Ocular transient receptor potential channel function in health and disease

Out of the 28 mammalian TRP genes, 6 different thermosensitive TRP isotypes are expressed in the eye. They include TRPV1, 2, 3 and 4 as well as TRPM8 and TRPA1 whose temperature sensitivities cover most of the environmental conditions that mammals encounter. TRPV4 and TRPV3 are activated by temperatures from 25 to 31 °C, respectively, whereas TRPV1 is activated at 43 °C and TRPV2 at a noxious temperature of 51 °C [30]. TRPM8 and TRPA1 on the other hand sense cooling once the temperature drops below 25 and 17 °C, respectively. It is unclear whether one of these three suggested mechanisms can account for the origin of the remarkably steep temperature sensitivity of the thermosensitive TRPs (thermo-TRPs). They include: 1) steep specific temperature dependence for ligand binding; 2) temperature-induced channel rearrangement; 3) temperature-dependent membrane tension changes controlling channel opening [31]. A definitive explanation for why only certain TRPs are thermosensitive remains somewhat elusive. Nevertheless, a recent report suggests that there are specific TRP channel molecular determinants endowing thermal sensitivity. In Drosophila, different TRPA1 isoforms are expressed and the requirement for TRPA1 thermal sensitivity was linked to the expression of a specific 37-amino-acid sequence within its intracellular region (encoded by a single exon) [32].

Cataloging which TRP subtypes elicit specific responses can be problematic if the characterization is based solely on drug-induced effects. This limitation stems from the fact that their effects are not restricted to interacting with a single subtype. Accordingly, transgenic animals are used to assess the impact of either a gain or loss of TRP function on an induced response. Even such a precaution may be confounded by compensatory upregulation of another gene replacing the functional loss resulting from a deleted gene. Despite these possible caveats, current assignments of functional roles of TRP channel subtypes appear to be accurate.

Initially, it was realized that either exogenous small organic synthetic compounds or natural products can activate TRPs. Besides capsaicin, [33] other (synthetic) agonists are icilin (TRPM8) [34] and camphor (TRPV3) [35, 36] which are super cooling agents. Furthermore, endogenous lipids or products of lipid metabolism are also ligands of TRPs. For example, anandamide is an endogenous lipid [37]. Inorganic ions such as Ca²⁺ or Mg²⁺ can also (directly) activate TRPs (e.g. TRPM6 for Mg²⁺) [38] (e.g. TRPA1 for Ca²⁺) [39]. However, it is not yet known if the TRP conformational changes induced by Ca²⁺ or Mg²⁺ are the same as those induced by a thermal transition known to activate these different TRP channel subtypes. Overall, classical activation of TRPs and in particular thermo-TRPs are mostly related to direct activation via mechanical stimuli, channel phosphorylation, certain exogenous and endogenous agents or inorganic ions.

Studies in Drosophila photoreceptors by Devary et al. demonstrated for the first time that the light activated TRP channels and TRPL are targets of G protein-coupled receptor (GPCR) activated rhodopsin, which lead to activation of Gq, PLC and PIP₂ hydrolysis [40]. Another study by Hardie et al. showed for the first time that the DAG branch of PI-signaling (and PUFAs) also activates in Drosophila TRP and TRPL channels [23]. Therefore, TRPs can also be (indirectly) activated by GPCRs and receptor tyrosine kinases (RTKs) activating phospholipase C (PLC). This can occur in different ways such as generation of inositol (1,4,5) trisphosphate (IP₃) and subsequent Ca²⁺ release from intracellular stores, which in turn activates store-operated Ca²⁺ channels (SOCs) composed of TRP subunits such as TRPC1 and TRPC4 [41]. Another way which GPCRs can modulate TRP channel activity is via the generation of diacylglycerol (DAG) (e.g. diacylglycerol-sensitive TRPC3/6/7) [42]. Finally, GPCRs can modulate TRPs via hydrolysis of phosphatidylinositol (4,5) bisphosphate (PIP₂) (TRPM8) [43] (TRPC4) [44].

