(A and G) Examples of transduction currents in frog saccule hair cells (A) and mouse utricle hair cells (G) in response to mechanical displacements of 150 ms (frog) or 250 ms (mouse). Color scheme of currents is same as in (B) and (H).
(B and H) Extent of adaptation as a function of time [X_e(t)] from currents in (A) and (G) using the inferred-shift method. Data were fit with double-exponential functions.
(C–F) Analysis of averaged data from 15 frog saccule hair cells.
(I–L) Analysis of averaged data from 31 pooled CD-1 and C57BL/6 mouse utricle hair cells.
(C and I) Average instantaneous current-displacement curves derived by converting the peak transduction current into P_o. Data were fit with Equation S9 (Supplemental Data), a three-state Boltzmann function. Displacements were −0.4 to +0.8 µm ([C], frog) or −1 to +2 µm ([I], mouse); only P_o values from positive displacements are displayed. Frog parameter values: Z₀₁ = 67 fN, X₀₁ = 0.13 µm, Z₀₂ = 20 fN, X₀₂ = 0.019 µm. Mouse parameter values: Z₀₁ = 22 fN, X₀₁ = 0.28 µm, Z₀₂ = 8.0 fN, X₀₂ = 0.51 µm.
(D and J) Average final extent of adaptation. Total extent was fit with a straight line ([D], frog; slope = 0.85) or a second-order polynomial ([J], mouse). For (D), fast extent was fit with Equation S5 (Supplemental Data); X_{e(fast) ∞} was the only free parameter and was 0.29 µm. For (J), fast extent was fit with Equation S5, yielding an X_{e(fast) ∞} of 0.76 µm. Lines describing slow adaptation in (D) and (J) are not fits, but instead are the total extent fit minus fast extent fit. (E and K) Average time constants for adaptation. Slow adaptation time constants were fit with a flat line (for frog, τ_slow = 26 ms; for mouse, τ_slow = 56 ms). Fast adaptation time constants were fit with Equation S6 (Supplemental Data).
(F and L) Average initial rate of adaptation (X_e/τ). Fast adaptation data were fit with an equation of the form rate = k_fast × X_e(fast), where k_fast is the rate constant for fast adaptation and depends on displacement (E and K). Slow adaptation data were fit with Equation S8. For (F), k_slow was 42 s⁻¹. For (L), k_slow was 22 s⁻¹.

(A) Negative adaptation in a representative frog hair cell, assessed with negative steps of −0.8 µm for 10–110 ms in 10 ms increments (indicated below). Channels closed completely (dotted line) during the negative steps; upon return of the bundle to rest, a large rebound current developed that was proportional to the extent of negative adaptation that occurred during the stimulus.
(B) Frog negative adaptation (average of 12 cells). Overshoot currents were converted to adaptation extent by comparison with the current-displacement curve. Data points were fit with sum of exponential and linear functions.
(C) Representative mouse (C57BL/6) hair cell subjected to increasing-duration protocol (−1 µm step).
(D) Mouse (C57BL/6) negative adaptation (average of 12 cells). Overshoots were converted to extent and fit as in (B).

(A) Examples of transduction currents in wt and Y61G hair cells. In both cases, a representative example (top) and the fastest cell in the dataset (middle) are shown for cells filled with control dialysis solutions. In the bottom row, representative cells dialyzed with 250 µM NMB-ADP are shown.
(B) Total transduction currents for Y61G hair cells early (1–3 min) and late (>3 min) during dialysis, with or without NMB-ADP. **p < 0.005.
(C) Rate constants for fast positive adaptation using linear displacement-rate fits. B6 = C57BL/6. *p = 0.11; **p < 0.001.
(D) Rate constants for slow positive adaptation using linear displacement-rate fits. ***p < 0.0001.

(A) Negative adaptation from nine Y61G hair cells dialyzed with a control solution and seven Y61G hair cells dialyzed with 250 µM NMB-ADP. Overshoots were converted to extent and fit with summed exponential and linear components as in Figure 4. Gray line is C57BL6 control data from Figure 4.
(B) Fast (exponential) negative adaptation initial rates. **p < 0.05.
(C) Slow (linear) negative adaptation rates. Y61G, 0.62 ± 0.17 µm/s (n = 9); Y61G+NMB-ADP, 0.12 ± 0.07 µm/s (n = 7).

