Harmonin inhibits presynaptic Ca_v1.3 Ca²⁺ channels in mouse inner hair cells

Abstract

Harmonin is a scaffolding protein required for normal mechanosensory function in hair cells. Here, we describe a novel presynaptic association of harmonin and Ca_v1.3 Ca²⁺ channels at the mouse inner hair cell synapse, which limits channel availability through a ubiquitin-dependent pathway.

Voltage-gated Ca_v1.3 (L-type) Ca²⁺ channels mediate Ca²⁺ influx and exocytosis at the inner hair cell (IHC) synapse and are required for hearing^1,2. The C-terminus of the Ca_v1.3 α₁ subunit (α₁1.3) binds to PDZ (PSD-95 (Postsynaptic density-95)/Discs large/ZO-1 (Zona occludens-1) domains, which affect neuronal Ca_v1.3 localization and function³. In screening for PDZ-proteins that interact with Ca_v1 α₁-subunits, we discovered that harmonin (variant a), a PDZ-protein expressed in IHCs⁴, binds to α₁1.3 via the second of its three PDZ domains (Fig. 1a,b).

Figure 1.

Harmonin inhibits Ca_v1.3 channels and is localized at inner hair cell synapses.

(a) Myc-harmonin coimmunoprecipitates with FLAG-α₁1.3 in cotransfected HEK293T cells (lane 1) but not in cells transfected with myc-harmonin alone (lane 2). (b) Western blot (upper panel) from pull-down assay shows GST-1.3CT (CT) but not GST (G) binds his-tagged PDZ2 of harmonin. Input (I) represents ~5% protein His-PDZ protein input. Ponceau staining (lower panel) shows amounts of GST-proteins used. Full length blots are presented in Supplementary Fig.5. (c) Harmonin inhibits Ca_v1.3 I_Ba density in transfected HEK293T cells. Ca_v1.3 alone, n=11; +harmonin, n=11. (d) Representative traces showing gating currents (I_gating) of Ca_v1.3 ± harmonin measured at the I_Ba reversal potential (+60 mV). (e) His-PDZ2 of harmonin binds to α₁1.3 CT but not with L-A mutation. Pull-down assay was done as in b. (f) No effect of harmonin on I_Ba density for Ca_v1.3 with L-A mutation (Ca_v1.3_L-A). Ca_v1.3_L-A alone, n=8; Ca_v1.3+harmonin, n=7. (g) Confocal projections of whole mounts of Organ of Corti from wild-type (WT, P7 or P15) or Ca_v1.3^−/− mice (P7) double-labeled for CtBP2 (red) and Ca_v1.3 (green) or harmonin (green). Synaptic labeling of CtBP2 appears as spots basal to the nucleus, which is also labeled by these antibodies. Areas of colocalization (arrows) are yellow in the merged images. Scale bars, 10 μm. Graph shows distribution of harmonin-positive synapses in individual IHCs from P6–8 and P14–16 IHCs. In c and f, error bars represent s.e.m.

Like the PDZ-protein erbin⁵, harmonin promoted voltage-dependent facilitation of Ca_v1.3 in transfected HEK293T cells (not shown). However, harmonin also significantly suppressed peak Ca_v1.3 Ba²⁺ current (I_Ba) density (~66%, p<0.001; Fig. 1c) without major effects on voltage-dependent activation (Supplementary table 1). Harmonin significantly decreased maximal “on” gating charge (Q_on) of I_Ba and tail current amplitudes at the reversal potential (Fig. 1d, Supplementary Fig. 1), which suggests that harmonin reduces the number of available Ca_v1.3 channels rather than the open probability. Disrupting the type I PDZ binding sequence of α₁1.3 by substituting the C-terminal leucine for alanine (L-A) prevented harmonin binding (Fig. 1e) and inhibition of Ca_v1.3 peak current density (p=0.93; Fig. 1f).

As described previously ^6-8, harmonin is strongly localized in the apical hair bundles of IHCs (Supplementary Fig.2). However, closer scrutiny revealed harmonin clusters at the base of IHCs, which colocalized with the ribbon synapse protein ribeye/CtBP2 at a subset of synapses (Fig. 1g). Comparisons before (P6–8) and after (P14–16) hearing onset revealed a developmental increase in the number of harmonin-positive IHC synapses (Fig. 1g). In most immature IHCs (30 of 35), harmonin was localized at ~10–30% of synapses per IHC. However, in mature IHCs (18 of 31), ~40–60% of synapses per IHC were harmonin-positive (Fig. 1g). Ca_v1.3 channels may be necessary for the synaptic localization of harmonin: the fraction of harmonin-positive synapses was significantly reduced in IHCs from Ca_v1.3^−/− mice (~26% of harmonin-positive synapses in WT, P6-8, n=84 IHCs, vs. ~10% in Ca_v1.3^−/−, P6-8, n=82 IHCs, p<0.001; Fig. 1g). Harmonin also coimmunoprecipitated with Ca_v1.3 from cochlear extracts (Supplementary Fig. 2). These findings support a presynaptic association of harmonin with Ca_v1.3, primarily in mature IHCs.

