Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells Laura F. Cornsa,1, Stuart L. Johnsona,1, Corné J. Krosb,c, and Walter Marcottia,2 aDepartment of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom; bSussex Neuroscience, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom; and cDepartment of Otorhinolaryngology, Head and Neck Surgery, University Medical Center Groningen, University of Groningen, 9700 RB, Groningen, The Netherlands Edited by A. J. Hudspeth, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, and approved August 22, 2014 (received for review May 28, 2014) Mechanotransduction in the auditory and vestibular systems deflecting their hair bundles using a piezo-driven fluid jet, which is depends on mechanosensitive ion channels in the stereociliary believed to produce a more uniform deflection of the hair bundles bundles that project from the apical surface of the sensory hair (20–23) compared with the piezo-driven glass rod (19, 24). cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the Results + excitatory direction, Ca2 entry through the open MET channels MET Currents Recorded in Mouse Outer and Inner Hair Cells. MET causes adaptation, rapidly reducing their open probability and re- currents were elicited by displacing the hair bundles of both outer setting their operating range. It remains uncertain whether such hair cells (OHCs) and inner hair cells (IHCs) with a piezoelectric + Ca2 -dependent adaptation is also present in mammalian hair fluid jet stimulator (20–22). The fluid jet was preferred to a rigid cells. Hair bundles of both outer and inner hair cells from mice probe (17, 19) because it does not require any direct contact with were deflected by using sinewave or step mechanical stimuli ap- the stereocilia, which could affect their resting position and also plied using a piezo-driven fluid jet. We found that when cochlear damage some of the interciliary links (16). Unless the shape of 2+ hair cells were depolarized near the Ca reversal potential or their the probe is perfectly matched to that of the V- or U-shaped 2+ hair bundles were exposed to the in vivo endolymphatic Ca bundle of the OHCs or IHCs, respectively, this method would μ concentration (40 M), all manifestations of adaptation, including also lead to nonuniform displacement of individual stereocilia the rapid decline of the MET current and the reduction of the avail- (16, 25, 26). Such uniformity is achieved with fluid jet stimulation able resting MET current, were abolished. MET channel adaptation 2+ (Fig. S1). MET currents were elicited from 14 OHCs and 7 IHCs was also reduced or removed when the intracellular Ca buffer of postnatal day 6 (P6) to P9 mice (Fig. 1 A–C) in response to N N N′ N′ 1,2-Bis(2-aminophenoxy)ethane- , , , -tetraacetic acid (BAPTA) a sinewave stimulus. Upon moving their bundles in the excitatory was increased from a concentration of 0.1 to 10 mM. The findings direction (i.e., toward the taller stereocilia using positive driver show that MET current adaptation in mouse auditory hair cells is + + voltages), large inward currents flowed at negative membrane modulated similarly by extracellular Ca2 , intracellular Ca2 buffer- potentials (−121 mV; Fig. 1 A–C). The resting MET current was ing, and membrane potential, by their common effect on intracel- + shut off in the inhibitory phase of the stimulus. With membrane lular free Ca2 . depolarization, the MET current decreased in size at first and then reversed near 0 mV (Fig. 1C), in agreement with the earing and balance depend on the transduction of me- Hchanical stimuli into electrical signals. This process depends Significance on the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter of pairs of adjacent stereocilia (1), which are specialized microvilli-like structures that form the In the inner ear, the sensory receptor cells (hair cells) signal hair bundles that project from the upper surface of hair cells reception of sound. They do so by converting mechanical input, due to sound waves moving the hair bundles on these cells, (2,3). Deflection of hair bundles in the excitatory direction (i.e., into electrical current through ion channels situated at the tips toward the taller stereocilia) stretches specialized linkages, the of the bundles. To keep the receptors operating at their max- tip-links, present between adjacent stereocilia (3–5), opening the imum sensitivity, the current declines rapidly, a process known MET channels. In hair cells from lower vertebrates, open MET + as adaptation. In nonmammalian vertebrates, Ca2 ions en- channels reclose during constant stimuli via an initial fast ad- tering the mechanosensitive ion channels drive adaptation, but aptation mechanism followed by a much slower, myosin-based + it has been