Depolarizing bipolar cell dysfunction due to a Trpm1 point mutation

Abstract

Mutations in TRPM1 are found in humans with an autosomal recessive form of complete congenital stationary night blindness (cCSNB). The Trpm1(-/-) mouse has been an important animal model for this condition. Here we report a new mouse mutant, tvrm27, identified in a chemical mutagenesis screen. Genetic mapping of the no b-wave electroretinogram (ERG) phenotype of tvrm27 localized the mutation to a chromosomal region that included Trpm1. Complementation testing with Trpm1(-/-) mice confirmed a mutation in Trpm1. Sequencing identified a nucleotide change in exon 23, converting a highly conserved alanine within the pore domain to threonine (p.A1068T). Consistent with prior studies of Trpm1(-/-) mice, no anatomical changes were noted in the Trpm1(tvrm27/tvrm27) retina. The Trpm1(tvrm27/tvrm27) phenotype is distinguished from that of Trpm1(-/-) by the retention of TRPM1 expression on the dendritic tips of depolarizing bipolar cells (DBCs). While ERG b-wave amplitudes of Trpm1(+/-) heterozygotes are comparable to wild type, those of Trpm1(+/tvrm27) mice are reduced by 32%. A similar reduction in the response of Trpm1(+/tvrm27) DBCs to LY341495 or capsaicin is evident in whole cell recordings. These data indicate that the p.A1068T mutant TRPM1 acts as a dominant negative with respect to TRPM1 channel function. Furthermore, these data indicate that the number of functional TRPM1 channels at the DBC dendritic tips is a key factor in defining DBC response amplitude. The Trpm1(tvrm27/tvrm27) mutant will be useful for elucidating the role of TRPM1 in DBC signal transduction, for determining how Trpm1 mutations impact central visual processing, and for evaluating experimental therapies for cCSNB.

Fig. 1.

A: sequences from control and Trpm1^{tvrm27/tvrm27} mice. B: schematic denoting the location of the p.A1068T mutation. C: alignment of murine TRPM1 amino acid sequence (1051–1087, accession no. NP_001034193.2) with the orthologous region of TRPM1 in 17 other vertebrate species. This region, including the A1068 residue (yellow), is highly conserved.

Fig. 2.

Comparison of retinal anatomy of control and Trpm1^{tvrm27/tvrm27} mice. A: by light microscopy, no difference between control and Trpm1^{tvrm27/tvrm27} retinas was observed. Scale bar, 15 μm. B and C: electron micrographs show that rod (B) and cone (C) ribbon synapses in Trpm1^{tvrm27/tvrm27} mice are present and have a morphology similar to those in wild-type (WT) mice. Scale bars, 500 nm. B, bipolar cell; H, horizontal cell.

Fig. 3.

Immunohistochemistry for GRM6 (red) and TRPM1 (green) in WT (A–C), Trpm1^{tvrm27/tvrm27} (D–F), and Trpm1^−/− (G–I) retinas. Note that TRPM1 expression is comparable in WT and Trpm1^{tvrm27/tvrm27} but absent in Trpm1^−/− retina. Scale bars, 5 μm.

Fig. 4.

Immunohistochemistry for TRPM1 (green) and RIBEYE (red) in WT (A), Trpm1^{tvrm27/tvrm27} (B), and Trpm1^−/− (C) retinas. Note that the apposition of pre- and postsynaptic elements are comparable to WT in Trpm1^{tvrm27/tvrm27} retina. Scale bar, 5 μm. Larger box displays the field contained in the small box at ×2 magnification.

Fig. 5.

Comparison of electroretinogram (ERG) waveforms of WT (black traces) and affected Trpm1^{tvrm27/tvrm27} (blue traces) mice. A: dark-adapted ERGs. The b-wave response present in WT ERGs is missing in Trpm1^{tvrm27/tvrm27} mice, revealing the electronegative slow PIII component. B: light-adapted ERG waveforms of Trpm1^{tvrm27/tvrm27} mice are abnormal. Values indicate flash luminance in log cd s/m².

Fig. 6.

Comparison of dark-adapted ERGs recorded from Trpm1 heterozygotes. A: representative waveforms obtained from WT, Trpm1^+/tvrm27, and Trpm1^+/− mice. The amplitude of the b-wave is selectively reduced in Trpm1^+/tvrm27 mice. Values indicate flash luminance in log cd s/m². B: luminance-response functions for a-wave and b-wave amplitude in WT, Trpm1^+/tvrm27, and Trpm1^+/− mice. Data points indicate the average ± SE of 7 WT and Trpm1^+/tvrm27 and 3 Trpm1^+/− mice. C: luminance-response functions for WT, Grm6^+/nob3/Gpr179^+/nob5, and Nyx^+/nob mice. For each mouse tested, the amplitude of the b-wave amplitude is expressed relative to the amplitude of the maximum a-wave. Data points indicate the average ± SE of 19 WT, 8 Grm6^+/nob3Gpr179^+/nob5, and 5 Nyx^+/nob mice.

Fig. 7.

Patch-clamp responses of rod depolarizing bipolar cells (DBCs). A: representative responses of WT, Trpm1^+/tvrm27, and Trpm1^{tvrm27/tvrm27} littermates to the mGluR6 agonist LY341495 or the TRP channel antagonist capsaicin. B: average peak response amplitude for WT (n = 7), Trpm1^+/tvrm27 (n = 4), and Trpm1^{tvrm27/tvrm27} (n = 5) rod DBCs with a holding potential of +40 mV. Error bars indicate SE. **P = 0.002; *P = 0.01.