Meglumine

Recordings were made with borosilicate patch electrodes filled with a solution containing the following (mM): 135 CsCl, 3 MgATP, 10 Tris phosphocreatine, 1 EGTA, and 10 Cs-HEPES, pH 7.2 (∼298 mOsm/l), connected to an amplifier (Axopatch 200A; Molecular Devices) with an output filter at 10 kHz. Membrane potentials were corrected for liquid junction potentials and for voltage drop across the uncompensated series resistance. Most voltage-clamp protocols are referred to a holding potential of −84 mV. Hair bundles were mechanically stimulated with a fluid jet from a pipette (tip diameter of 10–15 µm) driven by a 25-mm-diameter piezoelectric disc (Johnson et al., 2011 (link)). The position of the stimulating pipette was adjusted to elicit a maximal MT current. The stimulus was usually a 35-Hz sinusoid, but sometimes the fluid jet was driven with voltage steps filtered at 1 kHz (Kros et al., 2002 (link)). In some experiments, the bundle motion produced by fluid stimulation was calibrated by projecting an image of the bundle onto a pair of photodiodes (LD 2–5; Centronics) at a total magnification of 340. Current–voltage relationships and MT current–displacement curves were determined from automated protocols generated by a Cambridge Electronic Design (CED) Power1401 interface driven by a PC. Data were digitized with the CED interface, and the results were analyzed with IGOR Pro v6 (WaveMetrics). Unless otherwise stated, values are given as mean ± 1 SEM, and P < 0.02 indicates statistical significance on a two-tailed Student’s t test. All experiments were performed at room temperature of 21–25°C.
Reversal potentials (V_REV) and relative Ca²⁺ permeability (P_Ca/P_Cs) were determined (Kim and Fettiplace, 2013 (link)) by bathing cells in an extracellular solution containing the following (mM): 100 CaCl₂, 20 N-methylglucamine, 6 Tris, and 10 glucose, adjusted with HCl to pH 7.4, in both the fluid jet and the puffer pipette. Reversal potentials were corrected for a liquid junction potential of 9 mV. The relative Ca²⁺ permeability, P_Ca/P_Cs, was calculated from the Goldman–Hodgkin–Katz equation: ${\text{P}}_{\text{Ca}}/{\text{P}}_{\text{Cs}}=\left\{{\text{a}}_1\left[{\text{Cs}}^{+}\right]/{\text{4a}}_2\left[{\text{Ca}}^{2+}\right]\right\}\times \left\{\text{exp}\left({\text{V}}_{\text{REV}}\text{F}/\text{RT}\right)\right\}\times \left\{1+\text{exp}\left({\text{V}}_{\text{REV}}\text{F}/\text{RT}\right)\right\},$ where RT/F has its usual meaning, with a value at room temperature of 25.7 mV; [Cs⁺] and [Ca²⁺] are the concentrations of Cs⁺ intracellularly (142 mM) and Ca²⁺ extracellularly (100 mM), and a₁ and a₂ are activity coefficients for Cs⁺ (Partanen, 2010 (link)) and Ca²⁺ (Rard and Clegg, 1997 (link)), respectively.
To isolate single MT channels in a whole-cell recording mode, saline with submicromolar free Ca²⁺ (composition [mM]: 140 NaCl, 6 KCl, 1 CaCl₂, 5 1,2-bis(o-aminophenoxy) ethane-N,N,N9,N9-tetraacetic acid [BAPTA], 8 glucose, and 10 Na-HEPES, pH 7.4; free Ca²⁺ of 0.05 µM) was pressure-ejected onto the hair bundles for 10–20 s to sever almost all of the tip links (Beurg et al., 2006 (link)). Single-channel responses were elicited by hair bundle deflections with a glass stylus, polished to ∼3-µm tip diameter, and driven by a piezoelectric stack actuator (PA8/12; piezosystems jena GmbH). The driving voltage to the actuator was filtered at 3 kHz, yielding a step displacement with a rise time of ∼100 µs. Single-channel currents were filtered at 5 kHz.

MET currents were recorded from OHCs and inner hair cells (IHCs) in isolated organs of Corti of mice between P0 and P10 with methods described previously (10 (link), 30 (link)). Excised cochlear turns were immobilized in a recording chamber on a fixed-stage microscope (Leica DMFS) and perfused with saline of composition (in mM): 152 NaCl, 6 KCl, 1.5 CaCl₂, 2 Na–pyruvate, 8 D-glucose, and 10 Na–Hepes, pH 7.4, at room temperature of 21° to 23 °C. Electrical recordings were made with patch electrodes filled with the following solution (in mM): 128 CsCl, 3.5 MgCl₂, 5 Na₂ATP, 10 Tris phosphocreatine, 1 BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetracetic acid), 10 Cs–Hepes, pH 7.2, and connected to an Axopatch 200B amplifier; recording time constants were 25 to 50 µs. Stereociliary bundles were stimulated with a fluid jet or a stiff glass probe driven by a piezoactuator and bundle displacements were calibrated (31 (link)). Single MET-channel events were characterized as previously described (12 (link), 13 (link)). Single-channel amplitudes were also determined from MET-channel nonstationary current noise (32 (link), 33 (link)). MET currents in response to prolonged near-maximal bundle stimuli were low-pass-filtered at 10 kHz; currents were analyzed by subtracting the mean current, I, from each of 40 or more individual traces and squaring the difference to yield the current variance, σ_I², which was then corrected by subtracting the variance attributable to the background noise. A plot of σ_I² against I was fit with the following parabolic equation: σ_I² = i_o·I − I²/N_MET, where i_o is the single-channel current and N_MET is the number of channels (33 (link)). The Ca²⁺ selectivity of the MET channel was determined (5 (link), 10 (link)) by measuring the Ca²⁺ reversal potential (V_rev) using the following CsCl-based intracellular solution of composition (in mM): 135 CsCl, 3 MgATP, 10 Tris phosphocreatine, 1 EGTA–CsOH, 10 Hepes (pH 7.2) and an extracellular solution (in mM): 100 CaCl₂, 20 N-methylglucamine, 6 Tris, 10 D-glucose (pH 7.4). MET current reversal potentials were corrected for a −9-mV junction potential, and the permeability ratio, P_Ca/P_Cs, was calculated from the Goldman–Hodgkin–Katz equation using activity coefficients for Cs⁺ and Ca²⁺ as described previously (10 (link)).