tmc2a (transmembrane channel-like 2a) is one of two zebrafish co-orthologs of vertebrate TMC2, the paralog tmc2b being the other; both lie on chromosome 5, tmc2b next to tmc1. Tmc2a is a multi-pass membrane protein of the TMC family and a pore-forming subunit of the hair-cell mechanoelectrical transduction (MET) channel, a non-selective cation channel with high calcium permeability that opens when tip-link tension deflects the hair bundle. Its cytoplasmic N-terminus binds the cytoplasmic domain of the tip-link protein Pcdh15a. tmc2a is expressed only in sensory hair cells, from 1 dpf in the inner ear, where it is enriched in central (striolar) hair cells and in the upper layer of the cristae, and at low levels in a subset of lateral-line hair cells that respond to posterior-to-anterior deflection. Tmc2a is the main Tmc subunit for hearing through the saccule, is required for mechanotransduction in upper-layer crista hair cells and for high-frequency utricular sensitivity, and together with Tmc2b carries all lateral-line transduction; in neuromasts it enlarges the responses of the hair cells that express it.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005245 voltage-gated calcium channel activity | IBA GO_REF:0000033 | MODIFY | Summary: Voltage-gated calcium channel activity, propagated from the PANTHER TMC2 node (mouse Tmc2 donor). Reason: The ion-channel part is right but the gating stimulus is wrong. TMC channels are opened by tension in the tip link that accompanies hair-bundle deflection, not by membrane voltage, and they conduct cations non-selectively with high calcium permeability. The same row was modified to the mechanically gated cation channel term in the human TMC1 review; the same argument applies to every member of this node. In zebrafish the Tmc2a N-terminus acts in a complex with the tip-link protein Pcdh15a, and loss of Tmc2a reduces mechanically evoked hair-cell responses. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: MGI:MGI:2151017 · Tmc2 (mouse) SOURCE BAD QuickGO shows mouse Tmc2 carries GO:0005245 by IMP/IGI from PMID:23871232, a study of calcium permeability and single-channel currents of the mechanically gated hair-cell channel, not of voltage gating (as traced in the human TMC1 review). PANTHER:PTN002761430 SUPPORTS SOURCE BUT NOT TARGET The node correctly groups vertebrate TMC2 orthologs including both zebrafish copies; only the gating mechanism in the chosen term is wrong. Proposed replacements: mechanosensitive monoatomic cation channel activity Supporting Evidence: PMID:25114259 Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a–Tmc complex is critical for mechanotransduction. PMID:39773557 Significant progress in our understanding of MET has been recently made by identifying the transmembrane channel-like proteins 1 and 2 (TMC1 and TMC2) as the pore-forming subunits of the MET channel (Kawashima et al., 2011; Pan et al., 2018). |
| GO:0005262 calcium channel activity | ISS GO_REF:0000024 | ACCEPT | Summary: Calcium channel activity transferred by UniProt curators from mouse Tmc2 (Q8R4P4). Reason: The MET channel formed by TMC subunits is a non-selective cation channel that is highly permeable to calcium, so calcium conduction is part of the activity. In zebrafish, neuromast hair bundles that express Tmc2a in addition to Tmc2b give larger calcium signals than bundles with Tmc2b alone. The term does not capture the mechanical gating, which is covered by GO:0008381 and the proposed GO:0140135. Supporting Evidence: PMID:36905930 This idea is supported by our results, as we show that the calcium signals in hair bundles expressing both Tmc2b and Tmc2a are larger compared to those expressing just Tmc2b (Figure 6). |
| GO:0005515 protein binding | IPI PMID:25114259 Tip-link protein protocadherin 15 interacts with transmembra... | MODIFY | Summary: Tmc2a N-terminus binds the cytoplasmic domains (CD1 and CD3 isoforms) of the tip-link protein Pcdh15a in yeast two-hybrid and co-immunoprecipitation assays. Reason: The interaction is well supported (yeast two-hybrid, co-IP in HEK293T cells, and a dominant-negative effect of the Tmc2a N-terminus on Pcdh15a localization in hair bundles), but 'protein binding' is uninformative. The partner is protocadherin 15a, a cadherin-superfamily tip-link protein, so the more informative binding term is cadherin binding. The functional consequence, coupling of the channel to the tip link, is captured