In 1969, a Drosophila melanogaster mutant was described that exhibits a defect in the response of photoreceptor cells to sustained light stimulation of moderate to high intensities, which was later named “transient receptor potential” or trp. The cDNA of the mutated gene was cloned in 1989 and subsequently shown, by patch-clamping Drosophila photoreceptor cells, to code for ligand-gated and Ca2+-permeable ion channels. As the first identified member of such a class of ion channels, up to then completely elusive, these discoveries triggered an intense search for mammalian homologues of the Drosophila TRP channel. After the initial, quick discovery of the seven members of the TRPC (C for canonical or classical) subfamily (displaying the highest homology with the original Drosophila TRP protein), more distantly related subfamilies were discovered (Minke 2010; Hardie 2011; Montell 2011). The mammalian TRPM (M for melastatin, the original name of the first identified member of this group) subfamily was found to contain eight members, and TRPM3 was the last of them to be cloned and functionally investigated (Montell et al. 2002).
TRPM3 Gene Structure, Isoforms, and Expression
The TRPM3 gene is the largest gene on the human chromosome 9 and exhibits very high homology among mammals. Homology also exists to invertebrate trpm genes; most notably, mammalian TRPM3 is the closest homologue of the sole trpm gene in the genome of Drosophila melanogaster. The human and murine TRPM3 genes have 28 known exons (Oberwinkler and Philipp 2014) that are translated into a multitude of different proteins due to alternative splicing, alternative exon usage, and the usage of different start codons (Oberwinkler and Philipp 2014). Only two splice events (out of at least eight sites, at which isoform diversity is generated) have been investigated functionally. Exon 13 appears to be indispensable for channel function and has thusly been termed ICF-region (Frühwald et al. 2012); exon 24 impacts the ionic permeability (see below). In the intron between exons 8 and 9, the TRPM3 gene harbors a micro-RNA (miR-204). This arrangement is paralleled in the TRPM1 gene, which is the closest relative of TRPM3. The micro-RNA inside the human TRPM1 gene (miR-211) also has very high homology in its sequence to miR-204. The high sequence similarity of both the host gene and the micro-RNAs, together with the similar genomic arrangement, strongly argues that TRPM1 and TRPM3 arose out of a sequence duplication event.
Up to now, the expression pattern of miR-204 has been found to be identical to the exonic host TRPM3 sequence, indicating that miR-204 is transcribed together with TRPM3 and subsequently spliced out of the resulting TRPM3 pre-mRNA. While it should be noted that one publication reported that miR-204 negatively regulates TRPM3 expression (Hall et al. 2014), the function and the biological effects of miR-204 will not be covered by this chapter.
TRPM3 expression is widespread, but not ubiquitous. Cell types, in which TRPM3 expression has been reported, include: epithelial cells of the choroid plexus, dermal papillar cells, synovial fibroblasts, vascular smooth muscle cells, prostate, and kidney cells. Also, TRPM3 expression has been demonstrated in hormonal cells, such as pancreatic β cells and cells from the anterior pituitary. Strong expression has been reported from neurons of various brain regions (including the hippocampus/dentate gyrus, hypothalamus, and cerebellum) and also from oligodendrocytes. TRPM3 seems to be expressed in odontoblasts and periodontal ligament cells, but most prominently, TRPM3 expression has been detected in sensory organs, such as the inner ear (organ of Corti), several structures in the eye (ciliary body, iris, lens epithelial cells, retinal pigment epithelium, and various cell types of the retina, including some types of bipolar cells, retinal ganglion cells, and Müller cells), and small-diameter cells of the somatosensory system (dorsal root ganglia and trigeminal ganglia). A more complete list with references can be obtained from recent reviews (Oberwinkler and Philipp 2014; Held et al. 2015a). It must be emphasized that often TRPM3 expression has only been demonstrated on the mRNA level (either by PCR-based techniques or by in situ hybridization). Localization of TRPM3 in tissues has been hampered by the lack of good and/or appropriately validated antibodies (although convincing data have been produced with some antibodies using Western blots). Only in very few tissues, expression of functional TRPM3 channels has been demonstrated with physiological techniques.
