ATP Hydrolysis Is Critically Required for Function of Ca_V1.3 Channels in Cochlear Inner Hair Cells via Fueling Ca²⁺ Clearance

Abstract

Sound encoding is mediated by Ca²⁺ influx-evoked release of glutamate at the ribbon synapse of inner hair cells. Here we studied the role of ATP in this process focusing on Ca²⁺ current through Ca_V1.3 channels and Ca²⁺ homeostasis in mouse inner hair cells. Patch-clamp recordings and Ca²⁺ imaging demonstrate that hydrolyzable ATP is essential to maintain synaptic Ca²⁺ influx in inner hair cells via fueling Ca²⁺-ATPases to avoid an increase in cytosolic [Ca²⁺] and subsequent Ca²⁺/calmodulin-dependent inactivation of Ca_V1.3 channels.

Introduction

Neurotransmission at the inner hair cell (IHC) synapse is driven by Ca²⁺ influx (I_Ca) through Ca_V1.3 channels (Platzer et al., 2000; Brandt et al., 2003; Dou et al., 2004) that cluster at the active zones (AZs) (Brandt et al., 2005). Within IHCs, this L-type channel activates at low voltage and displays only weak Ca²⁺ dependent inactivation (CDI) (Yang et al., 2006; Cui et al., 2007). At least two mechanisms of inhibiting CDI (Lee et al., 1999; Peterson et al., 1999) of Ca_V1.3 in IHCs are currently considered: (1) autoregulation involving the distal and proximal C-terminal domains (Singh et al., 2008) and (2) competition of Ca²⁺ binding proteins (CaBPs) with calmodulin (Yang et al., 2006; Cui et al., 2007; Schrauwen et al., 2012). However, a unifying picture of CDI regulation during physiological signaling in IHCs has yet to be established.

Here we studied the role of ATP in Ca²⁺ signaling and Ca_V1.3 channel regulation in IHCs. Decay (“rundown”) of I_Ca is commonly observed in whole-cell patch-clamp recordings, suggesting a failure of the channel regulation and/or function upon washout of cell constituents. Adding ATP to the pipette can partially prevent I_Ca rundown (Chad and Eckert, 1986; Armstrong and Eckert, 1987). Besides being used by kinases and phosphatases, ATP supports the function of ATP-driven pumps and is therefore required for cellular Ca²⁺ homeostasis and low basal [Ca²⁺]_i (for review, see Mammano et al., 2007). Elevations in basal [Ca²⁺]_i could affect the Ca²⁺ channel behavior (e.g., via CDI). The regulation of [Ca²⁺] at ribbon synapses involves Ca²⁺ buffering and diffusion (Roberts, 1993; Tucker and Fettiplace, 1995; Issa and Hudspeth, 1996; Frank et al., 2009) as well as Ca²⁺ clearance via Ca²⁺ ATPase (PMCA) and Na⁺/Ca²⁺ exchange (Zenisek and Matthews, 2000; Kennedy, 2002).

Here, we combined patch-clamp recordings and Ca²⁺ imaging of IHCs during dialysis with different [ATP] or the poorly hydrolyzable analog ATP-γ-S to probe the requirement of ATP hydrolysis for Ca²⁺ homeostasis and Ca_V1.3 channel regulation. We demonstrate that interference with ATP hydrolysis dramatically increases [Ca²⁺]_i because of failure of PMCA-mediated Ca²⁺ clearance and consequently decreases the presynaptic I_Ca via Ca²⁺/calmodulin-mediated CDI.

Materials and Methods

Electrophysiology.

IHCs from the apical coil of organs of Corti from C57 Bl/6 mice of either sex (postnatal day 14 [P14] to P16) were patch-clamped (at 20°C–25°C) as described previously (Moser and Beutner, 2000). The pipette solution contained the following (in mm): 134–140 Cs-gluconate, 10 tetraethylammonium-Cl (TEA-Cl), 10 4-AP, 10 CsOH-HEPES, 1 MgCl₂, 0.3 NaGTP, 0.5 or 10 EGTA or 10 BAPTA and 0–4 MgATP or 2 Li₄-ATP-γ-S, pH 7.2, osmolarity: 295 mOsm/L. CaMKII 290–309, H-89 (both Merck), carboxyeosin, trifluorocarbonylcyanide phenylhydrazone (FCCP) (both Sigma-Aldrich), and fura-2 and Fluo-4FF (both Invitrogen) were dissolved in H₂O. KN-93, CaMKII inhibitor XII, KT5720 (all Merck), CGS-9343B (Sigma-Aldrich), and berbamine E₆ (Santa Cruz Biotechnology) were dissolved in DMSO. The extracellular solution contained the following (in mm): 110 NaCl, 35 TEA-Cl, 10 HEPES, 1 CsCl, 1 MgCl₂, 2 CaCl₂, 11.1 glucose, pH 7.2, osmolarity: 300 mOsm/L. BayK 8644 (Biotrend) was added to the extracellular solution. The liquid junction potential was numerically estimated as 14 mV and subtracted. Leak currents were subtracted using the P/10 protocol. The series resistance was typically <15 mΩ.

