong, persistent channel stimulation. Although the extreme carboxyl terminal domain of TRPV1 was not included in the structures solved to date, previous work suggests that phosphorylation of this domain, or its interaction with calmodulin, phosphoinositides, and other regulators [41], offers further opportunities for coupling with the inner and outer gates, most likely via the allosteric linker domains described earlier. Mutagenesis studies further suggest that similarly multiple, parallel but interconnected mechanisms for channel activation exist in other TRP channels [42]. Moreover, TRPV1 subunits, and thus probably subunits in other TRP channels, are clearly not autonomous of one another within the tetrameric holochannel. Rather, their regulatory and allosteric coupling domains are intricately interlaced with one another [5,6].

This remarkable interconnectedness among channel domains and subunits renders plausible a view of TRP channels as being exceptionally well poised to respond to diverse environmental signals at the slightest provocation and to function as coincidence detectors of multiple stimuli. This view is consistent with the observation that point mutations leading to constitutively active TRP channel function have been observed in many of the regulatory domains described earlier [32]. It also provides a potential explanation for how disease-related changes in the multitude of TRP channel regulators might manifest as disease. Given the numerous structural interactions required to maintain such allosteric complexity and thereby avoid unfettered calcium influx into the cell, it is amazing that, outside of a few well-conserved regions, the primary sequences of the many TRP channel family are actually quite divergent. For the same reason, it is a wonder that there are not more TRP channelopathies. Perhaps there are, but their incompatibility with life masks their true frequency.

Therapeutic Strategies Based on TRP Channel Modulation

The prevalence of TRP channel involvement in human disease creates numerous opportunities, in principle, to develop therapeutic strategies based on these channels. A number of these strategies are described in detail in later chapters. In general, they fall into several categories outlined here.

(1) TRP channel antagonists. In the case of diseases arising from TRP channel hyperactivity, specific small molecule antagonists offer the prospect of directly suppressing this activity. This strategy has been greatly facilitated by the fact that many TRP channels are druggable, in that they possess binding sites for small molecules such as the natural products (e.g., capsaicin, resiniferatoxin, cannabinoids, menthol) that have been used to characterize their functions. High throughput screens, often based on TRP channel-mediated influx of calcium in response to chemical activation, have enabled the isolation of numerous small molecules with high affinity and, in some cases, exquisite selectivity for particular TRP channel subtypes. As will be seen in later chapters, one potential complication associated with the therapeutic application of TRP channel antagonists, even if they are highly target specific, is interference with desirable physiological functions such as regulation of body temperature [8]. One approach to circumventing this potential hazard has been to develop peptides that prevent TRP channels from interacting with sensitizing binding partners. For example, A-kinase anchoring protein (AKAP) is an adaptor protein that facilitates the phosphorylation of TRPV1 by protein kinase C or protein kinase A. In animal models, peptides that mimic the TRPV1-recognition motif of AKAP have been shown to interfere with this interaction and thereby suppress hyperalgesia without impairing normal acute nociceptive function [43]. As an alternative strategy to avoid side effects, the complexity of TRP channel regulation described earlier also creates the potential opportunity to finely tune antagonists to interfere only with selected modalities of channel activation [8].