Permeation of Ions Through TRPM3 Channels
TRP proteins, in general, have been shown to form tetrameric ion channels, where each subunit possesses six transmembrane domains, between the fifth and the sixth of which is located the ion-permeating pore. The same arrangement is assumed (and generally accepted) for TRPM3 proteins, although no experimental proof of these assumptions has been reported for these channels.
The first studies on TRPM3 function (using heterologous TRPM3 overexpression in HEK293 cells) found that TRPM3 channels display a measurable spontaneous activity (Grimm et al. 2003; Lee et al. 2003), allowing to identify TRPM3 as cation-selective, Ca2+-permeable ion channel. However, one splice event (at exon 24) of TRPM3 affects the pore region of TRPM3, resulting in two proteins that form ion channels with very different properties. The inclusion of 12 amino acids (resulting in a “long-pore” isoform) leads to the formation of channels that are highly active under standard conditions but have only little permeability for divalent cations. On the contrary, after overexpression of the short-pore isoform of TRPM3 (termed TRPM3α2), ion channels are found that exhibit only little spontaneous activity (partly due to a block by extracellular Na+ ions) but are highly permeable to divalent cations (Oberwinkler et al. 2005; Wagner et al. 2010; Vriens et al. 2014). Subsequent studies have exclusively focused on the divalent-permeable, short-pore TRPM3 variant, completely ignoring the existence of long-pore TRPM3 isoforms. With the exception of the choroid plexus (Frühwald et al. 2012), it is not yet known where (and under which conditions) mRNAs for long-pore TRPM3 proteins are produced.
The short-pore variant TRPM3α2 has about ten times larger relative permeability for Ca2+, Mg2+, and Zn2+ than for the monovalent cations Na+ and K+ (Wagner et al. 2010). It has been reported that under approximately physiological conditions, this high divalent permeability translates to ∼40% of the current through TRPM3α2 being carried by Ca2+ ions (Vriens et al. 2014).
Addition of clotrimazole to TRPM3α2 channels activated by steroid agonists (see below) leads to a profound shift in biophysical behavior of these channels: The I/V relationship changes from outwardly rectifying to sigmoidal or doubly rectifying, and the relative permeability for Ca2+ decreases sharply, as does the fraction of current being carried by Ca2+. Equally, the single channel events of TRPM3 channels display profound alterations, indicating a second mode of operation (Vriens et al. 2014). Astonishingly, pharmacological and mutational analysis of these phenomena strongly indicated that TRPM3 channels form two separable and thus independent ionic pathways through the plasma membrane, one being highly Ca2+ permeable and outwardly rectifying, while the other, termed ω-pathway, being monovalent-selective and inwardly rectifying (Vriens et al. 2014). Recently, a synthetic compound was described (CIM0216) that activates both pathways simultaneously (Held et al. 2015b). Ω-pores have been described in a variety of voltage-gated ion channels, but so far, TRPM3 is the only TRP channel with this property. At the moment, the ω-pore of TRPM3 can only be observed after activation by exogenous, pharmacological substances and physiological conditions leading to the activation of this pathway still await discovery.
In the remainder of this chapter, the term TRPM3 is used for short-pore, divalent-permeable TRPM3 isoforms, such as TRPM3α2, or for endogenously expressed TRPM3 proteins, which typically have not been characterized with respect to the precise isoforms involved.
Activation of TRPM3 Channels by Physical Stimuli
Like many other TRP channels (such as TRPV1, TRPV3, or TRPA1, collectively termed “thermoTRPs”), TRPM3 channels are activated by elevated temperatures (Vriens et al. 2011); this has been recently reviewed by Held et al. (2015a). This behavior has been described by a two-state model that assumes that the opening and closing rates are temperature dependent. TRPM3 channels, again like the other thermosensitive TRP channels, also show voltage dependence, which can be incorporated in the same model (Vriens et al. 2011). Since the single-channel conductance of TRPM3 has been shown to be linear with voltage (Vriens et al. 2014), the strong outwardly rectifying I/V relationship observed in whole-cell recordings appears to be a reflection of the voltage dependence of TRPM3. Activating TRPM3 with chemical agonists (see below) then can be conceptualized as shift of the voltage and temperature dependence to lower values, i.e., the chemical and physical stimuli act synergistically to activate TRPM3 channels. It is noteworthy that the temperature at which TRPM3 channels are activated half-maximally is higher compared to TRPV1 channels (Held et al. 2015a).