(A) Mechanical model for transduction apparatus. Four mechanical elements are included: an elastic gating spring, a channel with a swinging gate, a release element, and an adaptation motor. Note that, although the gating spring is depicted here to be the tip link, present evidence locates it elsewhere (Gillespie et al., 2005), although still mechanically in series. When the channel is open, the mechanical chain is lengthened; when it is closed, the chain is shortened. The release element lengthens the mechanical chain when unfolded. The motor is modeled as a purely viscous element; force production by the adaptation motor is assumed to be constant and is not depicted. After Howard and Hudspeth (1987).
(B) Channel-reclosure model. A positive mechanical stimulus (X_s) stretches the gating spring to open the channel. Fast adaptation occurs when Ca²⁺ enters, binds on or near the channel, and forces the channel to close. Increased tension in the gating spring occurring due to channel closing raises the tension felt by the adaptation motor and hence increases the rate of slow adaptation. (Bottom) Hypothetical adaptation rates. Over most of the displacement range, because the adaptation motor exhibits purely viscous behavior, the rate is linear with displacement; over the range where fast adaptation develops, however, the rate of slow adaptation is relatively higher for small displacements because of increased tension (*).
(C) Release model. Ca²⁺ entering an open channel binds to the release element, lengthening the mechanical chain in series with the channel and gating spring. Reduced tension decreases the channel’s P_o. (Bottom) Because the release reduces the force applied to the adaptation motor, for small displacements, the slow adaptation rate is reduced relative to purely viscous behavior (*). Models where Ca²⁺ changes the stiffness of a mechanical element in series with the channel are formally similar to the release model.

(A) No correlation between fast and slow adaptation rates measured in individual cells.
(B) Inverse correlation between fast and slow adaptation extent slopes in individual cells. For each cell, fast and slow extents were determined over a range of displacements; the slopes (dimensionless) of the resulting fast and slow displacement-extent relations were plotted for each cell. A linear fit to mouse data (all genotypes: Y61G, C57BL/6, and CD-1) is shown: slope, −1.18; y-intercept, 0.94; R = 0.90. Legend applies to (A) and (B).

(A) Targeting strategy. Sequence coding for the Y-to-G mutation at amino acid residue 61 was inserted, resulting in conversion of a Bgl II restriction site to BamH I. The loxP-flanked neomycin-resistance cassette was introduced into a large intron between exons 4 and 5.
(B) Southern blot demonstrating successful targeting and Neo removal. DNA from homozygous wild-type, Y61G/Neo, or Y61G mice was digested with BamH I and detected with a 3′ probe (indicated in [A]).
(C) Immunoblot analysis of Myo1c expression in tissues of wild-type and Y61G mice. Equal amounts of total protein from wild-type or homozygous Y61G mice from the indicated tissues were loaded. Myo1c position (~120 kDa) indicated.
(D) Immunoblot analysis with anti-Y61G-Myo1c, selective for the mutant sequence. Equal amounts of total protein from wild-type or homozygous Y61G mice were loaded on the same gel. Cochlea lanes were exposed to film for 20 s and vestibule lanes for 120 s.
(E) Inhibition of ATPase activity by NMB-ADP. Wild-type and Y61G Myo1c were expressed with baculovirus and purified on Ni²⁺-NTA agarose; ATPase assays were carried out with 10 µM ATP.
(F) In vitro motility of Myo1c. Wild-type and Y61G Myo1c were expressed with baculovirus, then purified with Ni²⁺-NTA agarose and actin cycling. Motility assays were carried out using the following solutions: ATP, 2 mM ATP, 1 mM EGTA; NMB-ADP, 2 mM ATP, 250 µM NMB-ADP, 1 mM EGTA; Ca²⁺, 2 mM ATP, 0.1 mM CaCl₂; NMB-ATP, 2 mM NMB-ATP, 1 mM EGTA. Other components of motility solution were as described previously (Gillespie et al., 1999). Filament rates were quantified and binned. Data were fit assuming one population of immobile filaments and another population of moving filaments, with Gaussian-distributed velocities.

