An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal BCs [34
]. The release of glutamate from photoreceptors modulates the photoresponse of ON BCs [35
] via metabotropic glutamate receptor 6 (mGluR6) and the G-protein (Go
) that regulates a cation channel [36
]. We recently reported that we identified a mouse trpm1
long form (trpm1-L
) and found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in co-localization with mGluR6 [11
null mutant mice completely lose the photoresponse of ON BCs. TRPM1-L channel activity is negatively regulated by activated Go
in the mGluR6 cascade. These results suggest that TRPM1-L is a component of the ON BC transduction channel. These observations led us to examine whether the gene is mutated in human patients with complete CSNB in the current study.
To identify human TRPM1
gene mutations in CSNB patients, we analyzed four separate Japanese patients with complete CSNB in whom previous molecular examination revealed no mutation in either the NYX
genes. In the current study, we identified five different mutations in TRPM1
: IVS2–3C>G, IVS8+3_6delAAGT, R624C (c.1870C>T), S882X (c.2645C>A), and F1075S (c.3224T>C). Mutations were present in three unrelated patients with complete CSNB (#373, #437, and #484). Using a minigene expression assay, we showed that two intron mutations, IVS2–3C> and IVS8+3_6delAAGT, can cause splicing abnormalities leading to defects in protein production. Since these two mutations are located in the N-terminus region of TRPM1, these mutations are likely to produce a loss-of-function allele of TRPM1
. The nonsense mutation S882X (c.2645C>A) is located between TM2 and TM3. Thus, the truncated TRPM1 protein is likely to be non-functional as a channel. Regarding the two missense mutations, R624C (c.1870C>T) and F1075S (c.3224T>C), we observed failure of the transportation of the missense mutant channels to the dendritic tips. However, ~20% of ON BCs could transport the mutant forms of TRPM1, suggesting that these ON BCs are still active. Considering that human patients carrying these mutations still have cone ERG responses, this fraction might be cone ON BCs converging inputs from cone photoreceptors. Since these two amino acid residues are evolutionarily conserved among the TRPM subfamily, these amino acid residues are indispensable for the physiologic functions of TRPM channels. For example, R671Q mutation in the Drosophila TRPM1
) resulted in a significant reduction of ERG response [39
]. This mutation is located between the 6TM and the TRP domain [40
], which is close to the F1075 residue of human TRPM1. Although this region is not in the functional domains, it still could be responsible for the physiologic function of the TRPM channel.
The scotopic ERG b-waves recorded using Ganzfeld stimuli were nonrecordable, and the photopic ERG showed normal a-wave amplitude, but the amplitude of the b-wave is reduced or absent in the three affected human individuals. There seems to be no apparent genotype-phenotype correlation in our patients with TRPM1
mutations. Full-field ERG results showed no detectable post-receptoral ON-pathway function for the three patients. The ERG amplitudes of patient #484 were smaller than those of the other two patients, but the reductions were considered to be due to high myopia (−12 to −13 D) [41
] or other unknown reasons. The function of the inner retina was further analyzed using L-M cone ERGs elicited by rectangular, 100–125 msec duration light stimuli under light-adapted conditions in one patient, and as a result, the amplitude of the b-wave at light onset (ON-response) was significantly reduced, but the d-wave amplitude (OFF-response) at light offset was unaffected with normal OPs on the OFF-responses (data not shown). These electrophysiological results indicated that the pathology in complete CSNB with TRPM1
mutations lay in the dysfunction of the depolarizing ON BCs because it is generally considered that the positive ON-response (b-wave) reflects the depolarizing ON BCs. The impairment in the ON-response was also observed in the trpm1–/–
mice in which b-wave amplitude for both scotopic and photopic conditions were completely defective. These results in human and mice strongly indicated that TRPM1 is essential for the function of ON BCs in the visual pathway.
The clinical features of patients with complete CSNB are similar even when the causative gene is different. Most patients complain of night blindness while showing normal fundi accompanied by highly myopic refractive errors [4
]. The best-corrected visual acuity is mildly reduced, or sometimes normal. We could not clarify any difference in ERG responses that were recorded according to ISCEV protocol among those with different gene defects. To date, the only phenotypic difference among complete CSNB patients with different gene mutations has been detected using a scotopic 15 Hz-flicker ERG with increasing intensities [42
]. It was reported that patients with TRPM1
mutations showed 15 Hz-flicker ERG responses similar to those with NYX
mutations, but different from those with GRM6
mutations or normal control subjects, suggesting some difference in the rod pathway [21
Very recent studies reported that TRPM1
gene mutations were the major cause of AR complete CSNB in patients with Caucasian ancestors [20
]. We previously identified NYX
mutations in five families and GRM6
mutations in two families among 11 Japanese families with complete CSNB [43
], and in the current study, we found TRPM1
mutations in three of the remaining four families. From these results we confirmed that the gene was responsible for patients with complete CSNB. It is likely that one of the three genes that is localized at the dendritic terminals of ON BCs and contributing the cell activity, TRPM1
, or NYX
, would responsible for most patients with complete CSNB.
In conclusion, we identified five different mutations in the TRPM1 gene in three unrelated Japanese patients with complete CSNB. TRPM1 is essential for the depolarizing ON BC function in humans as well as in mice.