Besides the TRP channel subtypes mediating retinal phototransduction in the Drosophila eye [1, 2, 45, 46], other subtypes identified in the mouse retina include: TRPC1-4 TRPM1/3/7, TRPML1, TRPP2, TRPV2, TRPV4 [47, 48]. In mouse retinal ganglion cells (RGCs), TRPV4 modulates calcium flux, spiking rate, and apoptosis of these cells [48]. TRPV1 in rat retina contributes to eliciting RGC apoptosis and increased intracellular Ca²⁺ levels during exposure to elevated hydrostatic pressure [49]. Furthermore, TRPC1 and TRPC4 are expressed in chicken retina [50]. In the human retina, TRPM1 expression was detected on ON-bipolar cell dendrites. This suggests a dual function for TRPM1 in the ON-pathway [51]. More specifically, TRPM1 is required for the photoresponse in mouse retinal ON-bipolar cells and it is regulated by the metabotropic glutamate receptor 6 (mGulR6) cascade in ON-bipolar cells [52‐56]. Many human mutations affected this system [53]. Other TRPs like TRPV1, TRPM8 and TRPA1 are expressed in retinal tumor cells (retinoblastoma). On the other hand, TRPA1 is completely suppressed in a retinoblastoma cell line, which is resistant to the cytostatic agent etoposide [57]. In addition, TRPV1-4, TRPM8 and TRPA1 were identified in retinal pigment epithelial (RPE) cells [58, 59]. Increasing the ambient temperature or insulin like growth factor-1 (IGF-1) enhanced vascular endothelial growth factor-A (VEGF-A) secretion rate in RPE cells [58]. TRPs are involved in the IGF-1 induced response [58]. Regarding the human uvea, there is only one study demonstrating gene expression of TRPV1, TRPM8 and TRPA1 [59]. In contrast, in human uveal melanoma cells, the gene expression of TRPM8 is at lower levels whereas the TRPA1 expression is at high levels in healthy uvea [59].

TRPC4 is the first TRP channel subtype identified in human corneal epithelial cells (HCEC). Activation by epidermal growth factor of its cognate receptor, EGFR, transactivates TRPC4. The resulting increases in intracellular trigger mitogen activated protein kinase (MAPK) cascade signaling leading to rises in cell proliferation and migration in vitro and in vivo [60]. Conversely, TRPV1 activation by capsaicin, induces increases in cell proliferation and migration through mediating increases in release of heparin bound EGF, which transactivates EGFR [61]. TRPV1-4 vanilloid are expressed in rat, mice and HCEC [62‐64]. Thermal transitions were used to delineate functional TRPV1-4 [65‐72] and TRPM8/TRPA1 [73, 74] expression.

TRPV1 expression in HCEC has potential clinical relevance since in TRPV1 knockout mice re-epithelialization and restoration of tissue transparency following debridement was delayed in these mice compared to their wild type counterpart. This effect of TRPV1 in promoting cell proliferation and migration is associated with increases in IL-6 and substance P expression, which are coactivators of growth factor induced wound healing [62]. TRPV4 expression in intact human corneal epithelium and its activation by exposure to a hypotonic challenge is required for inducing regulatory cell volume decrease behavior [75]. Furthermore, Pan et al. [76] found that hypertonic stresses identical to those described in some dry eye patient tears elicited TRPV1 channel stimulation leading to rises in proinflammatory cytokine levels through MAPK and NF-kB activation [64, 77]. TRPM8 cold receptor gene, protein and functional expression was detected in HCEC. On the other hand, TRPM8 activation by either temperature lowering or icilin inhibits TRPV1-induced increases in intracellular Ca²⁺ levels (Türker, Mergler et al. unpublished observation 2015).