(2) TRP channel agonists. In certain disease states, it may be therapeutically desirable to stimulate, rather than inhibit, one or more TRP channels. Sometimes, the goal might be to exaggerate the normal physiological function of the channel for positive cellular benefit. For example, as described in Chapter 14, activators of TRPM8 might be useful to treat chronic pain because primary afferent inputs from cool-sensitive neurons expressing TRPM8 can apparently antagonize, at the spinal circuit level, inputs related to noxious heat [44]. In other cases, the goal is to achieve cytotoxicity through TRP channel-mediated calcium influx and thereby remove or neutralize a particular cell type. In the case of TRPV1, humans have been practicing this latter strategy for millennia through the regular consumption of spicy foods [45]. Whereas, acutely, exposure to capsaicin causes pain, prolonged exposure to this compound, or a more potent TRPV1 agonist, resiniferatoxin, desensitizes nociceptive neurons and eventually results in degeneration of nociceptor terminals (Chapter 6). Indeed, it was this observation that first led to the recognition that nociceptors constitute a neurochemically distinct subset of sensory neurons. As described in Chapter 7, this strategy has been explored not only for the treatment of pain, but also for the treatment of hyperactive bladder. Of note, this strategy is not necessarily aimed at specifically reducing the activity of the target TRP channel, but rather at more generally eliminating the function of the cell in which it is expressed. Another proposed use of cytotoxic hyperstimulation of TRP channels is to bring about apoptosis in tumor cells (Chapter 22).

(3) TRP channel cargos. An especially clever TRP channel based therapeutic strategy exploits the unusual ability of some TRP channels, mentioned earlier, to mediate the influx of relatively large cations (mol. wt. > 600 Da). The best example of this strategy again involves TRPV1 and is based on the coadministration of capsaicin with a relatively large cationic molecule (QX314) that inhibits voltage-gated sodium channels through action at the intracellular end of the sodium channel pore [46]. Because capsaicin facilitates the entry of this cation through TRPV1, nociceptive neurons that express TRPV1 can be selectively loaded with relatively high concentrations of the sodium channel blocker, achieving therapeutically useful local concentrations without subjecting the recipient to potentially toxic systemic doses.

(4) TRP channel gene therapy. An admittedly more ambitious approach that might prove useful to treat diseases resulting from TRP channel gain-of-function is genetic manipulation of TRP channel expression or sequence. For example, one could introduce an exogenous copy of a TRP channel cDNA, by viral transduction or other methods, to either rescue TRP channel hypofunction or to selectively drive ectopic expression of cytotoxic TRP channels in target cells to enhance agonist-stimulated elimination of those cells. RNA interference, antisense cDNAs, might be used to selectively reduce the expression of gain-of-function mutant TRP channels, whereas dominant negative TRP channels could be used to suppress the function of hyperfunctional or overly abundant endogenous TRP channels. Alternatively, gene editing using Cas9/CRISPR [47] or related tools might be used to repair mutated TRP channel genes or manipulate their promoters to modify channel expression.

The diversity of TRP channels, their extraordinary physiological and pathophysiological importance, and the plethora of review articles and book chapters on these topics have inspired the invention of many variations of the TRP acronym. As will be evident throughout this book, TRP channels can fairly be viewed as both the problem and the potential solution in many human disease states. In keeping with the acronym tradition, it is therefore suggested that the reader view this incredible family of ion channels through the lens of their promise as Targets for the Resourceful Physician.

Acknowledgment

M.J.C. is an inventor on a patent on the use of products related to TRPV1 and TRPV2, which is licensed through UCSF and Merck. This conflict is being managed by the Johns Hopkins Office on Policy Coordination.

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Chapter 2

Transient Receptor Potential Dysfunctions in Hereditary Diseases

TRP Channelopathies and Beyond

Balázs István Tóth¹,²,*; Bernd Nilius¹,*    ¹ Katholieke Universiteit of Leuven, Department of Cellular and Molecular Medicine, Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), Campus Gasthuisberg, Leuven, Belgium

² DE-MTA Lendület Cellular Physiology Research Group, Department of Physiology, University of Debrecen, Medical and Health Science Center, Research Center for Molecular Medicine, Debrecen, Hungary

* Corresponding authors: [e-mail], [e-mail]

Abstract

TRP channels are important in the maintenance of the normal cellular homeostasis, monitor the external and internal environment sensing various physical and chemical stimuli, and also play a significant role in the pathomechanism of various acquired and inherited diseases. In the last decade, an emerging number of mutations in the 28 mammalian TRP channel coding genes were described as a primary cause of hereditary diseases called TRP channelopathies. In this review, we not only focus on those primary TRP channelopathies but also discuss the potential etiological role of TRP channels in various additional hereditary diseases.