Apart from voltage and temperature sensitivity, it was reported that TRPM3 channels are activated by exposure to hypotonic extracellular solutions (Grimm et al. 2003). Because these early data have not been repeated, it is unclear how they relate to the newer concepts outlined above or to the pharmacological findings on TRPM3 discussed below. This clearly is an area in urgent need of more investigation.
Pharmacology of TRPM3 Channels
The pharmacology of TRPM3 channels has recently been reviewed in detail (Oberwinkler and Philipp 2014; Held et al. 2015a). Four chemically different substances have been reported to activate TRPM3 channels: D-erythro-sphingosine (Grimm et al. 2005), nifedipine, the steroid pregnenolone sulfate (Wagner et al. 2008) and, recently, CIM0216 (Held et al. 2015b). Of these substances, only D-erythro-sphingosine and pregnenolone sulfate are endogenous substances produced in the body. However, fairly high concentrations (in the micromolar range, which is likely higher than the endogenous concentration) are needed to activate TRPM3 channels, casting doubt whether these substances are indeed physiologically regulating the activity of TRPM3 channels. For pregnenolone sulfate, it has been shown that the aforementioned synergism between temperature and agonist-induced activity leads to activation of TRPM3 at body temperatures already at much lower pregnenolone sulfate concentrations (∼100 nM), making a physiological regulation of TRPM3 activity by pregnenolone sulfate more conceivable (Vriens et al. 2011). The dihydropyridine and the steroid pregnenolone sulfate have been shown to act allosterically on different binding sites of TRPM3 (Drews et al. 2014). As an experimental tool, pregnenolone sulfate has been the most popular choice for activating TRPM3 channels, likely because this substance is chemically reasonably stable, acts rapidly from the extracellular side, and can quickly be washed out again (Wagner et al. 2008).
A large list of substances from diverse chemical groups have been described to inhibit TRPM3 channels (reviewed in Oberwinkler and Philipp (2014)), including steroids, such as cholesterol and progesterone (Naylor et al. 2010; Majeed et al. 2012), fenamates (Klose et al. 2011), and dihydropyridines, such as nimodipine (Drews et al. 2014). The most potent of the inhibitors described so far is the flavonoid isosakuranetin (Straub et al. 2013). Recent additions to the list of inhibitors are U73122, a compound often used to inhibit phospholipases C (Leitner et al. 2016), the analgesic diclofenac (Suzuki et al. 2016), and the antiepileptic drug primidone (Krügel et al. 2017).
Regulation of TRPM3 by Intracellular Signaling Molecules
TRPM3 channels are inhibited by intracellular Mg2+ ions with an IC50 value of ∼1 mM that might lie in the physiological range, indicating that changes of the Mg2+ concentration could have an impact on TRPM3 channel activity (Oberwinkler et al. 2005). However, it is unknown if this regulatory mechanism is physiologically relevant.
More recently, TRPM3 channels have been shown to be regulated by PI(4,5)P2 and possibly other polyphosphated phosphoinositides such as PI(3,4,5)P3 (Badheka et al. 2015; Tóth et al. 2015; Uchida et al. 2016). This finding indicates that TRPM3 channel activity could be controlled by receptors coupling to phospholipases or phosphokinases. Again it has not yet been established if this regulatory mechanism is relevant in vivo. Interestingly, TRPM3 channels activated by nifedipine have been reported to be less (or not at all) sensitive to PI(4,5)P2 depletion, in contrast to the same channels activated by pregnenolone sulfate (Uchida et al. 2016). Although the mechanistic details of this difference are not understood, it reinforces the idea that the dihydropyridine nifedipine and the steroid pregnenolone sulfate act differently on TRPM3 channels.
