The transient receptor potential (TRP) channels play a diverse range of essential roles in the sensory systems of various species, including both invertebrates and vertebrates. The TRP channel was first identified as a molecule required for proper light response in Drosophila melanogaster. Thereafter, a series of TRP family proteins have been identified. Recently, TRPM1, the founding member of the melanoma-related transient receptor potential (TRPM) subfamily, was shown to be required for the photoresponse in mouse retinal ON bipolar cells. It further proved that TRPM1 is a component of the transduction cation channel negatively regulated by the metabotropic glutamate receptor 6 (mGluR6) cascade in ON bipolar cells through a reconstitution experiment using CHO cells expressing TRPM1, mGluR6, and Goα. In addition to the function of visual transduction cation channel, human TRPM1 is associated with hereditary and acquired diseases in which the retinal ON pathway is selectively affected. Mutations of human TRPM1 are associated with congenital stationary night blindness (CSNB), whose patients lack rod function and suffer from night blindness starting in early childhood. In some patients with melanoma-associated retinopathy (MAR) and cancer-associated retinopathy (CAR), autoantibodies to TRPM1 are generated resulting in progressive retinal dysfunction. In this section, we focus on the physiological functions and regulatory mechanisms of the TRPM1 channel in retinal ON bipolar cells and the association of TRPM1 with human diseases including CSNB, MAR, and CAR.
Identification of TRPM1
TRPM1 belongs to the transient receptor potential channel (TRP) gene family, which encodes non-voltage-gated Ca2+-permeable cation channels. The first TRP channel was identified in Drosophila melanogaster as a necessary molecule for photoreceptor response to light (Montell et al. 1985). However, whether or not a TRP channel plays an essential role in the vertebral visual systems had not been determined until recently.
TRP family proteins participate broadly in sensory reception in a variety of living organisms, including both invertebrates and vertebrates. TRP superfamily members are categorized into seven subfamilies, TRPC, TRPM, TRPV, TRPA, TRPP, TRPML, and TRPN, based on amino acid sequence homology, although TRPN family genes have not been identified in mammals (Venkatachalam and Montell 2007). TRP channel proteins share a common structure of six predicted transmembrane domains and a reentrant pore loop, which is involved in forming the pore of the channel, as it does in voltage-gated ion channels (Venkatachalam and Montell 2007). In addition to these features, a highly conserved 23–25 amino acid residue TRP domain containing an “EWKFAR” TRP box is located near the sixth transmembrane domain in the cytoplasmic region of TRPV, TRPC, TRPM, and TRPN channels (Venkatachalam and Montell 2007). In mammals, TRP channels are mainly involve in sensing activities, including taste, olfaction, hearing, and touch, in addition to thermosensation and osmosensation.
TRPM family genes are well conserved through evolution, both in invertebrates and vertebrates, and encode nonselective cation channels involved in many biological functions. There are eight TRPM genes in human and mouse, four genes in C. elegans, and a single gene in Drosophila melanogaster. Phylogenetic analysis of TRPM channels classified the TRPM family into four groups: TRPM1 and TRPM3, TRPM6 and TRPM7, TRPM4 and TRPM5, and TRPM2 and TRPM8 (Fig. 1). Similar to other TRP channels, TRPM channels possess intracellular N- and C-terminals, six transmembrane regions, and a hydrophobic pore loop between transmembrane regions 5 and 6. The N-terminal regions of this family protein contain four TRPM homology regions (MHRs), whereas the C-terminal regions contain a TRP domain and coiled-coil domain (Fig. 2). The coiled-coil domain in the C-terminal region is involved in the assembly of subunits and the regulation of cellular localization of channel proteins (Tsuruda et al. 2006).
ON and OFF Pathway of Visual Processing in the Retina
Functional separation of neuronal signaling into the ON and OFF pathways contributes visual contrast recognition of the vertebrate visual system. Under the dark condition, photoreceptor cells are constitutively depolarized and release glutamate, which in turn hyperpolarizes ON bipolar cells and depolarizes OFF bipolar cells. On the other hand, under the light condition, photoreceptor cells are hyperpolarized and release less glutamate. This causes ON bipolar cells to lose their inhibition and become depolarized, while OFF bipolar cells lose their excitation and become hyperpolarized. In mammalian retinas, rod photoreceptors form synapses with ON-type rod bipolar cells, and cone photoreceptors connect with cone bipolar cells, which are subdivided into ON and OFF types.