Camera-based Ca²⁺ imaging.

IHCs were loaded with 100 μm fura-2 and imaged alternately at 340 and 380 nm using a polychrome IV light source and an Imago VGA CCD operated by Tillvision software (all, Tillphotonics-FEI). Fura-2 measurements were calibrated in vivo and in vitro (Neher, 2013), and [Ca²⁺] was calculated according to the following equation (Grynkiewicz et al., 1985):

In our experiments, calibration coefficients determined in vivo were R_min = 0.24, R_max = 5.15, and R_med = 1 (yielding K_eff of 2457 nm). The K_D for fura-2 was found to be 243.5 nm.

Confocal Ca²⁺ imaging.

Confocal Ca²⁺ imaging was performed using an Olympus FV300 confocal microscope essentially as described previously (Frank et al., 2009) using 400 μm Fluo-4FF in the pipette solution described above. In brief, carboxytetramethyl-rhodamine-conjugated RIBEYE-binding dimer peptide (10 μm) (Francis et al., 2011) was used to identify synaptic ribbons, and changes in Fluo-4FF (400 μm) fluorescence were repeatedly observed with line scans through the center of the same ribbon during (20 ms) depolarizations to −7 mV.

Data analysis and statistics.

Data analysis and statistics were done in IgorPro and MATLAB. Wilcoxon rank test was used to compare data (with non-normal distribution and/or unequal variances). Correlation was tested using Pearson's correlation. Regression lines were compared among each other by one-way analysis of covariance (ANCOVA) test. Data are presented as mean ± SEM.

Results

IHC Ca_V1.3 channels require hydrolyzable ATP for proper function

To determine the requirement of ATP for Ca²⁺ influx, IHCs were dialyzed with 2 mm ATP, ATP-γ-S, or pipette solution lacking ATP. We first assessed the I_Ca properties (current–voltage relationship, IV) 1–2 min after break-in (Fig. 1A) and thereafter ran a series of depolarization pulses (P1–P3, 20 ms; P4, 100 ms) to the peak I_Ca potential. Taking into account the molecular weight (MW), series resistance R_S, and the estimated cell volume of 2.2 pl, the diffusion time constant for ATP (MW = 507.18 g/mol, R_S = 12 mΩ) and ATP-γ-S (MW = 546.98 g/mol, R_S = 10 mΩ) was calculated as 67 and 57 s, respectively (Pusch and Neher, 1988). Based on these calculations, the diffusional exchange should have been complete after 3–4 min, when P1 was applied. The cells infused with ATP-γ-S or 0 ATP may not have been completely devoid of ATP because of further ATP supply by oxidative metabolism, glycolysis, or phosphocreatine.

Figure 1.

ATP hydrolysis is required for maintaining IHC I_Ca. A, Unaltered amplitude of I_Ca revealed by the current–voltage relationship (IV) of IHCs dialyzed with 2 mm ATP (n = 26), 2 mm ATP-γ-S (n = 15), or without exogenous ATP (n = 8) at 1–2 min after break-in. B, Representative I_Ca in response to 20 ms depolarization to peak I_Ca potential after 3, 4.5, and 6 min dialysis with ATP, ATP-γ-S, or without ATP. C, Time course of the I_Ca reduction upon ATP manipulation. Top, The I_Ca of individual IHCs dialyzed with ATP (n = 21), ATP-γ-S (n = 13), or without ATP (n = 8). Bottom, I_Ca normalized to the first 20 ms depolarization of each cell. Mean normalized values with SEM displayed as overlay that were binned by time, with a bin size of 120 s. Statistical comparison was performed between 1 min and 11 min after loading. D, The concentration-dependent effect of ATP on Ca²⁺ channels. Normalized I_Ca values of IHCs dialyzed with 0.5 (n = 4), 0.75 (n = 5), or 4 mm ATP (n = 5) over time. For comparison, the mean normalized I_Ca values of IHCs dialyzed with 2 mm (black dashed line) and without ATP (gray dashed line) are displayed. E, The I_Ca rundown is partially reversible. IHCs initially infused without ATP (n = 3) were repatched with a solution containing 2 mm ATP after 8–13 min. ***p < 0.001.