Keywords

TRP channels

Channelopathies

Hereditary diseases

Mutation

Pathophysiology

Altered channel properties

Outline

Introduction   13

TRPC Channelopathies   14

TRPV Channelopathies   17

TRPM Channelopathies   20

TRPA Channelopathies   23

TRPML Channelopathies   23

TRPP Channelopathies   24

Conclusions   25

Acknowledgements   26

References   26

Acknowledgments

We thank all members of the Laboratory of Ion Channel Research, KU Leuven, Department Cellular and Molecular Medicine for constructive discussion. We thank especially Grzegorz Owsianik (Leuven) for his input in an early phase of this project. For the work on this review, B. I. T. was supported by the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA Grant agreement no. 330489. B. N. was supported by the KU Leuven in his position as Emeritus met opdracht.

Introduction

In the broadest sense, channelopathies can be defined as diseases associated with malfunction of ion channels or their regulatory proteins. Although this definition covers both congenital and acquired forms, generally only hereditary diseases are referred to as channelopathies in which disease mutations in genes encoding ion channel subunits or regulatory proteins play an etiological role [1]. In hereditary transient receptor potential (TRP) channelopathies, a TRP channel is affected by the mutation. Recently, several hereditary TRP channelopathies have been described, and they have been discussed in many comprehensive reviews [2–5]. The increasing number of TRP channel-related diseases highlights these channels as novel pharmaceutical targets and also provide insight into its physiological function [6]. In this review, we describe hereditary channelopathies and also mention examples with available genetic evidence to explain several putative pathological conditions in which TRP dysfunction is suggested, although the primary mutations affect other genes. We refer also for in-depth information the OMIM link channelopathies.

TRPC Channelopathies

TRP channels are linked to diseases since their first description. The discovery of the founding member of the TRP superfamily, the Drosophila TRP channel, was already due to a drosophila channelopathy disrupting the phototransduction and resulting in blindness of the fruit fly [7]. The closest mammalian relatives of the Drosophila TRP, members of the canonical (TRPC) subfamily, have been linked to many acquired diseases affecting, among else, cardiovascular and respiratory systems, skin, inflammatory processes, and probably neurodegenerative diseases [8], but there are only few examples for real hereditary TRPC channelopathies. In this review, we will not refer to hereditary diseases linked to store-operated (STIM/ORAI/TRPCs ?) Ca² + channels, although evidence has been reported for their involvement in several disease (e.g., severe combined immune deficiency [9], primary Sjogren’s syndrome [10], and tubular-aggregate myopathy [11]).

TRPC1 can play a role in several skin diseases [12], including a few hereditary ones. Recently, it was discussed to be involved in the Gorlin (or Gorlin-Goltz) syndrome, a rare basal cell nevus syndrome with autosomal dominant hereditary (OMIM 109400). The syndrome has 100% penetrance and variable expressivity characterized by odontogenic keratocysts of the mandible, postnatal tumors, and multiple basal cell carcinomas (BCCs). Although it is mostly linked to mutations in the tumor suppressor gene PTCH1, a member of the patched gene family and receptor for sonic hedgehog, in some cases the TRPC1 gene was suggested to be involved in the development of many postnatal tumors [13]. Indeed, the lack of TRPC1 (and TRPC4) was also correlated with failure of differentiation in BCC cells [14]. The autosomal-dominant inherited skin malady, Darier(-White) disease (DD) or keratosis follicularis, characterized by hyperkeratotic papules, might also be connected to TRPC1 malfunction, although the primary causes are mutations in the atp2a2 gene encoding the SERCA2b endoplasmic reticulum Ca² + pump [15]. In DD patients’ keratinocytes, increased protein expression and TRPC1-mediated Ca² + influx were detected, which can contribute to the augmented proliferation and survival of DD keratinocytes [16]. Beyond the skin, TRPC1 can be associated with other hereditary diseases. For example, a novel spliced isoform of TRPC1 with exon 9 deletion (TRPC1E9del) was reported in a human ovarian adenocarcinoma cell line, and its role (together with other TRPC isoforms) in the proliferation and differentiation is also discussed [17]. In a genome-wide association study, SNPs in TRPC1 were discovered that were associated with type 2 diabetes [18]. The role of TRPC1 and ORAI1 might also be implicated in several angiogenesis syndromes leading to tumor neovascularization, which are frequently due to mutations in the Von Hippel Lindau tumor suppressor gene [19].

