The tetraspan LHFPL5 is critical to establish maximal force sensitivity of the mechanotransduction channel of cochlear hair cells

Qiu, Xinwei; Liang, Xiaoping; Llongueras, José P.; Cunningham, Christopher L.; Müller, Ulrich

doi:10.1016/j.celrep.2023.112245

Cited by 9 publications

(15 citation statements)

References 57 publications

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“…The existence of a direct, physical connection between the channel complex and the tip link is supported by two lines of results: First, TMC1/2, TMIE, and LHFPL5 can all bind in vitro to PCDH15, which constitutes the lower part of the tip link [10][11][12][13]33,35 . Second, from dominant negative experiments in zebrafish stereocilia: endogenous PCDH15 is redistributed broadly after the overexpression of membrane-bound TMC2a-N-terminal fragments in zebrafish 33 .…”

Section: Introductionmentioning

confidence: 92%

“…Previous in vitro and electrophysiological studies have reported the existence of multiple molecular interactions between the pore channel subunit TMC1 and the lower part of the tip link, PCDH15: TMC1 can interact directly with PCDH15 33 or indirectly via TMIE and LHFPL5, which can also interact between themselves [10][11][12]31,32 . However, we found that the combination of all these interactions is insufficient to maintain TMC1 connected to the tip link in the absence of LOXHD1.…”

Section: The Mature Auditory Met Channel Complex Requires a 5th Subun...mentioning

confidence: 99%

“…Molecular solutions to MET are radically different between these senses: touch relies mainly on a single molecule forming a mechanosensitive ion channel, PIEZO2 18,19 . Contrarily, hearing requires the assembly of at least four distinct proteins to form a functional ion channel complex: TMC1/TMC2, TMIE, CIB2, and LHFPL5 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] . To sense sound-induced forces, MET proteins must be transported, positioned, and maintained at precise sites within the hair bundle (HB).…”

Section: Introductionmentioning

confidence: 99%

“…Each channel complex protein interacts with multiple other subunits, and many complex proteins depend on other subunits for proper localization 5,[8][9][10][11][12][13][31][32][33][34][35][36][37] . For instance, in Tmie KO and Cib2 KO hair cells, TMC1 and TMC2 do not localize to the HB 8,11,31 , a phenotype that also progressively develops in Lhfpl5 KO 12 .…”

Section: Introductionmentioning

confidence: 99%

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LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission

Wang,

Miller,

et al. 2024

Preprint

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Hearing is initiated in hair cells by the mechanical activation of ion channels in the hair bundle. The hair bundle is formed by stereocilia organized into rows of increasing heights interconnected by tip links, which convey sound-induced forces to stereocilia tips. The auditory mechanosensitive channels are complexes containing at least four protein-subunits – TMC1/2, TMIE, CIB2, and LHFPL5 – and are located at the tips of shorter stereocilia at a yet-undetermined distance from the lower tip link insertion point. While multiple auditory channel subunits appear to interact with the tip link, it remains unknown whether their combined interaction alone can resist the high-frequency mechanical stimulations owing to sound. Here we show that an unanticipated additional element, LOXHD1, is indispensable for maintaining the TMC1 pore-forming channel subunits coupled to the tip link. We demonstrate that LOXHD1 is a unique element of the auditory mechanotransduction complex that selectively affects the localization of TMC1, but not its close developmental paralogue TMC2. Taking advantage of our novel immunogold scanning electron microscopy method for submembranous epitopes (SUB-immunogold-SEM), we demonstrate that TMC1 normally concentrates within 100-nm of the tip link insertion point. In LOXHD1’s absence, TMC1 is instead mislocalized away from this force transmission site. Supporting this finding, we found that LOXHD1 interacts selectively in vitro with TMC1 but not with TMC2 while also binding to channel subunits CIB2 and LHFPL5 and tip-link protein PCDH15. SUB-immunogold-SEM additionally demonstrates that LOXHD1 and TMC1 are physically connected to the lower tip-link complex in situ. Our results show that the TMC1-driven mature channels require LOXHD1 to stay coupled to the tip link and remain functional, but the TMC2-driven developmental channels do not. As both tip links and TMC1 remain present in hair bundles lacking LOXHD1, it opens the possibility to reconnect them and restore hearing for this form of genetic deafness.