Finally, up to four binding sites of the Ca2+-binding protein calmodulin have been predicted bioinformatically. Two of them in the N-terminal region of TRPM3 have been verified by biochemical binding assays of calmodulin to peptide fragments of TRPM3. Interestingly, these calmodulin-binding sites have also been proposed to be able to bind the protein S100A and/or to PI(4,5)P2, suggesting competition between these binding partners (Holakovska et al. 2012; Holendova et al. 2012). If this proposed binding actions and competition occurs in vivo is unknown.
Function of TRPM3 in Pancreatic β Cells
Using pregnenolone sulfate to probe for functional TRPM3 channels, these channels were found to be endogenously expressed in the insulin-secreting β cells of mouse Langerhans islets, but not in the α cells (Wagner et al. 2008; Klose et al. 2011; Mayer et al. 2011). Subsequently, also human pancreatic islets were found to express TRPM3 (Marabita and Islam 2017). Activation of TRPM3 channels in these cells induces Ca2+ influx that in turn increases insulin secretion and the activation of a signal transduction cascade (Wagner et al. 2008; Mayer et al. 2011; Thiel et al. 2013). However, both of these effects require supraphysiological concentrations of TRPM3 agonists. Additionally, TRPM3-deficient mice do not display altered glucose homeostasis at baseline (Vriens et al. 2011). These points show that TRPM3 channels are not necessary for the normal function of pancreatic β cells, indicating that the function of TRPM3 channels in these cells is not yet understood.
Function of TRPM3 in Somatosensory Primary Neurons
The cell bodies of somatosensory neurons are found in the dorsal root ganglia or the trigeminal ganglia. As judged by in situ hybridization, ∼80% of these cells express TRPM3 mRNA in mice, and in ∼60% of these cells, functional TRPM3 channels could be demonstrated (Vriens et al. 2011). TRPM3 channels were mainly found in small-diameter, nociceptor neurons. The analysis of TRPM3-deficient mice indicated that TRPM3 channels play a role in the detection of noxious heat stimuli, consistent with the temperature sensitivity of these channels (Vriens et al. 2011). Similar data were obtained by inhibiting TRPM3 channels pharmacologically (Krügel et al. 2017). Additionally, strong activation of TRPM3 channels leads to the release of neuropeptides (e.g., CGRP) from nociceptor nerve endings (Held et al. 2015b).
Interestingly, TRPM3-deficient mice, or mice in which TRPM3 channels were pharmacologically blocked, exhibit reduced thermal hyperalgesia under inflammatory conditions (Vriens et al. 2011; Krügel et al. 2017). Liquiritigenin, a TRPM3-inhibiting flavonoid, reduced neuropathic pain in a rat model (Chen et al. 2014). These data indicate that TRPM3 plays an important role in thermal nociception and hyperalgesia.
Function of TRPM3 in Other Tissues
In other tissues, the function of TRPM3 channels has been less well studied compared to pancreatic β cells and somatosensory nociceptor neurons. In vascular smooth muscle cells, TRPM3 activity has been reported to reduce interleukin-6 secretion but to increase contractility (Naylor et al. 2010). In the ductus arteriosus, hypotonicity-induced contraction of vascular smooth muscle cells has been implicated in the closure of this duct, and TRPM3 channels have been shown to be strongly expressed in these cells. The reported sensitivity of TRPM3 channels to hypotonic solutions makes them strong candidates to play a role in this process (Aoki et al. 2014). In synovial fibroblasts, TRPM3 activity has been reported to reduce secretion of hyaluronan (Ciurtin et al. 2010).
TRPM3-deficient mice display a deficient light-induced pupillary contraction (Hughes et al. 2012). In this system, it is unclear whether this phenotype stems from altered detection of light in the retina or from deficient contraction of the iris, as both structures are known to express TRPM3.