The initial IVs revealed comparable I_Ca amplitudes, reflecting the largely unaltered physiological state of the IHCs briefly after break-in. IHCs dialyzed with ATP-γ-S displayed a significantly faster rundown of I_Ca compared with controls with 2 mm ATP in the pipette (p < 0.001; Fig. 1B,C). For better comparison, I_Ca values were normalized to the response upon P1 (Fig. 1C). Without exogenous ATP or ATP-γ-S, the I_Ca rundown was even more pronounced (p < 0.001 for comparison to ATP-γ-S; Fig. 1B,C). Interestingly, 4 mm ATP in the pipette prevented the mild rundown observed with 2 mm ATP (Fig. 1D). On the contrary, lowering [ATP] below 2 mm caused the onset of a fast rundown after a few minutes (Fig. 1D).

We then tested whether the effects of the lack of hydrolyzable ATP on Ca²⁺ channels is reversible. IHCs were initially dialyzed with a solution lacking ATP, the pipette was gently pulled off enabling resealing of the IHC membrane, and the cell was then repatched with an internal solution containing 2 mm ATP after 8–13 min (Fig. 1E). As observed, I_Ca could partially be restored and remained stable over another 10 min. Similar results were obtained in IHCs initially dialyzed with ATP-γ-S (data not shown).

Finally, we examined the effects of DHP agonist BayK 8644 on the I_Ca of ATP-γ-S-treated IHCs because BayK 8644 has been shown to partially overcome inhibition of I_Ca by lack of hydrolyzable ATP in pituitary GH₃ and smooth muscle cells (Armstrong and Eckert, 1987; Ohya and Sperelakis, 1989). We observed a twofold increase in the maximum steady-state I_Ca amplitude of the initial IV (Fig. 2A) consistent with the augmenting effects of BayK 8644 on IHC Ca_V1.3 channels (Brandt et al., 2005). However, regardless of BayK 8644, ATP-γ-S still caused a significant rundown of I_Ca (p = 0.43, compared with 2 mm ATP-γ-S without BayK 8644; Fig. 2B).

Figure 2.

Probing the requirement of IHC I_Ca for phosphorylation by PKA and CaMKII. A, The IV of IHCs dialyzed with ATP-γ-S in the absence (n = 15) and presence of 5 μm extracellular BayK 8644 (n = 15). B, BayK 8644 did not significantly prevent the I_Ca rundown (p = 0.43, statistical comparison was performed between 1 min and 13 min after loading). Mean normalized I_Ca values of IHCs dialyzed with ATP-γ-S (n = 13) in the presence of BayK 8644 (n = 9). For comparison, we display the mean normalized I_Ca of IHCs dialyzed with ATP. C, The effect of PKA inhibition on IHC I_Ca. The IVs of IHCs dialyzed with ATP in the absence (n = 5) or presence of intracellular H-89 (n = 14) or KT5720 (n = 5) are comparable. D, The normalized mean I_Ca values of IHCs dialyzed with low (1, 2, and 4 μm, n = 6), middle (10 and 20 μm, n = 5), or high (100 μm, n = 3) concentration of H-89 or KT5720 (2 μm, n = 6) over time. Statistical comparison was performed between 3 min and 19 min after loading. E, CaMKII inhibition shows no effect on IHC I_Ca. The IVs of IHCs dialyzed with ATP in the absence (n = 5) or presence of intracellular CBD (n = 4), CaMKII Inhibitor XII (n = 3), or KN-93 (n = 4). F, The rundown of the mean normalized I_Ca in IHCs dialyzed with CBD (n = 4), inhibitor XII (n = 4), and KN-93 (n = 3) is similar to controls. In comparison, the mean normalized I_Ca of IHCs dialyzed with ATP or ATP-γ-S is displayed. ***p < 0.001.

