TRPV6, TRPM6 and TRPM7 Do Not Contribute to Hair-Cell Mechanotransduction

Abstract

Hair cells of the inner ear transduce mechanical stimuli like sound or head movements into electrical signals, which are propagated to the central nervous system. The hair-cell mechanotransduction channel remains unidentified. We tested whether three transient receptor channel (TRP) family members, TRPV6, TRPM6 and TRPM7, were necessary for transduction. TRPV6 interacted with USH1C (harmonin), a scaffolding protein that participates in transduction. Using a cysteine-substitution knock-in mouse line and methanethiosulfonate (MTS) reagents selective for this allele, we found that inhibition of TRPV6 had no effect on transduction in mouse cochlear hair cells. TRPM6 and TRPM7 each interacted with the tip-link component PCDH15 in cultured eukaryotic cells, which suggested they might be part of the transduction complex. Cochlear hair cell transduction was not affected by manipulations of Mg²⁺, however, which normally perturbs TRPM6 and TRPM7. To definitively examine the role of these two channels in transduction, we showed that deletion of either or both of their genes selectively in hair cells had no effect on auditory function. We suggest that TRPV6, TRPM6 and TRPM7 are unlikely to be the pore-forming subunit of the hair-cell transduction channel.

Keywords: TRP channels; auditory brainstem response (ABR); hair cells; mechanotransduction; stereocilia.

Figure 1

Characterization of TRPV6. (A) Seven transient receptor channel (TRP) channels (of 33) with candidate PDZ-binding interface motifs. Yeast two-hybrid (Y2H) results examining interactions with USH1C (harmonin) are indicated. (B) The 33 terminal amino acids of the TRPV6 PBI interact with USH1C, but deletion of the C-terminal seven amino acids abolishes binding (top row). The interaction of TRPV6 is through PDZ1 of USH1C (bottom row). (C) RT-PCR (with reverse transcription primed either with oligo dT or random hexamers) showing that mRNA for TRPV6 is present in vestibular RNA.

Figure 2

Targeted mutations in mouse Trpv6, Trpm6 and Trpm7 genes. (A) Top, Trpv6 gene structure. Horizontal gray line indicates scaled length of gene with coding exons, which are shown as vertical black rectangles. Middle, magnification of targeted exon. The loxP site remaining after excision of the neo cassette is shown in blue. Bottom, nucleotides and protein translation for region targeted. The M527C mutation is indicated. (B) Structure of the KcsA ion channel (image from https://commons.wikimedia.org/wiki/File:1K4C.png), along with the locations of the residue analogous to M527C-Trpv6. The pore and transmembrane domain 2 (TM2) are indicated as well. Two of the four subunits are shown. Color coding: protein, green; backbone carbonyl groups, oxygen in red and carbon in green; potassium ions (occupying S2 and S4), purple spheres; and oxygen atoms of water molecules (S1 and S3), red spheres. (C) Structure of Trpm6^fl allele; exon 7 is flanked by loxP recombination sites; a residual FRT site remaining after excision of the Flp cassette is indicated. (D) Structure of Trpm7^fl allele; exon 21 is flanked by loxP sites.

Figure 3

Methanethiosulfonate (MTS) reagents do not inhibit hair-cell transduction in M527C-Trpv6 hair cells. (A,B) Hair-cell transduction currents from wild-type (A) and heterozygous M527C-Trpv6 (B) mice before and after treatment with 1 mM MTSET, applied in the extracellular solution. Transduction currents were recorded with a holding potential of −80 mV. (C,D) Summarized data; each line connects a cell’s control maximum current (before application of inhibitor) and its maximum current after application of 1 mM MTS reagents (MTSET or MTSES).

