Biophysical Study Of Sensory Transduction Mechanisms

National Institute On Deafness And Other Communication Disorders

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Abstract

Hearing of mammals depends on hair cells, both inner and outer, which convert mechanical signal into electrical signal to transmit to the brain. Outer hair cells are of particular significance in the ear's sensitivity and frequency selectivity, critical factors for communication. The role of outer hair cells stems from their property that these sensory cells are also motors to amplify weak signals. We have previously established that the hair cell motor uses electrical energy available at the plasma membrane in a manner similar to piezoelectricity, based on the coupling of electric charge transfer across the membrane with membrane area changes. Specifically, this motility can be reasonably explained by a simple two state model in which two states differ in charge and membrane area. The area difference is determined by tension dependence of the motor activity. It has been shown that the major component of this membrane motor is prestin, a member of the SLC26 superfamily. Prestin is the membrane protein in outer hair cells that harness electrical energy by changing its membrane area responding to changes in the membrane potential. To examine the effect of membrane thickness on this protein, phosphatidylcholine with various acyl chain length were incorporated into the plasma membrane by using gamma-cyclodextrin. Incorporation of short chain PCs increased the linear capacitance and positively shifted the voltage dependence of prestin, up to 120 mV, in cultured cells. phosphatidylcholines with long acyl chains had the opposite effects. Since the linear capacitance is inversely related to the membrane thickness, these voltage shifts are attributable to membrane thickness. The corresponding voltage shifts of electromotility were observed in outer hair cells. These results demonstrate that electromotility is extremely sensitive to the thickness of the plasma membrane, presumably involving hydrophobic mismatch. These observations indicate that the extended state of the motor molecule, which is associated with the elongation of outer hair cells, has a conformation with a shorter hydrophobic height in the lipid bilayer. To examine the morphological basis for the physiological function, we imaged the cytosolic surface of the lateral plasma membrane of outer hair cells from guinea pigs'inner ear, using atomic force microscopy. We used a "cell-free" preparation, in which a patch of plasma membrane was firmly attached to a substrate and the cytoplasmic face was exposed. The membrane patches contained densely packed particles whose diameter, after correcting for the geometry of the probing tip, was about 10 nm. The particles were predominantly aligned unidirectionally with spacing of about 36 nm. The density of the particle was about 850 per square m, which could be an underestimate presumably due to the method of sample preparation. Antibody-labeled specimens showed particles more elevated than unlabeled preparation indicative of primary and secondary antibody complexes. The corrected diameters of these particles labeled with anti-actin were about 12 nm while that with anti-prestin were about 8 nm. The alignment pattern in anti-prestin labeled specimens resembled that of the unlabeled preparation. Specimens labeled with actin antibodies did not show such alignment. We interpret that the particles observed in the unlabeled membranes correspond to the 10 nm particles reported by electron microscopy and that these particles contain prestin. We also investigated some details in mechanoelectrical transduction in the stereocilia, the essential step of detecting the sound. While gating of transducer channels has been successfully described by assuming that one channel is associated with a tip link in the hair bundle, recent reports indicate that a single tip link is associated with more than one channel. To address the consistency of the model with the observations, gating of transducer channels is described here by assuming that each tip link is associated with two identical transducer channels, which are connected either in series or in parallel. We found that series connection does not lead to a single minimum of stiffness with respect to hair bundle displacement unless the minimum is above a certain positive value. Thus negative stiffness must appear in pairs in the displacement axis. In contrast, parallel connection of the two channels predicts gating compliance similar to that predicted by the one-channel-per-tip-link model of channel gating, within the physiological range of parameters. Parallel connection of transducer channels is, therefore, a reasonable assumption to explain most experimental observations. However, the compatibility with series connection cannot be ruled out for experimental data on turtle hair cells.