Functional TRPV1 expression was identified in human stromal fibroblast cultures [78, 79]. Transforming growth factor TGFβ-1 transactivates TRPV1 in these cultures through activation of its cognate receptor, TGFβR. In vivo, a murine corneal alkali burn compromises basement membrane integrity leading to TGFβ stromal infiltration and myofibroblast transdifferentiation, fibrosis and a proinflammatory cytokine, chemoattractant storm followed by immune cell infiltration. These injury-induced effects cause corneal opacification and ulceration [80]. This marked difference in the wound healing outcome resulting from epithelial and stromal TRPV1 activation suggests that TRPV1 antagonist usage in a clinical setting may need to be restricted to cases involving penetrating stromal injury rather than superficial epithelial injury. TRPM8 gene, protein and functional expression was validated in human corneal stromal cells (Türker, Mergler et al. unpublished observation 2015).

Thermosensitive TRPA1 and TRPM8 are also expressed on corneal afferent nerves [81]. Corneal nerve fibers are radially distributed within the stroma and the adjacent limbus [82]. TRPV1 and TRPM8 are highly expressed in mouse, guinea pig and human corneal sensory nerve fibers at levels similar to those in their non-corneal counterpart [81, 83‐85] and in non-corneal primary sensory neurons [30]. These channels desensitize during prolonged exposure to capsaicin (TRPV1) or menthol (TRPM8). In a clinical setting, nerve fiber TRPM8 activation by a super cooling agent may provide an option for treating dry eye syndrome since corneal cooling in humans increased lacrimation, whereas warming had an opposite effect [81]. Parra et al. found that TRPM8 functional expression is needed to maintain ocular surface hydration during exposure to a desiccating stress [81]. Taken together, these results suggest that drug-induced fibroblast TRPV1 and neuronal TRPM8 functional modulation may provide novel options to lessen corneal opacification and increase lacrimation subsequent to a penetrating corneal injury and in aqueous deficient dry eye disease, respectively.

Rae and Watsky [86] described in the corneal endothelium an outwardly rectifying thermosensitive current along with a K⁺-selective current. The origin of the thermosensitive component was not identified, but may be attributable to TRP activity [86]. More recently, thermosensitive TRPV1-4 channel activity was identified in human corneal endothelial cells [87, 88]. Besides voltage-operated Ca²⁺ channels (VOCCs) [27, 28], there is functional menthol receptor TRPM8 expression in human corneal endothelial cells because the super cooling TRPM8 agonist icilin [34] reversibly increased intracellular Ca²⁺levels [89]. Besides TRPM8 expression in human corneal endothelial cells, Mergler et al. [28] suggested that other TRP subtypes may be also expressed in human corneal endothelium since H₂O₂ induced significant rises in [Ca²⁺]_I levels. This Ca²⁺ rise could be due to activation of redox sensitive TRP subtypes mediating previously unexplained biological phenomena and are involved in various pathologies [90]. Corneal endothelial functional TRPM8 expression may help explain why storing isolated corneas in an eye bank setting at temperatures below those in-situ results in less swelling than at 37 °C [89, 91, 92]. TRPM8 stimulation could result in graded shifts of voltage-dependent activation to more negative membrane voltages increasing the electrical driving force for intracellular Ca²⁺ influx [31]. TRPM8 may be also regulated by G protein-coupled receptors (GPCRs) since both activation of a GPCR and a nerve growth factor receptor inhibited menthol- and cold-induced TRPM8 activity in non-corneal cells [20].

Intracellular Ca²⁺ regulation in lens epithelial and fiber cells is critical to lens transparency maintenance since rises in its level are cataractogenic. Functional TRPM3 and TRPV1 expression was identified in human lenses [93]. TRPM3 variants are associated with cataractogenesis [93]. TRPV4 expression in porcine lens epithelium regulates hemi-channel-mediated ATP release and Na⁺- K⁺-ATPase activity [94]. Plasma membrane associated store operated channel (SOC) activity is modulated through changes in the intracellular store (ICS) Ca²⁺ filling status. Such feedback control of SOC activity is mediated by TRPC1 and TRPC5 subtypes whose open time is prolonged following ICS Ca²⁺ depletion [76].

In conjunctival epithelial cells, there are temperature-sensitive TRPV1, TRPV2 as well as TRPV4 channels [95]. There are indications for crosstalk between TRPV1 and TRPM8 since TRPM8 activation suppressed TRPV1-induced Ca²⁺ increases as well as increases in proinflammatory cytokine IL-6 release [96].

