Neuron, Vol. 7, 985-994, December, 1991, Copyright 0 1991 by Cell Press Tip-link Integrity and Mechanical Transduction in Vertebrate Hair Cells John A. Assad,*+ Gordon M. G. Shepherd,* This suggests that the gating springs are not rigid ele- and David P. Corey*511 ments, but can be slack-that they can pull but not *Department of Neurobiology push on the channels (Corey and Hudspeth, 1983). *Program in Neuroscience The structural correlate of this process has not been Harvard Medical School well established. A simple model has evolved from Boston, Massachusetts 02115 several independent observations. First, measure- SDepartment of Neurology ment of current flow near moving bundles indicated Massachusetts General Hospital thatthetransductionchannelsareatornearthetipsof Boston, Massachusetts 02114 the stereocilia (Hudspeth, 1982). While this has been IINeuroscience Group challenged by measurements with a Ca*+ indicator Howard Hughes Medical Institute dye (Ohmori, 1988), two additional experirnents have corroborated the localization of the channels at the tips (Huang and Corey, 1990, Biophys. Sot., abstract; Summary Jaramillo and Hudspeth, 1991). Second, the discovery of fine filaments between the tips of adjacent ster- An attractive hypothesis for hair-cell transduction is that eocilia led to the suggestion that these “tip links”were fine, filamentous “tip links” pull directly on mechani- the actual mechanical linkages to the channels (Pick- cally sensitive ion channels located at the tips of the les et al., 1984). The geometry of the bundle is such stereocilia. We tested the involvement of tip links in the that excitatory displacements would stretch the tip transduction process by treating bundles with a BAPTA- links and apply tension to the channels; inhibitory buffered, low-Ca*+ saline (1O-v M). BAPTA abolished the displacements would relax them. All vertebrate hair transduction current in a few hundred milliseconds. cells so far examined-from different species and dif- BAPTA treatment for a few seconds eliminated the tip ferent organs whose stereociliary morphology may links observed by either scanning or transmission elec- otherwise vary-possess tip links. The tip-lr,nk hypoth- tron microscopy. BAPTA also eliminated the voltage- esis for transduction is thus extremely attractive, yet dependent movement and caused a positive bundle dis- direct evidence for it is limited. placement of 133 nm, in quantitative agreement with a In this paper we directly implicate the tip links in model for regulation of tension. We conclude that tip the transduction process, with the finding that a brief links convey tension to the transduction channels of hair treatment of low extracellular Ca2+ destroys both the cells. tip links and the mechanical sensitivity. In dissociated cells, moreover, low Ca*+ both abolishes the voltage- Introduction dependent bundle movement driven by the cells’ active regulation of gating spring tension and causes When the mechanosensitive bundle of a vertebrate the bundle to relax forward by 133 nm, 1ir-rquantita- hair cell is displaced in the positive direction (toward tive agreement with the idea that low Ca2+ destroys the taller stereocilia), transduction channels open and transduction by cutting the attachments to the ion allow the flow of positive ions into the cell. The open- channels. ing of a channel is thought to result from an increase of mechanical tension on the channel protein itself. Results The principal evidence for direct mechanical gating of these channels is that the transduction process is Abolition of Transduction by low Ca*+ extremely rapid (Corey and Hudspeth, 1979a), that the It has long been recognized that Ca*+ in the solution opening and closing rates depend on the size of the bathingthe hair bundles is required for hair-cell trans- stimulus (Corey and Hudspeth, 1983; Crawford et al., duction (Sand, 1975; Corey and Hudspeth, 1979b; 1989), and that the mechanical complianceof a bundle Crawford et al., 1991). While earlier experiments sug- includes a component that matches the opening of gested that Ca*+ might carry the receptor current the channels (Howard and Hudspeth, 1988). In this (Sand, 1975), more recent work views Ca2+ as a neces- view, the open probability is a direct function of ten- sary cofactor for the transduction apparatus (Craw- sion, conveyed by an elastic “gating spring.” A pecu- ford et al., 1991). We have reexamined the Ca2+ depen- liarity of the gating kinetics is that channel opening is dence with whole-cell voltage clamp, direct bundle progressively speeded by larger positive displace- stimulation, and rapid application of tes,t solutions. ments, whereas the closing rate is independent of the Figure 1 shows transduction currents of single cells in stimulus for sufficiently large negative displacements. response to a triangle-wave stimulus of 1.0 pm peak- to-peak amplitude; this was a saturating stimulus for these cells. The bath contained a normal frog saline + Present