(A and B) Progression of inhibition of adaptation in a Y61G hair cell dialyzed with 250 µM NMB-ADP. Responses to 1.8 µm stimuli.
(A) Transduction currents. Note slowing of adaptation. Times indicate minutes after establishment of the whole-cell configuration.
(B) Extent of adaptation determined from data in (A) using inferred-shift analysis. Double-exponential fits. Initial rates of fast adaptation: 60 µm/s (0.7 min), 9.4 µm/s (1.7 min), and 4.3 µm/s (2.3 min). Initial rates of slow adaptation: 15 µm/s, 8.6 µm/s, and 3.8 µm/s.
(C–J) Averaged data from 16 Y61G cells filled with a control solution (blue symbols) and 20 Y61G cells filled with 250 µM NMB-ADP. Thick gray lines in (A)–(H) correspond to mouse control fits from Figure 2.
(C) Current-displacement curves. Fits used Equation S9.
(D) Total extent of adaptation. Fits used second-order polynomial functions.
(E) Fast adaptation extent. Linear fits for Y61G (slope, 0.51; R = 0.99) and Y61G + NMB-ADP (slope, 0.29; R = 0.99).
(F) Slow adaptation extent. Lines correspond to total extent minus fast extent. Open symbols correspond to slow adaptation parameters (same color scheme as in [A]).
(G) Fast adaptation time constant. Data for Y61G cells without NMB-ADP were fit with a flat line (data not shown), giving a displacement-independent time constant of 13 ms; data with NMB-ADP were fit with a flat line (τ_fast = 34 ms). Data from the smallest stimulus (0.2 µm) were excluded due to uncertainty in assignment to fast or slow adaptation.
(H) Slow adaptation time constant. Data (except those from 0.2 µm stimulus) were fit with flat lines: for Y61G, τ_slow = 53 ms; for Y61G + NMB-ADP, τ_slow = 129 ms.
(I) Fast adaptation rate. Data were fit with an equation of the form rate = k_fast × X_e(fast). The decrease in τ for large displacements means that the fast rate cannot be described by a single parameter.
(J) Slow adaptation rate. As in Figure 2L, data were fit with Equation S8. In Y61G hair cells, k_slow (rate constant for slow adaptation) was 25 s⁻¹. For Y61G hair cells filled with NMB-ADP, k_slow was 11 s⁻¹.

(A) Cartoon model. (Inset) Force (downward in diagram) rotates the lever arm (extended IQ domains that bind calmodulin molecules [CaMs]). By reducing crossbridge stiffness, Ca²⁺ or NMB-ADP produces further rotation of lever arm under tension. During a positive deflection, tension in the gating spring increases; entering Ca²⁺ triggers lever arm movement. Tension decreases, allowing channels to close. Slow positive adaptation occurs as Myo1c molecules detach and reattach cyclically to further reduce tension (data not shown). During a negative deflection, reduced gating spring tension (and reduction in intracellular Ca²⁺) allows the lever arm to rotate to the zero-force state, producing fast negative adaptation. Tension increases modestly in the gating spring, but is not enough to reopen channels. Slow negative adaptation occurs as Myo1c molecules detach and reattach cyclically to further increase tension (data not shown).
(B) Mechanical model. After Shepherd and Corey (1994). K_g, gating spring stiffness; K_e, extent spring stiffness; K_Myo1c, myosin-actin cross-bridge stiffness (diagrammed as release element); Ξ_m, adaptation-motor viscous element; F_m, adaptation-motor force generator; X_s, bundle position; K_s, stereocilia spring stiffness; K_p, probe stiffness; X_p, probe displacement. F_m increases tension both on the gating spring and the stereociliary springs when active, moving the bundle (X_s) to the rest point. Release of K_Myo1c decreases tension in K_g, allowing channels to close.
(C) Predicted effect of Ca²⁺ or NMB-ADP on force-displacement relationship for K_Myo1c. We hypothesize that the spring becomes very compliant for small displacements, but larger displacements re-engage the stiff spring. This behavior will resemble a pure release. The offset between the two curves corresponds to X_e(fast), the release distance.