TRPC3 is mostly linked to the central nervous system by hereditary diseases. In mice, a gain-of-function mutation in TRPC3 (T635A) caused degeneration of cerebellar Purkinje cells and a loss of type II unipolar brush cells, resulting in a cerebellar ataxia, the moonwalker mouse phenotype [20,21]. A single base pair polymorphism (rs13121031) located within the CpG island in the alternative promoter of the human TRPC3 gene was also connected to cerebellar ataxia and heart hypertrophy [22]. Although the link between TRPC3 and cerebellar ataxia is fairly strong in the aforementioned mouse models, there has not been any evidence presented in humans. However, a genetic screen for TRPC3 mutations in patients with late-onset cerebellar ataxia does not support a contribution of TRPC3 mutants to this disease [23]. TRPC3 might be indirectly targeted in various inherited diseases affecting the nervous system. One of them is the autosomal-dominant Spinocerebellar ataxia type 14 (SCA14) primary caused by mutations in PKCγ. Wild-type PKCγ negatively regulated TRPC3 channels, whose regulation was impaired in cerebellar Purkinje cells transfected with the S119P mutant isoform resulting in increased postsynaptic current amplitudes. This alteration could contribute to disruptive synapse pruning disturbing synaptic transmission and plasticity found in SCA14 patients [24]. TRPC3 might also be involved in another neurodevelopmental disorder, the Williams-Beuren syndrome, which is associated with hypercalcemia and heart or blood vessel problems. The main genetic defect generally lays in the transcription factor IIi gene that encodes TFII-I, which normally suppresses cell-surface accumulation of TRPC3, i.e., mutations in TFII-I can cause a TRPC3 gain-of-function due to increased protein expression in the plasma membrane [25]. The pervasive developmental disorder Rett syndrome (RTT), affecting mostly female patients and causing mental retardation, is a progressive neurodevelopmental disorder that can also be linked to TRPC3. RTT is caused by mutations in the gene MECP2 (methyl CpG binding protein 2) encoding a transcriptional regulator protein with mostly repressive functions [26]. TRPC3 has been identified recently as target of MeCP2 transcriptional regulation, and it was suggested to be involved in the impaired brain-derived neurotrophic factor signaling in RTT [27]. An SNP in TRPC3 (rs6820068) was also found to be associated with the risk to develop immunoglobulin A-induced nephropathy (IgA nephropathy, IgAN) in women; the prevalence of the SNP was 23% vs. 12% in female patients and healthy controls, respectively [28]. Some pharmacological evidence proposed that excessive Ca² + influx via TRPC3 contributed to Ca² + toxicity in pancreas and salivary gland, whose symptoms are characteristic for acute pancreatitis and Sjögren syndrome, a systemic autoimmune disease, in which immune cells destroy exocrine cells in tear glands, pancreas, and salivary glands [29].

TRPC4 has not been directly connected to any channelopathy yet. However, a genetic association study has shown some link between TRPC4 SNPs and generalized photosensitive epilepsies and related symptoms [30]. Furthermore, a missense SNP caused gain-of-function mutation in TRPC4 (I957V) that was suggested to be protective against myocardial infarction [31].