Figure 4

Interaction of PCDH15 with TRPM6 and TRPM7. (A) Lysates were immunoprecipitated with anti-HA agarose, and probed with anti-HA (top panels) or anti-PCDH15 antibody (bottom panels). PCDH15 was only immunoprecipitated by anti-HA if TRPM6 or RPM7 were present. No PCDH15 was precipitated by the control antibody (V5). (B) Lysates were immunoprecipitated with protein A/G agarose and either PB811 antibody (for PCDH15) or HRP antibody (control), then probed for TRP channels with anti-HA antibody. TRPM6 and TRPM7 were only precipitated with co-expressed with PCDH15 and precipitated with an anti-PCDH15 antibody. (C) Lysates were immunoprecipitated with anti-HA agarose, and probed with anti-PCDH15 antibody (left panels) or CDH2 antibody (right). Two C-terminal deletion proteins of PCDH15, Δ1 and Δ2, did not immunoprecipitate with TRPM6/7. Chimeras that have N-terminal extracellular domains of PCDH15 (15–2–15 and 15–2–2) immunoprecipitated with TRPM6/7 more strongly than PCDH15. The chimera that does not contain extracellular domains of PCDH15 (2–15–2) did not immunoprecipitate with TRPM6/7. (D) Lysates were immunoprecipitated with anti-HA agarose and probed with an anti-PCDH15 antibody (PB811). The last two lanes are two controls: anti-V5 agarose as a control for non-specific binding of the agarose with PCDH15, and TRPC6 as a TRP channel outside the TRPM family. Both controls have much lower levels of PCDH15 immunoprecipitated. (E) Summary of PCDH15 constructs (N- to C-terminal) used in panel (C). Chimeras with CDH2 used the CD3 splice form of PCDH15. (F) Mass spectrometry spectral counts for TRPM6, TRPM7, and PCDH15 in large-scale immunoprecipitation experiments using ant-HA agarose. No PCDH15 was immunoprecipitated when it was expressed alone, but large amounts were co-precipitated with TRPM6 and TRPM7.

Figure 5

Localization of TRPM7 in the inner ear. (A) Single x-y slice through apical region of outer hair cells. One transfected cell, identified by ZsGreen signal, shows tdTomato signal at the lateral membrane. Box indicates region used for x-z reslice in (B). P6+1, cochlea dissected at P6 and maintained in culture for 1 day. (B) Reslice of stack shows lateral view of transfected hair cell (in row 2; OHC2) and two other untransfected cells. tdTomato-TRPM7 signal was located near apex of cell. Red signal outside of OHC2 is presumably background fluorescence. Panel full widths in (A,B) are 15 μm.

Figure 6

Novel splice forms of Trpm6 and Trpm7 interact with PCDH15. (A) Tissue expression profiles of mouse Trpm6, Dex27 and Dex26. Primer sets for Dex27 and Dex26 were designed such that they bind to the junctions of Trpm7 and Trpm6 exons 19 and 21; signal was only seen if exon 20 was absent. Both Dex27 and Dex26 were highly expressed in the ear (first lane on the left). (B) Both TRPM6/7 and DEX26/7 proteins traveled to the cell surface. HEK293T cells were transfected with TRPM6/7 or DEX26/7; before harvest, the cells were surface biotinylated. Cell lysates were then immunoprecipitated with neutravidin-agarose, then probed with anti-HA antibody. (C) DEX26 and DEX27 interacted with PCDH15 in a similar fashion with TRPM6 and TRPM7. (D) Predicted membrane topology for canonical TRPM7 sequence (left) and hypothetical inverted DEX27 sequence (right). The loss of TM2 when exon 20 is spliced out could force inversion of TM3–TM6; a cryptic transmembrane domain (S7 here) is predicted by some topology algorithms to be used, maintaining the N- and C-termini intracellular.

Figure 7

No evidence for contribution of TRPM6 or TRPM7 conductances to hair-cell transduction or auditory function. (A–C) No effect of internal Mg²⁺. (A) Representative transduction currents from hair cells that were dialyzed with internal solution containing 0 or 3 mM MgCl₂. (B) No difference in transduction current amplitudes were observed. Mean ± SEM are plotted and fit with a three-state Boltzmann relation; n = 5 for each. (C) Replotted as displacement-P_open relationship. Boltzmann fits are nearly identical. (D) Examples of auditory brainstem response (ABR) waveforms at 32 kHz for WT (Trpm6^fl/fl; Trpm7^fl/fl; Atoh1-CRE-negative) and Trpm6^CKO; Trpm7^CKO DKO mice. The bold red trace for each indicates the threshold (30 and 35 dB SPL, respectively). (E) Summarized ABR measurements for Trpm6^CKO, Trpm7^CKO, and Trpm6^CKO; Trpm7^CKO DKO mice. Mean ± SEM are plotted. Trpm6^CKO : WT, n = 11; Trpm6^CKO, n = 17. **p < 0.01. Trpm7^CKO: WT, n = 10; Trpm7^CKO, n = 14. Trpm6^CKO; Trpm7^CKO DKO: WT, n = 11; Trpm6^CKO; Trpm7^CKO DKO, n = 8.