Probing for a role of phosphorylation in the regulation of IHC Ca_V1.3 channels

Because ATP requirement may reflect phosphorylation events relevant to Ca²⁺ channel function, we tested for effects of the protein kinase A (PKA) inhibitors H-89 (Chijiwa et al., 1990) and KT5720 (Okada et al., 1995), and the calmodulin-dependent kinase II inhibitors CamKII 290–309 (calmodulin binding domain [CBD]) (Basavappa et al., 1999), CaMKII Inhibitor XII (Asano et al., 2010), and KN-93 (Sumi et al., 1991) applied via the pipette that also contained 2 mm ATP. Micromolar concentrations of drugs (see figure legends; K_i or IC₅₀ values in nm range) were chosen after observing no effects at submicromolar levels. None of the tested PKA or CaMKII inhibitors had an effect on the IV (Fig. 2C,E). Of all the kinase inhibitors (Fig. 2D,F), only KT5720 had an effect on the I_Ca measurements compared with control (p < 0.001).

Correlation between the rise of basal [Ca²⁺]_i and Ca²⁺ current rundown

Next, we considered the possibility that the lack of hydrolyzable ATP disables Ca²⁺ pumping and thereby Ca²⁺ clearance. Using simultaneous fura-2 imaging of [Ca²⁺]_i and whole-cell I_Ca recordings in IHCs dialyzed with ATP-γ-S (and 0.5 mm EGTA), we found that the I_Ca reduction coincided with a rise of basal [Ca²⁺]_i (Fig. 3A,B). Furthermore, IHCs that displayed a fast and pronounced elevation of basal [Ca²⁺]_i also displayed the most severe I_Ca rundown (Fig. 3B, dashed lines), also reflecting in the observed negative correlation between the rise of basal [Ca²⁺]_i and the I_Ca (Fig. 3D).

Figure 3.

IHC I_Ca requires intact Ca²⁺ homeostasis. A, Representative examples of IHC bulk [Ca²⁺]_i obtained by fura-2 imaging under different conditions: with 2 mm ATP, 2 mm ATP-γ-S, no ATP, 2 mm ATP + 10 μm CE, or 2 mm ATP + 5 μm CE + 10 μm FCCP. In each case, the pipette contained 0.5 mm EGTA. B, Simultaneous measurements of [Ca²⁺] and the normalized I_Ca of single IHCs dialyzed with 2 mm ATP (n = 5) or 2 mm ATP-γ-S (n = 11). The initial [Ca²⁺] ([Ca²⁺]_init) measured after sufficient loading of fura-2 was subtracted from subsequent [Ca²⁺] measurements. Dotted lines indicate two ATP-γ-S-cells with corresponding [Ca²⁺]_i values and I_Ca. C, Simultaneous measurements of [Ca²⁺] and the normalized I_Ca of single IHCs dialyzed with 2 mm ATP + 5 μm CE (n = 6) or with 2 mm ATP + 5 μm CE + 10 μm FCCP (n = 6). For comparison, the mean [Ca²⁺]_i of control IHCs (no drug) is shown (n = 5). D, [Ca²⁺]_i and I_Ca rundown are correlated for IHCs dialyzed with ATP-γ-S, no ATP (n = 3), ATP + CE, or ATP + CE + FCCP. Pearson correlation coefficient (r) and regression lines are displayed. There are different slopes for no ATP and ATP-γ-S.

We then tested whether the PMCAs are the main mechanism of ATP-dependent Ca²⁺ clearance and required for maintaining Ca²⁺ influx in IHCs. Application of the PMCA inhibitor carboxyeosin (CE) caused a rise of the basal [Ca²⁺]_i and a parallel decrease of I_Ca (Fig. 3A,C,D). Once again, the IHCs with the fastest and largest elevation of basal [Ca²⁺]_i displayed the most severe I_Ca rundown. We conclude that failure of PMCA-mediated Ca²⁺ clearance explains most of the I_Ca reduction observed in the presence of ATP-γ-S. The slope of the regression line of cells dialyzed without ATP was steeper than of the cells containing ATP-γ-S (p = 0.0012), suggesting the contribution of an additional ATP-dependent mechanism to maintenance of I_Ca that can use ATP-γ-S.

To test for a direct role of mitochondria in Ca²⁺ homeostasis and regulation of Ca²⁺ influx, the uncoupling agent FCCP was used. The combined application of FCCP and CE resulted in an elevation of [Ca²⁺] and corresponding Ca²⁺ current rundown similar to the one observed with CE alone. However, the slope of the regression line of I_Ca and basal [Ca²⁺]_i was significantly steeper in the IHCs treated with CE and FCCP compared with either CE or ATP-γ-S (p = 0.0007 for ATP-γ-S, p = 0.002 for CE vs ATP + CE + FCCP, ANCOVA), suggesting that application of FCCP enhances Ca²⁺ channel inactivation potentially via disruption of mitochondrial ATP generation and/or Ca²⁺ uptake affecting the clearance of synaptic Ca²⁺.