TRPC6, with other TRPC channels, was linked to infantile hypertrophic pyloric stenosis (IHPS) (OMIM 179010), the most common gastrointestinal obstruction disease in infancy with genetic background affecting the smooth muscle of the pylorus. A linkage analysis in IHPS identified SNPs in two genetic loci involving TRPC5 and TRPC6 [32] and later also SNPs affecting TRPC1. An SNP in the promoter region and a missense variant in exon 4 of TRPC6 are hypothesized as putative causal gene variants [33]. However, another study carried out on Chinese patients and healthy controls has not found association between IHPS and other SNPs in TRPC6 [34].

TRPC6 plays an important role in glomerular diseases in the kidney. Among them, several cases of focal and segmental glomerulosclerosis (FSGS type 2) (OMIM 603965) are considered as a real TRPC6 channelopathies; currently, at least 15 mutations in the N- and C-terminus of the TRPC6 gene have been described and linked to FSGS type 2 (for review, see Ref. [35], new mutations in Refs. [36,37]). FSGS is functionally characterized by proteinuria and progressive decline of renal function caused by malfunction or loss of podocytes. Podocytes are highly specialized epithelial cells lining the Bowman’s capsule and playing a key role in the function of the glomerular filtration barrier. Although it is not fully understood, yet, how mutations in TRPC6 lead to dysfunction or death of podocytes impairing glomerular permeability and filtration and finally resulting in FSGS, the investigation of the mutants’ phenotypes highlighted two most probably interdependent mechanisms: altered channel functions and impaired interactions with other proteins. The distorted protein-protein interaction can consequently alter regulation and/or trafficking of the channel, significantly influencing channel properties or expression. In podocytes, TRPC6 associates with the transmembrane protein nephrin, which is coupled to the nephrin-interacting adapter protein, CD2AP, and to podocin. This complex forms the slit diaphragm, the crucial component of the glomerular filter. Nephrin is known to negatively regulate the expression of TRPC6 in the plasma membrane ([38], for a review, see Ref. [39]). By the mechanism, nephrin was shown to inhibit TRPC6-PLC-γ1 interaction, which seems to be crucial in the membrane trafficking of the channel. Some of the described mutations (e.g., P112Q, N143S, S270T, R885C, E897K) may affect the nephrin binding site of the TRPC6, making it less sensitive for the nephrin-dependent negative regulation, which results in higher surface expression and enhanced TRPC6-mediated Ca² + entry [40]. Although it is a fact that most of the TRPC6 mutations described in FSGS are associated with a gain-of-function phenotype and TRPC6-mediated calcium entry was found to mediate both angiotensine-II and albumin overload-induced loss of podocytes [41,42], downstream mechanisms, i.e., how overactivation of TRPC6 destroys the slit, are still under discussion. A very likely mechanism is the activation of nuclear factor of activated T-cells (NFAT) found in TRPC6 mutants. This effect was blocked by inhibitors of calcineurin, calmodulin-dependent kinase II, and phosphatidylinositol 3-kinase, but was found to be independent of Src, Yes, or Fyn ([43,44]; see for a review, Ref. [45]). Moreover, angiotensin II-induced Ca² + entry via TRPC6 further increased the expression of the channel via calcineurin-NFAT signaling forming a positive feedback loop [41]. Recently, the Wnt/β-catenin and the MAP kinase ERK1/ 2-associated signaling pathways have also been suggested to be involved in the pathogenesis of TRPC6-mediated diabetic podocyte injury [46,47]. Interestingly, vitamin D downregulated the enhanced TRPC6 expression in podocytes through a direct effect on TRPC6 promoter activity, which might contribute to the antiproteinuric effect of vitamin D [48]. It has to be mentioned that the effect of TRPC6 overactivation can be context dependent: for example, acute activation of TRPC6, at least in mice, rescues podocytes from complement-mediated damage; however, chronic overactivation seems to play an etiological role in FSGS [49]. TRPC6 is also involved in the steroid-resistant nephrotic syndrome (SRNS) (OMIM 600995). Three mutations and an intronic nucleotide substitution were described in the sporadic form of this disease [50]. An additional SNP in the promoter region of TRPC6 was also described, which resulted in enhanced transcription in vitro and correlated with an increased protein expression in the kidney of SRNS patients [51]. Mutations in TRPC6 may also contribute to the idiopathic pulmonary arterial hypertension, where an SNP in the promoter region was found more frequently in a cohort of patients. This mutation facilitated the binding of the inflammatory and carcinogenic transcription factor nuclear factor-κB and resulted in abnormally enhanced TRPC6 transcription [52]. TRPC6 is also mentioned as a candidate gene for Head and Neck Squamous Cell Carcinoma [53], and its overexpression in leukocytes was also demonstrated in primary open-angle glaucoma [54].