address: Department of Physiology and Center for Vi- sual Science, University of Rochester, Rochester, New York with 4 mM Ca*+. Halfway through the record, a low- 14642. Ca*+ saline (lOmg M; buffered with 5 mM BAPTA) was Neur0n 986 I I I 1 Time (set) Figure 1. Abolition of Transduction by Low Ca’+ (A-C) Transduction current in 3 different cells elicited by a 1 pm peak-to-peak, tnangk-wave displacement ot the bundle (bottom trace) A 5 mM BAPTA solution was delivered by a pressure pipette positioned about 20 @m from the cell. at the time Indicated by the bar In all 3 cases, transduction current was abolished by the BAPTA treatment and did not return for the duration ot the recording, usually several minutes after the exposure to low Ca2+. Membrane potential was maintained at -80 mV by the patch clamp. The bath contained the external recording solution, with 4 mM Caz+. puffed onto the cell from a distance of about 20 pm. curve-which relates receptor current to displace- The bundlewas held firmly bythe stimulus probeand ments-toward more negative positions, so that more continued to be driven by the triangle-wave stimulus. channels are open at rest (Assad et al., 1989; Crawford As the wave of low Ca2+ reached the bundle, the trans- et al., 1989, 1991). In extreme cases, it is possible that duction currentwas initially increased by about lOO%, such a shift is so large that all the channels are open a consequence of relieving a voltage-dependent block with zerodisplacement and that a cell appears insensi- of the channels by Caz+ (Assad and Corey, unpub- tive to mechanical stimuli, while in fact a sufficiently lished data; Crawford et al., 1991). In as little as 50 ms, large negative displacement could close channels. however, the transduction currentwas abolished, and That does not appear to account for the loss of sensi- it was not restored even several minutes after the tivity in this experiment, because there was not evi- BAPTA solution diffused away. In Figures IA and IB dence of a progressive shift of the I(X) curve, because the total membrane current increased, at least tran- the stimulus included large negative excursions that siently, during the exposure to low Ca*+. This increase were ineffectual in closing channels, and because the is due, at least in part, to an observed shift in the effect was not reversible upon return to normal Ca*- activation of basolaterally situated voltage-dependent concentration. It seems more likely that some part of Ca2+ channels (Roberts et al., 1990) to more negative the transduction apparatus was actually broken by potentials- perhaps as a result of alleviation of screen- lowering the Ca*+ concentration. ing charge-coupled with an increased monovalent ion flux through these channels (Assad and Corey, Abolition of Tip links by low CaZ+ unpublished data; Hess et al., 1986). To determinewhat part of the transduction apparatus We and others have found that lowering the Ca2+ was broken, hair cells were similarly treated with low concentration inside the stereocilia shifts the I(X) Ca*+ and prepared for scanning electron microscopy Hair Cell Transduction 987 Control Figure 2. Scanning Electron Micrographs of Stereocilia Treated with CaZ+ or BAPTA Ca2+-treated sacculi (left) were dissected in a 0.1 mM Caz+ solution and then placed in 4 mM Ca*+ before fixation in 4 mM Caz+. BAPTA-treated sacculi (right) were dissected in the same way, but placed in a 5 mM BAPTA solution for 10 s before fixation in 4 mM Caz+. Bar, 500 nm. (SEM). Sacculi were dissected as for physiological ex- periments and treated to remove their otolithic mem- branes, but were not dissociated. Experimental sac- culi were transferred to a solution buffered with 5 mM BAPTA for about 10 s and then immediately returned to normal saline. Control sacculi were transferred to normal saline in the same manner. Both sets were fixed with glutaraldehyde and 0~0~ and processed for field emission SEM. In order to photograph a representative sample of both control and experimental bundles, maculae were viewed at low magnification (3000x) and bun- dles were chosen more or less randomly from all re- gions. At the low magnification it was not possible to observe tip links; thus, their presence could not bias the choice of a cell. Bundles were photographed at Figure 3. Intact Tip Links following Detergent Treatment high magnification (50,000x), from an angle approxi- Specimen was dissected in 0.1 mM CaI+ and then maintained in control saline containing 4 mM Ca2+ at all times prior to fixation. mately perpendicular to the bevel of the tips. Twelve Triton X-100 (2%) was added to the fixative for the final 20 min to20 bundles were photographed from each of 4 mac- of fixation. Bar, 500 nm. ulae. Figure 2 shows representative images of control (left) and BAPTA-treated (right) bundles taken with an accelerating voltage of 4 kV. The stereocilia in both ular, the bundles were not splayed or otherwise dis- samples appearwell preserved, with no bending.