by the channel activity and mechanosensory process terms. Proposed replacements: cadherin binding Supporting Evidence: PMID:25114259 Tmc2a interacted with both CD1 and CD3 isoforms of Pcdh15a, demonstrating that binding is not restricted to a particular Pcdh15a isoform. PMID:25114259 Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a–Tmc complex is critical for mechanotransduction. |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Plasma membrane location propagated across the TMC family. Reason: TMC proteins are multi-pass membrane proteins that act in the plasma membrane of hair-cell stereocilia. Correct but general; the specific location is the stereocilium tip membrane. Supporting Evidence: PMID:25114259 Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a–Tmc complex is critical for mechanotransduction. |
| GO:0005886 plasma membrane | IDA PMID:25114259 Tip-link protein protocadherin 15 interacts with transmembra... | ACCEPT | Summary: Plasma membrane location curated from Maeda et al. 2014. Reason: The location is correct for a multi-pass channel subunit that works in the hair-bundle membrane. The zebrafish imaging in this paper used an N-terminal Tmc2a fragment fused to GFP-CAAX, which is targeted to the membrane by the CAAX motif, so the direct evidence for full-length Tmc2a is weak; a full-length Tmc2a-mEGFP transgene later failed to express. Accepted because the location is supported by family-wide evidence and by the requirement for Tmc2a at the site of mechanotransduction. Supporting Evidence: PMID:25114259 Next, we determined whether endogenous Pcdh15a was correctly localized in hair cells expressing Tmc2a 1–117 -GFP-CAAX. PMID:32371604 The Tg(myo6b:tmc2a-mEGFP-pA) construct failed to express Tmc2a-mEGFP. |
| GO:0005886 plasma membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Plasma membrane from InterPro TMC family and UniProt subcellular location. Reason: Consistent with the IBA and with the role of TMC subunits in the stereocilia membrane. |
| GO:0008381 mechanosensitive monoatomic ion channel activity | IBA GO_REF:0000033 | ACCEPT | Summary: Mechanosensitive ion channel activity asserted at the PANTHER TMC node. Reason: This is the core molecular function of the family, and zebrafish genetics fit it: loss of Tmc2a reduces saccular microphonic potentials and abolishes transduction in the upper layer of the cristae, and loss of tmc1, tmc2a and tmc2b together abolishes all hair-cell transduction. The more precise child GO:0140135 (cation channel) is recorded in core_functions as a contribution to the MET channel complex. Supporting Evidence: PMID:32371604 Interestingly, the tmc2a single mutants ( n = 8) had significantly reduced microphonic signals compared with WT siblings ( n = 7) and were the only single-gene mutants with such a deficit PMID:32371604 Together, our experiments reveal that, in cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop-shaped cells, and a lower, Tmc1/2b-dependent tier of gourd-shaped cells. PMID:39773557 Significant progress in our understanding of MET has been recently made by identifying the transmembrane channel-like proteins 1 and 2 (TMC1 and TMC2) as the pore-forming subunits of the MET channel (Kawashima et al., 2011; Pan et al., 2018). |
| GO:0008381 mechanosensitive monoatomic ion channel activity | ISS GO_REF:0000024 | ACCEPT | Summary: Mechanosensitive ion channel activity transferred from mouse Tmc2. Reason: Same function as the IBA row; mouse TMC2 is a pore-forming MET channel subunit and zebrafish Tmc2a is required for hair-cell mechanotransduction. Supporting Evidence: PMID:32371604 Interestingly, the tmc2a single mutants ( n = 8) had significantly reduced microphonic signals compared with WT siblings ( n = 7) and were the only single-gene mutants with such a deficit |
| GO:0016020 membrane | IEA GO_REF:0000002 | ACCEPT | Summary: Membrane, from the InterPro TMC domain. Reason: Correct; less specific than plasma membrane. |
| GO:0034220 monoatomic ion transmembrane transport | IEA GO_REF:0000108 | MODIFY | Summary: Ion transmembrane transport inferred from the mechanosensitive ion channel activity. Reason: Correct in kind but under-specific: the MET channel conducts cations. The same modification was made for this row in the human TMC1 review. Proposed replacements: monoatomic cation transmembrane transport |
