Development and localization of reverse-polarity mechanotransducer channels in cochlear hair cells

Abstract

Cochlear hair cells normally detect positive deflections of their hair bundles, rotating toward their tallest edge, which opens mechanotransducer (MT) channels by increased tension in interciliary tip links. After tip-link destruction, the normal polarity of MT current is replaced by a mechanically sensitive current evoked by negative bundle deflections. The "reverse-polarity" current was investigated in cochlear hair cells after tip-link destruction with BAPTA, in transmembrane channel-like protein isoforms 1/2 (Tmc1:Tmc2) double mutants, and during perinatal development. This current is a natural adjunct of embryonic development, present in all wild-type hair cells but declining after birth with emergence of the normal-polarity current. Evidence indicated the reverse-polarity current seen developmentally was a manifestation of the same ion channel as that evident under abnormal conditions in Tmc mutants or after tip-link destruction. In all cases, sinusoidal fluid-jet stimuli from different orientations suggested the underlying channels were opened not directly by deflections of the hair bundle but by deformation of the apical plasma membrane. Cell-attached patch recording on the hair-cell apical membrane revealed, after BAPTA treatment or during perinatal development, 90-pS stretch-activated cation channels that could be blocked by Ca 2+ and by FM1-43. High-speed Ca 2+ imaging, using swept-field confocal microscopy, showed the Ca 2+ influx through the reverse-polarity channels was not localized to the hair bundle, but distributed across the apical plasma membrane. These reverse-polarity channels, which we propose to be renamed "unconventional" mechanically sensitive channels, have some properties similar to the normal MT channels, but the relationship between the two types is still not well defined.hair cells | mechanotransducer channels | calcium imaging | cochlea | transmembrane channel-like protein I on channels sensitive to mechanical deformation of the cell membrane are widely distributed in vertebrates and are integral to the function of specialized mechanoreceptors, such as those in the sensory neurons of the skin, vasculature, or inner ear. The molecular structure of these mechanically gated channels has been the subject of much recent research and speculation (1-4) because they are the last category of vertebrate ion-channel (after voltage-gated and ligand-activated channels) evading characterization. Although success was achieved by assigning piezo2 as a transduction channel in many cutaneous touch receptors (3), uncertainty persists about the composition of the mechanotransducer (MT) channel in hair cells of the inner ear (2, 4). There, the MT channels reside in the stereociliary (hair) bundle, where they are activated by deflections of the bundle toward the taller edge of the staircase, and so increasing tension in the oblique extracellular tip links connecting adjacent stereocilia. Transmembrane channel-like protein isoforms 1 and 2, TMC1 and TMC2 (5), have been suggested as candidates for the ha...

Mechanosensory hair cells express two molecularly distinct mechanotransduction channels

“…7A). Similar to previous reports using zebrafish and rodents 10-13, 37 we observed that currents in immature hair cells of wild-type mice at P0 revealed both normal- and reverse-polarity currents with normal-polarity currents not having reached full amplitude (Fig. 7A).…”

“…We stimulated OHCs from wild-type and mutants at a similar position in the medial part of the cochlea with a fluid jet, which allowed us to record forward-polarity and reverse-polarity currents. As reported earlier in rat 13 and mice 10, 37, 44 , transducer currents in wild-type mice matured in vitro over several days (Fig. 7A).…”

“…The appearance of reverse-polarity currents correlates with the disappearance of normal-polarity currents, which has led to the hypothesis that the two currents might depend on similar pore-forming ion-channel subunits 10, 15, 16, 37 . Since our data suggest that different ion channels carry reverse-polarity and normal-polarity currents, we asked whether ion channels carrying normal-polarity currents suppress the activity of ion channels responsible for reverse-polarity currents.…”

Stretching out the early steps in hearing

“…The appearance of reversetransduction as a sign that functional bundle remodeling is under way is a tempting avenue to explore. Breaking the tip-link in the transduction complex clearly leads to reverse-transduction within about 5 min in immature hair cells (1,7,12), but it is not known whether the same occurs in adult hair cells. Beurg et al (1) suggest that it does not, at least to any significant extent.…”

“…Breaking the tip-link in the transduction complex clearly leads to reverse-transduction within about 5 min in immature hair cells (1,7,12), but it is not known whether the same occurs in adult hair cells. Beurg et al (1) suggest that it does not, at least to any significant extent. Nevertheless, hair cells do show signs of membrane, and presumably protein, trafficking at their apical surface, particularly around the base of the bundle and the site where the primary cilium is found.…”