TRPV Channelopathies

Transient receptor potential vanilloid 1 (TRPV1) the best characterized TRP channel, is not yet clearly linked to any hereditary disease. Although its central integrator role in nociception is widely accepted, only a few polymorphisms in TRPV1 are suggested associating with development and maintenance of chronic pain syndromes. Genetic variants of human TRPV1 with M315I mutation were found more frequently in Caucasian females suffering from neuropathic pain [55], and an intronic variant SNP (rs222741) in TRPV1 was found to be associated with migraine in a Spanish population [56]. Interestingly, the aforementioned M315I variation also showed higher frequency in type 1 diabetes-affected patients than in healthy controls in an Ashkenazi Jewish population [57]. In a patient with Miller-Dieker lissencephaly syndrome, an autosomal-dominant congenital disorder characterized by a developmental defect of the brain as a consequence of incomplete neuronal migration, a chromosome 17p13.3 deletion syndrome was identified, which includes, among else, deletion of TRPV1 [58]. A missense (I585V) variant of TRPV1 gene, showing decreased channel activity, was reported to be a potential genetic risk factor of painful knee osteoarthritis [59], and the same substitution also associated with lower risk of the symptoms of active asthma [60]. Another genetic TRPV1 variant (G315C) was linked to a functional dyspepsia in a Japanese population via influencing the upper gastrointestinal sensation [61].

Altered channel function and/or expression of TRPV2 has been widely connected to Duchenne muscular myopathy, diabetes, childhood asthma, and several forms of cancer [3,62–64]. Currently, elevated expression of TRPV2 has been described in induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) of patients affected by Hutchinson-Gillford progeria syndrome (HGPS; OMIM 176670), a rare genetic disorder in which the premature aging in multiple organs leads to early death. Elevated TRPV2 expression might be involved in the pathomechanism: it caused a sustained [Ca² +]i elevation in HGPS iPSC-ECs induced by hypotonicity, which induced apoptosis. However, despite its potential role in several diseases, no real hereditary TRPV2 channelopathy has been detected so far.

TRPV3 is one of the most abundantly expressed TRP channels in epidermal and follicular keratinocytes of both human and rodent skin (for recent reviews, see Refs. [12,65,66]). Olmsted syndrome (OS) (also known as mutilating palmoplantar keratoderma with periorificial keratotic plaques or Polykeratosis of Touraine), (OMIM 614594) is the first described hereditary cutaneous TRP channelopathy and also the first real TRPV3 channelopathy. It is a rare inheritable skin disease characterized by the combination of periorificial, keratotic plaques, bilateral palmoplantar keratodermas, alopecia and associated with dermatosis and severe itching. In humans, three gain-of-function mutations (G573S, G573C, W692G) were identified causing OS (for recent reviews, see Refs. [65–68]). Recently, a new TRPV3 mutant, G573A, has been described in an OS patient, which also causes multiple immune dysfunctions, such as hyper-IgE, elevated follicular T-cells, and persistent eosinophilia [69]. These findings turn the attention toward the immunological alterations of Olmsted syndrome and the potential role of TRPV3 mutants in the immunological dysregulation. The etiological role of TRPV3 mutants is also supported by rodent models DS-Nh mice and WBN/Kob-Ht rats, where two gain-of-function mutations, partly identical with the above ones found in OS, caused autosomal-dominant hairless phenotype associated with dermatitis [70]. Moreover, the hair growth regulatory role of TRPV3 was also evidenced in human [71]. Further supporting the role of TRPV3 as an important channel in skin pathophysiology, an upregulation of TRPV3 was reported in Rosacea, a frequent chronic inflammatory skin disease [72]. Recent studies have suggested that the pathophysiological role of TRPV3 can go beyond skin disorders. Genetic association studies have highlighted a potential role of the channel in primary headache disorders (like migraine, tension-type headache, and cluster headache) with a genetic preposition [73]. TRPV3 SNPs has also been identified in congenital hyperinsulinism of infancy [74].