| GO:0048882 lateral line development | IEP PMID:25114259 Tip-link protein protocadherin 15 interacts with transmembra... | REMOVE | Summary: Lateral line development, inferred from tmc2a expression in lateral-line hair cells. Reason: An expression pattern does not show that the gene acts in development of the organ. Tmc2a is a channel subunit of mature hair cells, it is barely detectable in the lateral line by in situ hybridization, and hair bundles form normally in tmc triple mutants. Its lateral-line role is transduction, which is covered by the mechanosensory detection terms. Supporting Evidence: PMID:29269857 According to RNA in situ hybridization studies of zebrafish larva, tmc2b is robustly expressed in hair cells of the lateral line, but the mRNAs of the paralogs tmc2a and tmc1 are present at much lower levels and cannot be detected by this method. PMID:32371604 Together, these results suggest that hair bundles are intact and central components of the MET complex are properly localized in tmc triple mutants. |
| GO:0050910 detection of mechanical stimulus involved in sensory perception of sound | IBA GO_REF:0000033 | ACCEPT | Summary: Detection of mechanical stimulus involved in sensory perception of sound, from the PANTHER TMC2 node. Reason: Core process, and zebrafish data support it directly for this copy: tmc2a single mutants are the only single tmc mutants with reduced saccular microphonics, the authors rank Tmc2a first in a Tmc2a > Tmc2b > Tmc1 hierarchy for sound detection, and tmc2a/tmc2b double mutants hear only intermittently. The WITH list includes the zebrafish genes themselves, which is expected when their own experimental rows seeded the node. Supporting Evidence: PMID:32371604 Interestingly, the tmc2a single mutants ( n = 8) had significantly reduced microphonic signals compared with WT siblings ( n = 7) and were the only single-gene mutants with such a deficit PMID:32371604 Together, these results demonstrate a Tmc2a > Tmc2b > Tmc1 hierarchy in the detection of sound. PMID:32167554 By examining tmc2b cwr2 tmc2a cwr3 double and tmc2b cwr8 tmc1 cwr4 tmc2a cwr6 triple mutants, we demonstrate that the double mutant has attenuated hearing attributable to diminished mechanotransduction, but the triple mutant is deaf owing to extinguished mechanotransduction |
| GO:0050910 detection of mechanical stimulus involved in sensory perception of sound | IGI PMID:25114259 Tip-link protein protocadherin 15 interacts with transmembra... | ACCEPT | Summary: Hearing-related mechanosensory detection from the Maeda 2014 genotype (dominant-negative Tmc2a N-terminal fragment line). Reason: The paper shows that expressing the Tmc2a N-terminus in hair cells mislocalizes Pcdh15a and reduces mechanically evoked calcium responses (tested in lateral-line hair cells). The assay is a dominant-negative interference with the Tmc-Pcdh15a complex rather than loss of tmc2a alone. The process itself is independently confirmed for tmc2a by later loss-of-function alleles that reduce saccular microphonics, so the annotation stands. Supporting Evidence: PMID:25114259 Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a–Tmc complex is critical for mechanotransduction. PMID:32371604 Interestingly, the tmc2a single mutants ( n = 8) had significantly reduced microphonic signals compared with WT siblings ( n = 7) and were the only single-gene mutants with such a deficit |
| GO:0050910 detection of mechanical stimulus involved in sensory perception of sound | IGI PMID:29269857 A molecular basis for water motion detection by the mechanos... | MODIFY | Summary: Sound-detection term from the tmc2a/tmc2b double mutant of Chou et al. 2017. Reason: Chou et al. assayed the double mutant only in the lateral line, where microphonic potentials and dye uptake were abolished; the lateral line detects water motion, not sound. The finding is correct but belongs under the general mechanosensory detection term. Hearing in the double mutant was measured later (Chen et al. 2020, attenuated hearing) and is covered by the IBA row. Proposed replacements: detection of mechanical stimulus involved in sensory perception Supporting Evidence: PMID:29269857 Stimulus-evoked microphonic potentials were not detectable in neuromasts with A–P orientations in the PLL of tmc2a −/− /tmc2b −/− larvae (Fig. 8a, b) at 6 dpf, yet robust signatures were apparent in controls. PMID:29269857 Overall, these findings indicate that the residual activity in the tmc2b −/− mutant is dependent on Tmc2a. |