Role of TRPM3 Channels in Pathological Conditions or Diseases
In addition to the role of TRPM3 channels in thermal nociception, these channels have been strongly associated with inherited cataract and glaucoma (Bennett et al. 2014). Through the analysis of a five-generation pedigree of an affected family, a single point mutation leading to a single missense mutation in TRPM3 was linked to this condition, providing the only inherited human disease associated with TRPM3 identified so far. The mutation affected the patients in an autosomal dominant fashion. Since the amino acid exchange caused by the mutation introduces a novel methionine, the authors proposed that this mutation may add a novel translation initiation site and thus may alter the amino acid sequence of several TRPM3 isoforms expressed in the eye (Bennett et al. 2014). The consequences of these changes for TRPM3 expression and TRPM3 channel function are unknown.
Association of genetic polymorphisms in the TRPM3 gene with diseases has furthermore been suggested for conditions as diverse as chronic fatigue syndrome (Marshall-Gradisnik et al. 2016), aspirin-exacerbated respiratory disease (Narayanankutty et al. 2016), and, in mice, arthritis (Rai et al. 2015).
TRPM3 expression has been found to be altered in several cancers; typically, TRPM3 is upregulated in malignancies (Hall et al. 2014; Alptekin et al. 2015; Biasiotta et al. 2016; Doecke et al. 2016). In clear cell renal carcinomas, Ca2+ influx through TRPM3 channels has been directly implicated in promoting cancer growth (Hall et al. 2014).
TRPM3 channels were the last of the mammalian TRP channels to be cloned and characterized. In the meantime, considerable progress has been made in the development of pharmacological and genetic tools that aid in the elucidation of the physiological and pathophysiological roles of these interesting channels. The analysis of the function of TRPM3 channels remains challenging because of the large number of alternatively spliced isoforms. Nevertheless, TRPM3 channels have been shown to play an important role in the detection of noxious thermal stimuli and to participate in the generation of inflammatory hyperalgesia. A specific mutation of TRPM3 causes inherited cataract and glaucoma. Due to the widespread expression of TRPM3 proteins in various tissues, many more functions of TRPM3 channels are likely to be discovered in the future. For some of the pathological conditions in which TRPM3 channels have been implicated (e.g., for inflammatory hyperalgesia), pharmacological modulation of TRPM3 channel activity may become a therapeutically feasible treatment strategy.
- Frühwald J, Camacho Londoño J, Dembla S, Mannebach S, Lis A, Drews A, et al. Alternative splicing of a protein domain indispensable for function of transient receptor potential melastatin 3 (TRPM3) ion channels. J Biol Chem. 2012;287:36663–72. doi: 10.1074/jbc.M112.396663.PubMedPubMedCentralCrossRefGoogle Scholar
- Hughes S, Pothecary CA, Jagannath A, Foster RG, Hankins MW, Peirson SN. Profound defects in pupillary responses to light in TRPM-channel null mice: a role for TRPM channels in non-image-forming photoreception. Eur J Neurosci. 2012;35:34–43. doi: 10.1111/j.1460-9568.2011.07944.x.PubMedPubMedCentralCrossRefGoogle Scholar
- Marabita F, Islam MS. Expression of transient receptor potential channels in the purified human pancreatic β-cells. Pancreas. 2017;46:97–101. doi: 10.1097/MPA.0000000000000685.
- Marshall-Gradisnik S, Johnston S, Chacko A, Nguyen T, Smith P, Staines D. Single nucleotide polymorphisms and genotypes of transient receptor potential ion channel and acetylcholine receptor genes from isolated B lymphocytes in myalgic encephalomyelitis/chronic fatigue syndrome patients. J Int Med Res. 2016;44:1381–94. doi: 10.1177/0300060516671622.
- Mayer SI, Müller I, Mannebach S, Endo T, Thiel G. Signal transduction of pregnenolone sulfate in insulinoma cells: activation of Egr-1 expression involving TRPM3, voltage-gated calcium channels, ERK, and ternary complex factors. J Biol Chem. 2011;286:10084–96. doi: 10.1074/jbc.M110.202697.PubMedPubMedCentralCrossRefGoogle Scholar