Dataset Information

Rat Auditory Inner Hair Cell Mechanotransduction and Stereociliary Membrane Diffusivity Are Similarly Modulated by Calcium.

ABSTRACT: The lipid bilayer plays a pivotal role in force transmission to many mechanically-gated channels. We developed the technology to monitor membrane diffusivity in order to test the hypothesis positing that Ca²⁺ regulates open probability (P _o) of cochlear hair cell mechanotransduction (MET) channels via the plasma membrane. The stereociliary membrane was more diffusive (9x) than the basolateral membrane. Elevating intracellular Ca²⁺ buffering or lowering extracellular Ca²⁺ reduced stereociliary diffusivity and increased MET P _o. In contrast, prolonged depolarization increased stereociliary diffusivity and reduced MET P _o. No comparable effects were noted for soma measurements. Although MET channels are located in the shorter stereocilia rows, both rows had similar baseline diffusivity and showed similar responses to Ca²⁺ manipulations and MET channel blocks, suggesting that diffusivity is independent of MET. Together, these data suggest that the stereociliary membrane is a component of a calcium-modulated viscoelastic-like element regulating hair cell mechanotransduction.

SUBMITTER: George SS

PROVIDER: S-EPMC7689183 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Rat Auditory Inner Hair Cell Mechanotransduction and Stereociliary Membrane Diffusivity Are Similarly Modulated by Calcium.

George Shefin S SS Steele Charles R CR Ricci Anthony J AJ

iScience 20201105 12

The lipid bilayer plays a pivotal role in force transmission to many mechanically-gated channels. We developed the technology to monitor membrane diffusivity in order to test the hypothesis positing that Ca2+ regulates open probability (P o) of cochlear hair cell mechanotransduction (MET) channels via the plasma membrane. The stereociliary membrane was more diffusive (9x) than the basolateral membrane. Elevating intracellular Ca2+ buffering or lowering ext ...[more]

PMID: 33294782

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Project description:Transmembrane channel-like protein isoform 1 (TMC1) is a major component of the mechano-electrical transducer (MET) channel in cochlear hair cells and is subject to numerous mutations causing deafness. We report a new dominant human deafness mutation, TMC1 p.T422K, and have characterized the homologous mouse mutant, Tmc1 p.T416K, which caused deafness and outer hair cell (OHC) loss by the fourth postnatal week. MET channels showed decreased Ca2+ permeability and resting open probability, but no change in single-channel conductance or expression. Three adjacent deafness mutations are TMC1 p.L416R, p.G417R, and p.M418K, the last homologous to the mouse Beethoven that exhibits similar channel effects. All substitute a positive for a neutral residue, which could produce charge screening in the channel pore or influence binding of an accessory subunit. Channel properties were compared in mice of both sexes between dominant (Tmc1 p.T416K, Tmc1 p.D569N) and recessive (Tmc1 p.W554L, Tmc1 p.D528N) mutations of residues near the putative pore of the channel. Tmc1 p.W554L and p.D569N exhibit reduced maximum current with no effect on single-channel conductance, implying a smaller number of channels transported to the stereociliary tips; this may stem from impaired TMC1 binding to LHFPL5. Tmc1 p.D528N, located in the pore's narrowest region, uniquely caused large reductions in MET channel conductance and block by dihydrostreptomycin (DHS). For Tmc1 p.T416K and Tmc1 p.D528N, transduction loss occurred between P15 and P20. We propose two mechanisms linking channel mutations and deafness: decreased Ca2+ permeability, common to all mutants, and decreased resting open probability in low Ca2+, confined to dominant mutations.SIGNIFICANCE STATEMENT Transmembrane channel-like protein isoform 1 (TMC1) is thought to be a major component of the mechanotransducer channel in auditory hair cells, but the protein organization and channel structure are still uncertain. We made four mouse lines harboring Tmc1 point mutations that alter channel properties, causing hair cell degeneration and deafness. These include a mouse homolog of a new human deafness mutation pT416K that decreased channel Ca2+ permeability by introducing a positively-charged amino acid in the putative pore. All mutations are consistent with the channel structure predicted from modeling, but only one, p.D528N near the external face of the pore, substantially reduced channel conductance and Ca2+ permeability and virtually abolished block by dihydrostreptomycin (DHS), strongly endorsing its siting within the pore.

Project description:Membrane proteins, such as ion channels, interact dynamically with their lipid environment. Phosphoinositol-4,5-bisphosphate (PIP2) can directly or indirectly modify ion-channel properties. In auditory sensory hair cells of rats (Sprague Dawley) of either sex, PIP2 localizes within stereocilia, near stereocilia tips. Modulating the amount of free PIP2 in inner hair-cell stereocilia resulted in the following: (1) the loss of a fast component of mechanoelectric-transduction current adaptation, (2) an increase in the number of channels open at the hair bundle's resting position, (3) a reduction of single-channel conductance, (4) a change in ion selectivity, and (5) a reduction in calcium pore blocking effects. These changes occur without altering hair-bundle compliance or the number of functional stereocilia within a given hair bundle. Although the specific molecular mechanism for PIP2 action remains to be uncovered, data support a hypothesis for PIP2 directly regulating channel conformation to alter calcium permeation and single-channel conductance.SIGNIFICANCE STATEMENT How forces are relayed to the auditory mechanoelectrical transduction (MET) channel remains unknown. However, researchers have surmised that lipids might be involved. Previous work on bullfrog hair cells showed an effect of phosphoinositol-4,5-bisphosphate (PIP2) depletion on MET current amplitude and adaptation, leading to the postulation of the existence of an underlying myosin-based adaptation mechanism. We find similar results in rat cochlea hair cells but extend these data to include single-channel analysis, hair-bundle mechanics, and channel-permeation properties. These additional data attribute PIP2 effects to actions on MET-channel properties and not motor interactions. Further findings support PIP2's role in modulating a fast, myosin-independent, and Ca2+-independent adaptation process, validating fast adaptation's biological origin. Together this shows PIP2's pivotal role in auditory MET, likely as a direct channel modulator.

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Dataset Information

Rat Auditory Inner Hair Cell Mechanotransduction and Stereociliary Membrane Diffusivity Are Similarly Modulated by Calcium.

Publications

Rat Auditory Inner Hair Cell Mechanotransduction and Stereociliary Membrane Diffusivity Are Similarly Modulated by Calcium.

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