The TRPV4 coding sequence is a real hot spot of mutations causing channelopathies. Currently, more than 50 mutations in the trpv4 gene have been discovered, causing at least nine different channelopathies. By their symptoms, TRPV4 channelopathies cover skeletal dysplasias and peripheral neuropathies, although mixed forms are also well known, and a clear distinction between these two groups of TRPV4 channelopathies is not always possible. The first recognized TRPV4-related channelopathy, the brachyolmia type 3 (OMIM 113500), affects the skeletal system. It is a relatively mild, autosomal-dominant skeletal dysplasia characterized by short stature, flattened vertebrae (platysspondyly) especially in the cervical region, reduced intervertebral spaces, and scoliosis or kyphosis [75]. This surprising finding triggered intensive research focusing on the newly recognized relationship between TRPV4 and skeletal disorders and resulted in the discovery of new TRPV4 channelopathies among skeletal dysplasias. These diseases share the main symptoms like short stature, platysspondyly, defects in bone ossification, and abnormalities in joints, but their severity shows a high variation not only among the different diseases but also among the different mutations underlying the same symptoms. Despite the variability in the symptoms’ severity, all diseases are probably due to dysfunction and differentiation abnormalities in chondrocytes of the bone growth plate. In the spondyloepimetaphyseal dysplasia Maroteaux pseudo-Morquio type 2 (SEDM-PM2) (OMIM 184095), the manifestations of the preceding symptoms are limited to the musculoskeletal system [76]. In the spondylometaphyseal dysplasia Kozlowski type (OMIM 184252), mainly the vertebrae and the metaphyses are affected. Although, like in the previous cases, the body length is normal at birth, it shows short stature by shortening of the trunk during the development, which reaches the clinical significance mostly between ages 1 and 4 years. Generally, the symptoms are more severe than in brachyolmia type 3 and SEDM-PM2: a prominent feature is platyspondyly again, but severe scoliosis and defects in the distal metaphysis of the femur, the femoral neck, and trochanteric area are also observed. [77]. The most severe skeletal TRPV4 channelopathy is the metatropic dysplasia (OMIM 156530), which is sometimes combined with lethal fetal akinesia. The nonlethal forms are characterized by shortening of all long bones resulting in short limbs, serious enlargement of joints, heavy kyphoscoliosis, severe platyspondyly, and metaphyseal enlargement, as well as defects in ossification [77–79]. Parastremmic dysplasia (PD) (OMIM 168400) is characterized by severe dwarfism, thoracic kyphosis, and distortion and twisting of the limbs [parastremmic (Greek): twisted], contractures of the large joints, malformations of the vertebrae and pelvis, and it can also associate with incontinence [76]. A recently described mild form of skeletal dysplasia is the familial digital arthropathy-brachydactyly (OMIM 606835) which appears in the first decade of life. Short fingers, deviations in finger joints, and irregularities in the articular surfaces characterize this arthropathy [80].