The expression pattern of different glutamate receptors (GluRs) at the postsynaptic region determines the functional diversity of bipolar cells. ON bipolar cells express a metabotropic glutamate receptor 6 (mGluR6), which belongs to the class C G protein-coupled receptor (GPCR), on their dendrites (Nakanishi et al. 1998). The ON bipolar dendrite forms an invaginating single contact with a photoreceptor terminal. On the other hand, OFF bipolar cells express ionotropic GluRs (AMPA/kainate receptors), glutamate-gated cation channels, on dendrites (Morigiwa and Vardi 1999). The OFF bipolar dendrite makes multiple flat contacts with a photoreceptor terminal. Under the dark condition, the neurotransmitter glutamate is released at a high rate from rod terminals and depolarizes OFF bipolar cells through activation of ionotropic GluR, whereas glutamate hyperpolarizes ON bipolar cells through mGluR6 activation leading to the closure of downstream cation channels and a decrease in cationic conductance (Shiells et al. 1981). Until recently the downstream transduction cation channels of the mGluR6 pathway in retinal bipolar cells had been unknown, although Goα was reported to be required to close a downstream cation channel upon the activation of mGluR6 (Nawy 1999).
The Function of the TRPM1 Channel in the Retina
A mouse TRPM1-L cDNA that corresponds to the human TRPM1 long form was identified as being a highly expressed gene in the retina (GenBank Accession Number #AY180104) (Koike et al. 2007, 2010a). Northern blot analysis showed the presence of both TRPM1-L and TRPM1-S transcripts in the retina; however, only the latter was detected in the skin (Koike et al. 2010a). In situ hybridization (ISH) on the mouse retina revealed the presence of substantial TRPM1-L transcripts, specifically in the inner nuclear layer (INL), at postnatal stages. An antibody against TRPM1-L was raised and the localization of TRPM1-L was examined. At postnatal day 14 (P14), TRPM1-L proteins were observed diffusely in bipolar cell somata, and at 1 month after birth (1M), TRPM1 was localized at the tips of Goα- and mGluR6-expressing dendrites in the outer plexiform layer (OPL) (Koike et al. 2010a).
Analyses of TRPM1-deficient mice showed the essential function of TRPM1 in both rod and cone ON bipolar cells. Koike et al. generated TRPM1-null mutant (TRPM1 −/− ) mice by targeted gene disruption and revealed that neither their rod bipolar cells nor their cone ON bipolar cells showed photoresponses by using whole-cell patch-clamp analysis (Koike et al. 2007, 2010a). On the other hand, light stimulation on both wild-type (WT) and TRPM1 −/− mice cone OFF bipolar cells evoked photoresponses, and no significant differences were detected in either the amplitude of the light responses or the time for half-maximal amplitude after the light was turned off (Koike et al. 2010a). Examination of the optokinetic responses (OKRs) and electroretinograms (ERGs) of 2-month-old WT and TRPM1 −/− mice revealed optokinetic deficiencies similar to those of mice lacking mGluR6 (Iwakabe et al. 1997; Koike et al. 2010a). The ERGs evoked by light stimuli in WT mice show normal a-waves and b-waves, originating mainly from photoreceptors and rod bipolar cells, respectively. The ERG b-wave in TRPM1 −/− mice was absent, and their ERG waveforms were very similar to those of mGluR6 −/− mice (Masu et al. 1995). In the light-adapted state, the ERG b-wave was severely attenuated or absent leaving only the ERG a-wave in the TRPM1 −/− mice (Koike et al. 2007). These ERG results suggested that the function of both rod and cone bipolar cells was severely impaired in TRPM1 −/− mice.
TRPM1-L is a nonselective cation channel negatively regulated by the glutamate-activated mGluR6-Goα signaling cascade (Fig. 3). It was reported that upon activation of mGluR6 by glutamate, GTP-bound Goα dissociates from the Gβγ dimer and closes the downstream cation channel (Nawy 1999). To determine whether TRPM1-L satisfies the properties of a nonselective cation channel regulated by the mGluR6 cascade, TRPM1-L, mGluR6, and Goα were introduced to CHO cells, and ionic currents under a whole-cell voltage clamp were measured (Koike et al. 2010a). In this reconstitution system, which mimicked the postsynaptic membrane of retinal ON bipolar cells, constitutively active inward currents were detected. Even after replacing extracellular cations with Na+, K+, Ca2+, or Mg2+, constitutively active currents were observed in TRPM1-L-expressing cells (Koike et al. 2010a). Furthermore, in CHO cells expressing mGluR6, Goα and TRPM1-L, constitutively active cationic currents were suppressed by the administration of glutamate to the bath solution, while subsequent washout of glutamate restored the suppressed currents to levels comparable to those prior to glutamate administration (Koike et al. 2010a).