Worldwide, the number of people needing relief from ocular dysfunction is on the rise. One of the reasons for this increase is a change in lifestyle due to reliance on video display terminals and stresses imposed by urbanization. Corneal thermosensitive TRPV1-4, TRPA1 and TRPM8 channels in the cornea can be activated by stresses encountered in daily living and elicit responses that can contribute to ocular disturbances. In addition, 1) TRPV1 (capsaicin); 2) TRPV2/3 (camphor, laurel tree cinnamomum camphora); 3) TRPV4, bisandrographolide in Chinese herbal plant Andrographis paniculata; 4) TRPM8, menthol in green mint; 5) TRPA1, cinnamaldehyde, isothiocyanates and allicin in horseradish are selectively ligand gated [106]. An indication of the adaptive value of TRPV1 expression in the cornea is that application of capsaicin to the ocular surface elicits excruciating pain, which elicits an avoidance response to reduce tissue injury. Thermosensitive corneal TRP expression may also provide an adaptive advantage by eliciting responses that reduce disruptive noxious thermal effects on tissue homeostasis. Similar considerations are relevant to the posterior section of the eye such as retina, uvea and retinal epithelium (RPE) since in these tissues there is also thermosensitive TRP expression (e.g. TRPV2 in RPE) [58]. On the other hand, retinal TRPV1 expression level is low compared to that in the anterior section of the eye (e.g. lens) [107]. Furthermore, the menthol receptor, TRPM8, provides essential functions including contributing to controlling basal tear fluid secretion via corneal nerve fibers [81, 83]. In addition, human corneal endothelial TRPM8 expression may provide an adaptive advantage for preserving tissue function during temperature lowering [89]. In summary, functional thermosensitive TRP activation by environmental challenges elicits essential responses that have adaptive value in preventing or reducing tissue function compromise.

With the realization in the last decade of the polymodal regulatory roles of TRP channels, there is heightening interest in studying how they elicit such control. This is evident based on the ever-increasing number of reports appearing in Pub Med and their citation frequency. One of the realizations sparking such interest is that TRP gene mutations underlie numerous inherited diseases in humans including the eye [108]. TRP channelopathies were initially linked to cardiovascular, renal, skeletal and nervous system pathology [108‐110]. Regarding the eye, one of them deals with mucolipidosis type IV (MLIV) and another indicates that mutant TRPM3 expression is associated with cataractogenesis and glaucoma [93]. MLIV is an autosomal recessive, neurodegenerative lysosomal storage disorder, which is due to mutations in the gene MCOLN1. MLIV is clinically characterized not only by ophthalmologic abnormalities such as corneal opacity, retinal degeneration and strabismus, but also by other non-ocular abnormalities. MCOLN1 which encodes the protein mucolipin 1 (MNL1) is a non-specific cation channel (TRPML1) (review [111]). In addition, human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function and suffer from night blindness starting in early childhood [112].

Ocular tumor development is a rare occurrence compared to its prevalence in non-ocular tissues. In choroidal- or corneal neovascularization, this process is not directly related to tumor neovascularization. Nevertheless, neovascularization is a dangerous process whose progression determines tumor growth and metastasis. Endothelial cells (ECs) play a role in neovascularization and several different TRP subtypes have been detected in this tissue (review [123]). At this point, Ca²⁺ channel drug targeting is being considered in non-ocular tissues (review [124, 125]) and ocular tissues, but findings in these studies are unrelated to tumor neovascularization. One study showed that blockage of Ca²⁺ activated K⁺ channels inhibited angiogenesis induced by epidermal growth factor (EGF) [126]. In retinal pigment epithelium (RPE), activation of L-type Ca²⁺ channels is associated with increases in vascular endothelial growth factor (VEGF) secretion. Additionally, basic fibroblast growth factor (bFGF) increased L-type channel activity [127, 128]. A follow-up study demonstrated in the RPE that TRPV2 channels mediate increases in both heat-dependent and IGF-1 (via PI3-kinase activation)-induced VEGF secretion through rises in intracellular Ca²⁺ levels.