Following the description of an increasing number of TRPV4-caused skeletal dysplasias, the discovery of the causal role of TRPV4 in inherited neuropathies was a big surprise. As of today, three autosomal-dominant distal neuropathies are considered as hereditary TRPV4 channelopathies. Their main symptom is muscle atrophy caused by degeneration of the motoneurons in the spinal ventral horn, leading to muscle weakness and wasting in the distal limbs, but the respiratory system and the vocal cord can be also affected, and sometimes the motor symptoms are associated with sensory defects (for a review, see Ref. [81]). These diseases are congenital distal spinal muscle atrophy (CDSMA) (OMIM 600175), scapuloperoneal spinal muscle atrophy (SPSMA) (OMIM 181405), and hereditary motor sensory neuropathy type IIc (HMSN IIc or Charcot-Marie-Tooth neuropathy type 2C, CMT2C) (OMIM 606071). CDSMA is a nonprogressive lower motor neuron disorder restricted to the lower part of the body. It may associate with arthrogryposis (now also discovered in patients with mutations in the gene encoding the mechanosensory cation channel PIEZO2 [82]), bilateral talipes equinovarus, and flexion contractures of the knees and hips. Sometimes slight skeletal symptoms (e.g., lordosis, scoliosis, restricted joint movements) are also observed, but sensory defects are lacking [83]. SPSMA is a syndrome characterized by scapuloperoneal atrophy, scapular winging, muscle wasting in the lower limbs, absence of tendon reflexes, as well as laryngeal palsy and vocal-cord paralysis. Sometimes scoliosis and light sensory defects are reported [84–86]. In CMTC2C, a variable degree of muscle weakness of limbs, vocal cords, intercostal muscles, and sensoneurial hearing loss are the leading symptoms, but bladder urgency or incontinency are also common. It is often associated with slight skeletal or arthrial symptoms like club foot (talipes), congenital joint contractures (arthrogryposis), or scoliosis, but facial asymmetry, tongue fasciculations, and third and sixth cranial nerve palsies have also been reported. CMTC2 starts in infancy or childhood, and the life expectancy is shortened because of respiratory failure [85–88]. The exact pathomechanism by which mutations in TRPV4 are leading to the aforementioned diseases is vaguely understood, as are the reasons for the phenotype variability of TRPV4 channelopathies (i.e., why diverse mutations result in these different diseases) [89]. Although there are some exceptions and controversies, most of the disease-causing mutations show a gain-of-function phenotype, and there are speculations that the degree of channel overactivity might determine the severity of the disease ([90,91]; for reviews, see Refs. [89,92,93]). The increased, or at least altered, Ca² + signaling via TRPV4 can result in altered neurogenesis, altered gene expression, or even cell death. On the other hand, mutations can affect the association of TRPV4 subunits with each other or other molecules influencing channel formation, interaction with cytoskeletal elements, cellular trafficking, or spatial distribution; the latter can have a significant effect on the differentiation of polarized cells like osteocytes or neurons. Indeed, if we have a look at the distribution of the mutations along the amino acid sequence of the channel, three hot spots can be identified for disease-causing mutations: (1) the ankyrin-repeat-domain (ARD) on the N-terminus, (2) the transmembrane region S3-S5, and (3) a C-terminal region were the channel associates with several members of the cytoskeleton, such as tubulin, actin, and MAP7 [94]. Regarding the ARD, the neuropathy-causing mutations are mainly localized in the convex surface of the ARD, but mutations causing skeletal dysplasia, although scattering through the whole length of the channel protein, seem to be more frequently located in the concave surface of the ARD. Because of our limited knowledge, the puzzle created by the large number of mutations often located in the same domain of the channel and the consequent (at least) nine different diseases is still challenging [89]. To make the picture of TRPV4 channelopathies even more complex, we have to mention that TRPV4 is highly expressed in the inner ear and the urothelium; therefore, it is not surprising that some patients also have hearing problems or bladder symptoms such as overactive bladder and incontinence (for a review, see Ref.