questioned whether this mechanism applies to motor process, both of which are driven by Ca2 entry through – mammals. We show that adaptation in mammalian cochlear the channel itself (6 13). In mammalian auditory hair cells, MET hair cells is, as in other vertebrates, driven by Ca2+ entry, current adaptation seems to be mainly driven by the fast mech- – demonstrating the importance of this process as a fundamental anism (14 16), although the exact process by which it occurs is mechanism in vertebrate hair cells. still largely unknown. The submillisecond speed associated with the adaptation kinetics of the MET channels in rat and mouse Author contributions: L.F.C., S.L.J., C.J.K., and W.M. designed research; L.F.C., S.L.J., and 2+ cochlear hair cells (17, 18) indicates that Ca , to cause adap- W.M. performed research; L.F.C., S.L.J., and W.M. analyzed data; and L.F.C., S.L.J., C.J.K., tation, has to interact directly with a binding site on the channel and W.M. wrote the paper. or via an accessory protein (16). However, a recent investigation The authors declare no conflict of interest. + on rat auditory hair cells has challenged the view that Ca2 entry This article is a PNAS Direct Submission. is required for fast adaptation, and instead proposed an as-yet- Freely available online through the PNAS open access option. 2+ undefined mechanism involving a Ca -independent reduction in 1L.F.C. and S.L.J. contributed equally to this work. the viscoelastic force of elements in series with the MET chan- 2To whom correspondence should be addressed. Email: [email protected]. nels (19). In the present study, we further investigated the role of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2+ Ca in MET channel adaptation in mouse cochlear hair cells by 1073/pnas.1409920111/-/DCSupplemental. 14918–14923 | PNAS | October 14, 2014 | vol. 111 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1409920111 Downloaded by guest on September 27, 2021 nonselective permeability of MET channels to cations (27), to become outward, with a larger resting current at positive po- tentials. We then stimulated the stereocilia of hair cells using force steps to better investigate the adaptation properties of the MET current (Fig. 1 D–G). Upon moving the bundles in the excitatory direction and at −81 mV, adaptation- or time- dependent decline of the MET current in OHCs occurred for small driver voltages that produce nonsaturating bundle dis- placements (72 ± 7 nm, n = 14; approximately halfway through the bundle operating range shown in Fig. 1F). Adaptation exhibited a fast time constant (0.65 ± 0.08 ms; n = 14; contrib- uting 57.3% of the total extent of adaptation described by the exponential fit) and a slow time constant (16.9 ± 2.4 ms; con- tributing the residual 42.7%; n = 14; arrow in Fig. 1 D, Right). Inhibitory hair bundle deflection (negative driver voltage) shut off the small fraction of current flowing at rest, with the offset of large inhibitory steps causing a transient rebound inward MET current (downward dip indicated by the arrowheads in Fig. 1D). All of these manifestations of MET current adaptation were absent when stepping the membrane potential from –81 mV to + a positive value (+99 mV: Fig. 1E), which is near the Ca2 + equilibrium potential and strongly reduces Ca2 entry into the + MET channels. This finding is consistent with Ca2 entry driving Fig. 1. MET currents in mouse cochlear OHCs and IHCs. (A and B) Saturating adaptation as demonstrated in hair cells from lower vertebrates MET currents recorded from an OHC (A) and an IHC (B) in response to 50-Hz (9, 10). Additional evidence for the presence of MET current sinusoidal force stimuli to the hair bundles at membrane potentials of −121 adaptation is the channels’ increased resting open probability + ± + and 99 mV. The driver voltage (DV) signal of 35 V to the fluid jet is shown when Ca2 influx into the stereocilia is decreased (9, 12, 28, 29), above the traces (positive deflections of the DV are excitatory). The arrows + and arrowheads indicate the closure of the transducer channels, i.e., disap- which was evident in OHCs at 99 mV (Fig. 1 A and E; see also pearance of the resting current, during inhibitory bundle displacements at Fig. 2C). This finding is also highlighted by the different shapes –121 mV and +99 mV, respectively. Dashed lines indicate the holding current. of the plots describing the relation between the mean peak MET (C) Peak-to-peak MET current–voltage curves obtained from 14 OHCs and 7 IHCs current and bundle displacement (Fig. 1F; for driver voltage to + (P6–P9) using 1.3 mM extracellular Ca2 . The fits through the current–voltage bundle displacement conversion using a pair of photodiodes, see curves are according to a single-energy-barrier model (Eq.