The effect of Go activation on the TRPM1-L current was further evaluated by measuring whole-cell current in TRPM1-L expressing CHO cells transfected either with wild type of Goα or a constitutively active mutant of Goα (Goα-Q205L) (Koike et al. 2010a). The current density obtained in TRPM1-L-transfected cells was significantly larger than that in TRPM1-L- and Goα-co-transfected cells, while suppression of the current density in TRPM1-L- and Goα-Q205L-co-transfected cells was observed at a level comparable to that in TRPM1-L- and Goα-co-transfected cells intracellularly perfused with GMP-PNP, an unhydrolyzable analog of GTP (Koike et al. 2010a). To show the regulation of TRPM1-L by Go protein more directly, the effect of addition of the purified Go protein from the intracellular side on TRPM1-L activity was examined in the excised inside-out patch recordings (Koike et al. 2010a). Application of the purified Goα protein gradually, but strongly, suppressed open probability, whereas administration of heat-denatured Goα protein with GMP-PNP failed to suppress the channel activities. Application of Gβγ did not suppress the open probability of TRPM1-L (Koike et al. 2010b). Western blot analysis distinctly showed that the purified Goα protein fraction from the bovine brain did not contain Gβγ (Koike et al. 2010b).
On the other hand, the possibility that Gβγ dimer also can close TRPM1 channel was suggested. According to the report from Xu et al., TRPM1 interacts both with active mutant of Goα and with Gβγ, although the closure of TRPM1 channels by constitutively active mutant of Goα was confirmed by electrophysiological assay (Xu et al. 2016). They also showed that while Goα interacts with both the N- and the C-termini of TRPM1, Gβγ interacts only with the N-terminus by bioluminescent energy transfer assays (Xu et al. 2016). Shen et al. reported that Gβγ dimer but not Gαo closes TRPM1 in vitro (Shen et al. 2012). Therefore, it is still needed to be investigated whether and how Goα and/or Gβγ contribute to close the TRPM1channel.
TRPM1, mGluR6, and some other proteins form the macromolecular complex at the dendritic tips of ON bipolar cells. GPR179, which is a member of the orphan GPCR family, forms a complex with TRPM1, mGluR6, and the regulator of G protein signaling (RGS) proteins in the mouse retina (Orlandi et al. 2013). Nyctalopin (NYX), a small leucine-rich repeat protein, also directly interacts and forms a complex with TRPM1 and mGluR6 and functions as a scaffold for the correct localization of them at dendritic tips of ON bipolar cells (Pearring et al. 2011; Cao et al. 2011). Furthermore, Lrit3 is located on dendritic tips of ON bipolar cells, and its loss results in TRPM1 mislocalization at the dendritic tips (Neuillé et al. 2015).
TRPM1 and Congenital Stationary Night Blindness (CSNB)
Mutations of human TRPM1 are associated with congenital stationary night blindness (CSNB), which is a clinically and genetically heterogeneous group of retinal disorders. CSNB patients typically suffer from night blindness while their day visions are preserved. Based on the results of standard flash ERG, CSNB can be classified into two types: the complete form (cCSNB) and the incomplete form (icCSNB). The complete form, also known as type 1 CSNB or CSNB1, is characterized by a complete loss of the b-wave in response to a dim flash and an electronegative maximum response with a normal a-wave under scotopic conditions (Audo et al. 2008). The photopic single flash and 30Hz flicker ERG have a normal a-wave amplitude with a broadened trough and a sharply rising peak with no photopic oscillatory potentials and a reduced b/a ratio (Audo et al. 2008). The incomplete form (icCSNB), also known as type 2 CSNB or CSNB2, is characterized by a reduction of b-wave. The photopic responses of icCSNB patients are severely reduced and delayed both in response to a single flash or 30Hz flicker compared with CSNB1 (Audo et al. 2008).