| GO:0060005 vestibular reflex | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Vestibular reflex, from the PANTHER TMC2 node. Reason: Organism-level reflex downstream of vestibular hair-cell transduction. In zebrafish larvae the vestibular-induced eye movement is reduced in tmc2a/tmc2b and tmc1/tmc2b double mutants but not in tmc2a single mutants, so tmc2a contributes redundantly. Kept as a correct but non-core consequence of the channel function. Supporting Evidence: PMID:32371604 With regard to vestibular function of the utricle/anterior macula, we observed a reduction in VIEM in both tmc1/2b and tmc2a/2b double mutants, whereas all other genotypes yielded responses that were not statistically different from their siblings |
| GO:0060119 inner ear receptor cell development | IEP PMID:25114259 Tip-link protein protocadherin 15 interacts with transmembra... | REMOVE | Summary: Inner ear receptor cell development, inferred from tmc2a expression in nascent otic hair cells. Reason: Expression in developing hair cells is not evidence of a developmental role. Tmc2a is a transduction channel subunit; hair bundles and the other MET components are in place in tmc triple mutants. Supporting Evidence: PMID:25114259 We observed that tmc2a is expressed in the nascent hair cells of the developing ear as early as 1 dpf, and expression in the inner ear continues throughout development PMID:32371604 Together, these results suggest that hair bundles are intact and central components of the MET complex are properly localized in tmc triple mutants. |
| GO:0070588 calcium ion transmembrane transport | IEA GO_REF:0000120 | ACCEPT | Summary: Calcium ion transmembrane transport inferred from the channel activity terms. Reason: Calcium entry through the MET channel is a direct consequence of the channel activity; Tmc2a-expressing neuromast bundles show larger calcium signals. Supporting Evidence: PMID:36905930 This idea is supported by our results, as we show that the calcium signals in hair bundles expressing both Tmc2b and Tmc2a are larger compared to those expressing just Tmc2b (Figure 6). |
| GO:0071260 cellular response to mechanical stimulus | IMP PMID:33490059 Tmc Reliance Is Biased by the Hair Cell Subtype and Position... | ACCEPT | Summary: Cellular response to mechanical stimulus from tmc2a mutants (Zhu et al. 2020): short hair cells of the cristae lose mechanotransduction-dependent dye uptake. Reason: The paper measures cell-level transduction (styryl dye uptake) and shows that short hair cells need Tmc2a. The term is general but accurate for this assay; the more specific process, detection of mechanical stimulus in sensory perception, is recorded in core_functions. Supporting Evidence: PMID:33490059 These findings confirm that short hair cells mainly depend on Tmc2 isoforms, and Tmc2a plays a significant role in these cells. |
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Download this section (compressed HTML)Q: Does full-length Tmc2a localize to stereocilia tips and rescue tmc2a or tmc triple mutants when expressed from a hair-cell promoter, and can Tmc2a rescue the Tmc2b-only neuromast hair cells? The reciprocal Tmc2b transgene rescues all end organs, but the Tmc2a construct never expressed.
Q: Do Tmc2a and Tmc2b form heteromeric channels in the cells that express both, and does Tmc2a confer different conductance or calcium permeability (larger responses in P-to-A neuromast cells, broader frequency range in the utricle)?
Q: Is the tmc2a/tmc2b pair a product of the teleost genome duplication although both copies are on zebrafish chromosome 5? Are the medaka co-orthologs on different chromosomes, and does a gar-anchored double-conserved-synteny analysis place both in the same ancestral block?
Experiment: Express Tmc2b (and Tmc2a, if a stable construct can be made) under a tmc2a enhancer in tmc2a mutants and measure dye uptake in upper-layer crista hair cells and saccular microphonics.
Hypothesis: The Tmc2a-dependent crista and saccular phenotypes reflect expression, not a Tmc2a-specific protein property.
Type: cross-rescue
Experiment: Record MET currents from neuromast hair cells of tmc2b mutants (Tmc2a-only P-to-A cells) and tmc2a mutants (Tmc2b-only cells) and compare conductance and calcium permeability.
Hypothesis: Tmc2a changes single-channel conductance or calcium selectivity relative to Tmc2b.
Type: electrophysiology
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