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Section: Introductionmentioning

confidence: 92%

Section: The Mature Auditory Met Channel Complex Requires a 5th Subun...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission

Wang,

Miller,

et al. 2024

Preprint

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“…In accordance with these studies, the recently elucidated structures of the native C. elegans TMC-1 and TMC-2 complexes revealed a dimeric complex that is composed of two copies of TMC-1/2, TMIE, and CIB2/3, the latter of which is known as CALM-1 in C. elegans 23 . Further, while numerous immunoprecipitation and yeast two-hybrid experiments have suggested interactions between MT subunits 10,12,15,24,25 , these experiments have been hampered by the inability to express biochemically well-behaved TMC1 and TMC2 proteins. TMC1 and TMC2 do not migrate to the plasma membrane in heterologous cell lines 26 , preventing reconstitution of the vertebrate MT complex.…”

Section: Introductionmentioning

confidence: 99%

Single molecule studies of the native hair cell mechanosensory transduction complex

Clark,

Mitra,

Elferich

et al. 2023

Preprint

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Hearing and balance rely on the conversion of a mechanical stimulus into an electrical signal, a process known as mechanosensory transduction (MT). In vertebrates, this process is accomplished by an MT complex that is located in hair cells of the inner ear. While the past three decades of research have identified many subunits that are important for MT and revealed interactions between these subunits, the composition and organization of a functional complex remains unknown. The major challenge associated with studying the MT complex is its extremely low abundance in hair cells; current estimates of MT complex quantity range from 3-60 attomoles per cochlea or utricle, well below the detection limit of most biochemical assays that are used to characterize macromolecular complexes. Here we describe the optimization of two single molecule assays, single molecule pull-down (SiMPull) and single molecule array (SiMoA), to study the composition and quantity of native mouse MT complexes. We demonstrate that these assays are capable of detecting and quantifying low attomoles of the native MT subunits protocadherin-15 (PCDH15) and lipoma HMGIC fusion partner-like protein 5 (LHFPL5). Our results illuminate the stoichiometry of PCDH15- and LHFPL5-containing complexes and establish SiMPull and SiMoA as productive methods for probing the abundance, composition, and arrangement of subunits in the native MT complex.Impact StatementIn the present work, the authors develop and employ single molecule methods to detect, characterize, and quantitate attomole quantities of the hair cell mechanosensory transduction complex.

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LHFPL5 is a key element in force transmission from the tip link to the hair cell mechanotransducer channel

Beurg,

Schwalbach,

Fettiplace

2024

Proc. Natl. Acad. Sci. U.S.A.

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During auditory transduction, sound-evoked vibrations of the hair cell stereociliary bundles open mechanotransducer (MET) ion channels via tip links extending from one stereocilium to its neighbor. How tension in the tip link is delivered to the channel is not fully understood. The MET channel comprises a pore-forming subunit, transmembrane channel-like protein (TMC1 or TMC2), aided by several accessory proteins, including LHFPL5 (lipoma HMGIC fusion partner-like 5). We investigated the role of LHFPL5 in transduction by comparing MET channel activation in outer hair cells of Lhfpl5−/− knockout mice with those in Lhfpl5+/− heterozygotes. The 10 to 90 percent working range of transduction in Tmc1+/+; Lhfpl5+/− was 52 nm, from which the single-channel gating force, Z, was evaluated as 0.34 pN. However, in Tmc1+/+; Lhfpl5−/− mice, the working range increased to 123 nm and Z more than halved to 0.13 pN, indicating reduced sensitivity. Tip link tension is thought to activate the channel via a gating spring, whose stiffness is inferred from the stiffness change on tip link destruction. The gating stiffness was ~40 percent of the total bundle stiffness in wild type but was virtually abolished in Lhfpl5−/−, implicating LHFPL5 as a principal component of the gating spring. The mutation Tmc1 p.D569N reduced the LHFPL5 immunolabeling in the stereocilia and like Lhfpl5−/− doubled the MET working range, but other deafness mutations had no effect on the dynamic range. We conclude that tip-link tension is transmitted to the channel primarily via LHFPL5; residual activation without LHFPL5 may occur by direct interaction between PCDH15 and TMC1.

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The tetraspan LHFPL5 is critical to establish maximal force sensitivity of the mechanotransduction channel of cochlear hair cells

Cited by 9 publications

References 57 publications

LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission

LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission

Single molecule studies of the native hair cell mechanosensory transduction complex

LHFPL5 is a key element in force transmission from the tip link to the hair cell mechanotransducer channel

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