A clue to reveal the association of TRPM1 mutation with CSNB came from a study on horse spontaneous mutants. A single incomplete dominant gene, leopard complex (LP), causes the Appaloosa coat-spotting pattern in horses. Homozygous LP (LP/LP) causes CSNB in Appaloosa horses, characterized by a congenital and nonprogressive scotopic visual deficit (Bellone et al. 2008). Furthermore, Bellone et al. mapped a 6-cM LP candidate region where the TRPM1 gene is located. They found that in the retina of CSNB (LP/LP) horses, TRPM1 expression was downregulated, suggesting that TRPM1 is a strong candidate for LP; however, no TRPM1 mutation in LP/LP horses was reported (Bellone et al. 2008).
In an analysis of a large South Asian family with CSNB, Li et al. identified a large region of homozygosity on chromosome 15q which contains TRPM1 (Li et al. 2009). Screening identified a single homozygous mutation in the affected mother (IVS16+2T>C) (Li et al. 2009). In a Caucasian non-consanguineous family, the affected proband was found to have two likely disease-causing missense mutations in the TRPM1 gene (G138fs and Y1035X) (Li et al. 2009). The proband of non-consanguineous Caucasian family was found to harbor heterozygote for two missense mutations in TRPM1 (R74C and I1002F) (Li et al. 2009). Furthermore, Nakamura et al. identified five different mutations in the human TRPM1 gene, IVS2-3C>G, IVS8+3_6felAAGT, R624C (c.1870C>T), S882X (c.2645C>A), and F1075S (c.3224T>C)), in three unrelated patients (Nakamura et al. 2010). All three patients had compound heterozygous mutations. Biochemical and cell biological analyses revealed that the two intron mutations (IVS2-3C> and IVS8+3_6delAAGT) were likely to result in abnormal protein production from abnormal splicing, and the two missense mutations (R624C and F1075S) lead to the mislocalization of the TRPM1 protein in ON bipolar cells (Nakamura et al. 2010).
TRPM1 and Paraneoplastic Retinopathy
Paraneoplastic retinopathy (PR), including melanoma-associated retinopathy (MAR) and cancer-associated retinopathy (CAR), is a progressive retinal disorder caused by antibodies generated against non-ocular neoplasms. Patients with PR can suffer from night blindness, photopsia, ring scotoma, attenuated retinal arteriole, and abnormal electroretinograms (ERGs). Recently it was proved that TRPM1 is an autoantigen targeted by autoantibodies in some patients with MAR or CAR (Dhingra et al. 2011; Kondo et al. 2011). The ERGs of a patient with lung CAR exhibited a severely reduced ON response with normal OFF response, indicating that the defect is in the signal transmission between photoreceptors and ON bipolar cells (Kondo et al. 2011). Sera from MAR and CAR patients exhibited a significant immunoreactivity against TRPM1-transfected cell lysates by Western blot analysis (Kondo et al. 2011). These observations, the expression of TRPM1 in melanocytes, and its downregulation in melanoma cells suggest that TRPM1 is one of the retinal autoantigens in CAR or MAR, which is associated with retinal ON bipolar cell dysfunction (Duncan et al. 1998).
After years of considerable efforts to identify the ON bipolar transduction channel, TRPM1 was finally identified as a cation channel negatively regulated by the glutamate-activated mGluR6-Goα signaling cascade. This finding is based on TRPM1-deficient mouse phenotypes and a reconstitution system with TRPM1, mGluR6, and Goα, using cultured cells. TRPM1 is specifically expressed in ON bipolar cells in human retinas as well as in mouse retinas. Series of evidence from molecular analysis on CSNB patients or PR patients further support the concept that TRPM1 plays an essential role in mediating the photoresponse in retinal ON bipolar cells. Currently, it has been well established that mGluR6 activation leads to subsequent activation of heterotrimeric G proteins and closure of the TRPM1 cation channel; however, it has not been clearly understood which G protein subunit(s), Goα or Gβγ, mainly contributes to the closure of the TRPM1 channel. Therefore, further studies are still needed to solve this problem.
TRPM1-related work conducted in the authors’ laboratory is supported by the CREST from Japan Science and Technology Agency, Grant-in-Aid for Scientific Research (B), Takeda Science Foundation, Koyanagi Foundation, and Terumo Foundation for Life Science and Arts Life Science support program.
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