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. 2015 Feb 3;112(5):1589-94.
doi: 10.1073/pnas.1420906112. Epub 2014 Dec 30.

Subunit determination of the conductance of hair-cell mechanotransducer channels

Maryline Beurg  1 , Wei Xiong  2 , Bo Zhao  2 , Ulrich Müller  2 , Robert Fettiplace  3
Affiliations

Subunit determination of the conductance of hair-cell mechanotransducer channels

Maryline Beurg et al. Proc Natl Acad Sci U S A. .

Abstract

Cochlear hair cells convert sound stimuli into electrical signals by gating of mechanically sensitive ion channels in their stereociliary (hair) bundle. The molecular identity of this ion channel is still unclear, but its properties are modulated by accessory proteins. Two such proteins are transmembrane channel-like protein isoform 1 (TMC1) and tetraspan membrane protein of hair cell stereocilia (TMHS, also known as lipoma HMGIC fusion partner-like 5, LHFPL5), both thought to be integral components of the mechanotransduction machinery. Here we show that, in mice harboring an Lhfpl5 null mutation, the unitary conductance of outer hair cell mechanotransducer (MT) channels was reduced relative to wild type, and the tonotopic gradient in conductance, where channels from the cochlear base are nearly twice as conducting as those at the apex, was almost absent. The macroscopic MT current in these mutants was attenuated and the tonotopic gradient in amplitude was also lost, although the current was not completely extinguished. The consequences of Lhfpl5 mutation mirror those due to Tmc1 mutation, suggesting a part of the MT-channel conferring a large and tonotopically variable conductance is similarly disrupted in the absence of Lhfpl5 or Tmc1. Immunolabelling demonstrated TMC1 throughout the stereociliary bundles in wild type but not in Lhfpl5 mutants, implying the channel effect of Lhfpl5 mutations stems from down-regulation of TMC1. Both LHFPL5 and TMC1 were shown to interact with protocadherin-15, a component of the tip link, which applies force to the MT channel. We propose that titration of the TMC1 content of the MT channel sets the gradient in unitary conductance along the cochlea.