To determine if harmonin regulates presynaptic Ca_v1.3 channels in IHCs, we analyzed “deaf-circler” mice (dfcr)⁹. While the mutant (dfcr harmonin) retains PDZ2 that interacts with α₁1.3 (Fig. 1b), an internal deletion removes the coiled-coil domain and causes abnormal proximity of the PDZ domains. Dfcr harmonin did not bind to α₁1.3 (Fig. 2a) or affect peak Ca_v1.3 I_Ba density in transfected HEK293T cells (p=0.10; Fig. 2b), which demonstrates that dfcr harmonin does not functionally interact with α₁1.3. In agreement with a normally inhibitory role for harmonin, whole cell I_Ba density was greater in P16–18 dfcr IHCs than in control IHCs (~33%, p<0.01; Fig. 2c), despite otherwise normal Ca_v1.3 activation properties (Supplementary Table 2). This difference was not observed at P6-8 (p=0.59, Fig. 2d), consistent with sparse synaptic localization of harmonin at this age (Fig. 1g). Ca²⁺ microdomains, which primarily reflect presynaptic Ca²⁺ influx¹⁰, had higher amplitudes in mature dfcr than control IHCs (~53%, p=0.02; Fig. 2e, f). Taken together, these results show that harmonin directly inhibits synaptic Ca_v1.3 channel density in IHCs, which is disrupted by the dfcr mutation.

Figure 2.

Dfcr mutation prevents functional interaction of harmonin and Ca_v1.3

(a) Pull-down assays with GST or GST-α₁1.3 CT and increasing amounts of lysates from HEK293T cells transfected with myc-harmonin or myc-dfcr harmonin, as indicated. Left 2 lanes show input (~5%) and Ponceau staining (lower panel) shows GST or GST-α₁1.3 CT used in the assay. Full length blots are presented in Supplementary Fig.5. (b) Same protocol as in Fig.1c except in cells transfected with Ca_v1.3 alone, n=7, +harmonin, n=9, or +dfcr-harmonin, n=8. (c,d) Plots of I_Ba density vs. test voltage from whole-cell patch clamp recordings at P16-18 (c) and P6-8 (d) from control (+/−; n=23 in c, n=45 in d) or dfcr (−/−; n=31 in c, n=42 in d) IHCs. (e,f) Simultaneous whole-cell patch clamp recordings and fluorescent measurement of presynaptic Ca²⁺ microdomains. (e) Representative hotspots of Fluo-5N fluorescence in control (+/+) and dfcr (−/−) IHCs during depolarization. Scale bar, 1 μm. (f) Top, Whole-cell I_Ca recorded in control (black line and dark grey area, n = 14) and dfcr (grey line and light grey area, n = 16) IHCs. Bottom, Background-subtracted and normalized Fluo-5N fluorescence change (ΔF/F₀, mean±SD) in the center of the Ca²⁺ microdomain (41 domains in 14 control IHCs and 41 domains in 16 dfcr IHCs). In b-d, error bars represent s.e.m. Protocols for the use of mice were approved by the Institutional Animal Care and Use Committee at U. Iowa and U. Gottingen.

Cell surface density of ion channels can be regulated by ubiquitination and targeting for proteosomal or lysosomal degradation¹¹. Thus, we tested if harmonin enhanced ubiquitination of Ca_v1.3 in HEK293T cells. Following immunoprecipitation with α₁1.3 antibodies, western blotting revealed higher levels of ubiquitinated channels cotransfected with harmonin, which was intensified by the proteosomal inhibitor MG132 (Fig. 3a,b). In addition, harmonin altered the distribution of cell-surface Ca_v1.3 channels immunofluorescently labeled through an extracellular hemagglutinin epitope. While Ca_v1.3 channels alone showed a fairly continuous distribution, harmonin caused Ca_v1.3 channels to form large clusters in the plasma membrane (Fig. 3c, Supplementary Fig. 3). The patchier cell-surface distribution of Ca_v1.3 channels caused by harmonin was reversed by MG132 (Fig. 3c). Given that MG132 broadly affects the ubiquitin system and may affect both proteosomal and lysosomal protein pathways, the altered distribution of Ca_v1.3 channels caused by harmonin may result from improper trafficking and/or degradation of ubiquitinated channels. Although Ca_vβ subunits inhibit ubiquitination of Ca_v channels^12,13, the inhibition of Ca_v1.3 current density by harmonin did not require Ca_vβ (Fig. 3d) but was blocked by MG132 (Fig. 3e; MG132 did not affect current density in cells with Ca_v1.3 alone, Supplementary Fig. 4). We conclude that harmonin tags Ca_v1.3 channels for ubiquitination and alters their functional levels at the cell surface. This may constrain the number of available presynaptic Ca_v1.3 channels in IHCs, a parameter that varies between IHC synapses¹⁰ and during development¹⁴. Together with evidence that harmonin regulates mechanotransduction currents^8,15, our findings reveal harmonin as not simply a scaffold, but an integral component of ion channel complexes and fundamental regulator of electrical and Ca²⁺ signaling in auditory hair cells.

Figure 3.

Harmonin inhibits plasma membrane Ca_v1.3 channel density through a ubiquitin-dependent pathway

(a,b) Ubiquitination of Ca_v1.3 channels in HEK293T cells transfected with Ca_v1.3±harmonin. Ca_v1.3 channels were immunoprecipitated with α₁1.3 antibodies and western blotting was performed with anti-ubiquitin (Ub, top panel) or α₁1.3 antibodies (bottom panel). Cells were maintained with (+) or without (−) MG132 (5 μM, 12 h) prior to lysis. Ubiquitination of Ca_v1.3 was quantitated and plotted as relative intensities (b); *, p<0.05. Full length blots are presented in Supplementary Fig.5. (c) Confocal micrographs showing immunofluorescence of cell-surface labeled HA-Ca_v1.3 channels transfected alone or with harmonin. Pretreatment with MG132 was as in (a). Scale bars, 5 μm. (d,e) I_Ba density recorded as in Fig.1c in HEK293T cells cotransfected without Ca_vβ (d; n=12 for Ca_v1.3, n=11 for +harmonin) or with Ca_vβ (e; n=12 for Ca_v1.3 alone, n=14 for +harmonin, n=11 for +harmonin+MG132). In (e), cells were treated with MG132 (5 μM) 1-3 h before recording. In b, d, e, error bars represent s.e.m.