Keywords: LHFPL5; TMC1; cochlea; hair cell; mechanotransducer channels.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
MT currents in OHCs of mouse Lhfpl5 mutants. (A) MT currents evoked by sinusoidal fluid-jet stimuli in OHCs from the cochlear apex (d = 0.2; Left) and cochlear base (d = 0.75; Right) in Lhfpl5+/− heterozygotes. (B) MT currents were evoked by sinusoidal fluid-jet stimuli in OHCs from the cochlear apex (Left) and cochlear base (Right) in Lhfpl5−/−. The two-harmonic response included a reduced amplitude normal response and also a larger reverse-polarity current produced by deflections of bundle away from its taller edge. The driving voltage to the fluid-jet piezoelectric disk, VPIEZO, is shown at the top and was calibrated to give hair bundle motion with amplitude of ∼130 nm in Lhfpl5 heterozygotes and 80 nm in Lhfpl5 knockouts. (C) Peak MT current for the heterozygote, Lhfpl5+/− (open circles) and for the knockout Lhfpl5−/− (filled circles) is plotted against d, the cochlear location. Filled circles are for the conventional stimulus–polarity response. Each point is the mean ± 1 SEM, with numbers of cells, each from a different animal, indicated next to the points. Note the mean current increases toward the high-frequency base for the heterozygote, but not for the knockout. (D) Peak reverse-polarity currents (mean ± 1 SD) shown for apex and base as in C. The recordings at the base were from P2–P4 mice and those at the apex were from P4–P6 mice, the difference reflecting the two-day lag in development at the apex relative to the base; numbers of cells, each from a different animal, are indicated next to the points. Measurements were made at −84 mV holding potential.
Fig. 2.
Fig. 2.
Single MT channel currents in OHCs of Lhfpl5 mutants. (A–D) Each panel shows three single MT channel currents for step displacements of bundle, an ensemble average, and an amplitude histogram. (A) Lhfpl5+/− heterozygote, cochlear apex; ensemble average of 18 responses, unitary current, −6.3 pA, P5 mouse; (B) Lhfpl5−/− knockout, cochlear apex; ensemble average of 10 responses, unitary current, −5.4 pA, P6 mouse; (C) Lhfpl5+/− heterozygote, cochlear base; ensemble average of 10 responses, unitary current, −12.0 pA, P3 mouse; and (D) Lhfpl5−/− knockout, cochlear base; ensemble average of 15 responses, unitary current, −6.2 pA, P2 mouse. (E) Collected results of MT current amplitudes (mean ± 1 SEM) as a function of cochlear location for wild-type and Lhfpl5+/− heterozygotes (combined results, filled circles), and Lhfpl5−/− knockouts (open circles). Number of cells averaged is given beside each point, each cell being from a different animal. All measurements were made at −84 mV holding potentials in 1.5 mM extracellular Ca2+ saline. (F) Ratio of single-channel current in Lhfpl5 −/−, Ich(knockout), to single-channel current in control, Ich (control), versus cochlear location, filled triangles (mean ± 1 SEM, from measurements in E). Open squares are results for Tmc1 mutant, ratio of knockout to control, from ref. . Numbers of cells used to compute these ratios were (from apex to base) 9, 5, 9 (wild type), and 17, 3, 7 (Tmc1−/−), each cell from a different animal.
Fig. 3.
Fig. 3.
Immunolabelling with anti-TMC1 antibody. (A) Surface preparation of apical cochlea of P5 wild type, showing three rows of OHC hair bundles (Top) and one row of IHC bundles (Bottom). (Left) ESPN labelling; (Middle) anti-TMC1 antibody; and (Right) merge. Note the bundles, including the OHC kinocilia, are labelled for TMC1. (B) Apical cochlea of P4 Tmc1Δ/Δ mutant, as in A. Note that there is no TMC1 antibody label in the knockout. (C) Apical cochlea of P5 Lhfpl5−/− mutant shows no TMC1 label in the bundles. The hair bundles in the Lhfpl5−/− mutant were more fragile and disorganized and the ESPN label was weaker, hence the higher background. Each image is an average of three 0.3-μm-thick stacks. (D) Apical cochlear OHCs of P16 wild type exhibited punctate TMC1 labelling toward the Top of the bundle, although the resolution was insufficient to determine whether labelling was present in all three stereociliary rows. The five traces are confocal sections from the Top to the Bottom of the bundles (∼3 μm).
Fig. 4.
Fig. 4.
Interaction between LHFPL5 and TMC2. (A) Apical OHCs of P4 Lhfpl5+/− heterozygote cochlea, transfected with Tmc2-Myc and cultured for 1 d in vitro and labelled with myc antibody (red) and phalloidin (green). (B) Apical OHCs of P4 Lhfpl5−/− homozygote cochlea, transfected with Tmc2-Myc, and cultured for 1 d in vitro and labelled as in A. TMC2 was still targeted to bundle despite the absence of LHFPL5. (C) MT currents for large (200 nm) bundle displacement in apical OHCs of Lhfpl5+/− Tmc2+/+ (Top), Lhfpl5−/− Tmc2+/− (Middle), and Lhfpl5−/− Tmc2−/− (Bottom). (D) Collected MT currents (mean ± SEM) for the response component for positive displacement of hair bundle in the three mutants in C. The numbers of cells averaged are indicated next to the points, with recordings being collected from five Lhfpl5+/− Tmc2+/+, six Lhfpl5−/− Tmc2+/−, and three Lhfpl5−/− Tmc2−/− double knockout animals. The size of the reverse component was reduced from Tmc2+/+ to Tmc2+/− to Tmc2−/−.
Fig. 5.
Fig. 5.
Coimmunoprecipitations showing interactions between LHFPL5, TMC1, TMC2, and PCDH15. (A) HEK293 cells were transfected to express LHFPL5 either alone or together with Myc-TMC1. Immunoprecipitations were carried out with anti-Myc followed by Western blotting using anti-LHFPL5 for detection. The Bottom two lanes show input protein before immunoprecipitation. No protein interaction was in evidence. (B) HEK293 cells were transfected to express Myc-TMC1 or Myc-TMC2 alone, or together with PCDH15-CD1, -CD2, -CD3, or control GFP-tagged N-cadherin (N-Cd). Immunoprecipitations were carried out with anti-Myc followed by Western blotting with antibodies to the proteins indicated on the Right. The Top two panels show results from coimmunoprecipitations (Co-IP); the third row shows the amount of TMC1/2 proteins recovered in the immunoprecipitates (IP); the Bottom two rows show input PCDH15 and N-cadherin proteins in extracts before immunoprecipitation. Amounts of PCDH15 were consistently decreased by coexpressing TMC1 or TMC2, but coimmunoprecipitation was robustly observed.
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