Ca²⁺/calmodulin-mediated CDI of Ca²⁺ channels underlies the I_Ca rundown in the absence of hydrolyzable ATP

The most parsimonious interpretation of the I_Ca reduction caused by ATP-γ-S or CE is an increased CDI of Ca²⁺ channels resulting from the elevated resting [Ca²⁺]_i. To test this hypothesis, we studied the effect of adding a high concentration (10 mm) of the Ca²⁺ chelators EGTA or BAPTA to the ATP-γ-S-containing pipette solution. In both cases, the pronounced global rise in [Ca²⁺]_i as well as the I_Ca reduction were prevented (Fig. 4A,C).

Figure 4.

Ca²⁺ chelators and CaM inhibitors prevent I_Ca rundown; synaptic Ca²⁺ signaling requires ATP. A, Normalized I_Ca of single IHCs dialyzed with 2 mm ATP-γ-S and either 10 mm EGTA (n = 6) or 10 mm BAPTA (n = 9). Normalized mean I_Ca overlaid. For comparison, the mean normalized I_Ca of control IHCs is displayed (black solid line). B, The mean normalized I_Ca of IHCs dialyzed with 2 mm ATP-γ-S and 0.5 mm EGTA in the absence (n = 10) or presence of berbamine E₆ (n = 9) or CGS-9343B (n = 8) as well as control IHCs (2 mm ATP, n = 5)_. Statistical comparison was performed between 3 and 9 min after loading. C, Correlation between I_Ca and basal [Ca²⁺]_i of ATP-γ-S-infused IHCs is weaker in the presence of CaM inhibitors (compare with Fig. 3D) and absent with high buffer concentrations. D, Rundown of synaptic Ca²⁺ signals at IHC AZs in the absence of ATP. Normalized ΔF/F₀ (top) of Fluo-4FF at an AZ and normalized I_Ca (bottom) of the same IHCs in the presence (n = 5) and absence (n = 10) of 2 mm ATP in the pipette solution. Thick lines indicate mean ± SEM (bin size, 100 s); thin lines indicate individual traces. Statistical comparison was performed between 4 min and 12 min after break-in. The initial data points of ΔF/F₀ were omitted because of noise introduced by low F₀. E, Decreasing amplitude of AZ Ca²⁺ signal in the absence of ATP. Temporal profiles of Fluo-4FF fluorescence at two exemplary AZs in the presence (black) and absence (gray) of 2 mm ATP during 20 ms depolarization (black bars) at 2, 4, and 6 min after break-in. **p < 0.01. ***p < 0.001.

We further tested the hypothesis of an increased Ca²⁺/calmodulin-mediated CDI by applying the calmodulin inhibitors E₆ berbamine (Grant and Fuchs, 2008) and CGS-9343B (Norman et al., 1987). They significantly slowed down I_Ca rundown despite a comparable increase of the resting [Ca²⁺] (Fig. 4B,C), further supporting our notion that the lack of hydrolyzable ATP reduces the IHC I_Ca via a rise in resting [Ca²⁺] and consecutive CDI of the Ca²⁺ channels.

Finally, to test how the lack of ATP affects synaptic Ca²⁺ signals, we combined patch-clamp and confocal Ca²⁺ imaging that allows spatiotemporal characterization of submicrometer-sized Ca²⁺ domains at the fluorescently tagged ribbon-type AZs (Frank et al., 2009). We observed that the synaptic Ca²⁺ domains rapidly disappeared in the absence of ATP from the pipette solution, correlating in time with the rundown of whole-cell I_Ca (Fig. 4D,E).

Discussion

This study shows that ATP is required for maintaining operational Ca_V1.3 Ca²⁺ influx in IHCs via efficient Ca²⁺ clearance to secure sufficiently low basal [Ca²⁺]_i and avoid steady-state CDI.

ATP dependence of IHC Ca_V1.3

The role of ATP in the regulation of L-type Ca²⁺ channels varies among cells of different tissues. First, phosphorylation/dephosphorylation have been shown to regulate channel gating (Xu et al., 2004). Second, ATP may alter Ca²⁺-dependent proteases, which directly interact with the Ca²⁺ channel (Altier et al., 2011). Finally, lack of ATP leads to failure of ATP-dependent Ca²⁺ pumps and to cytosolic Ca²⁺ accumulation that may trigger CDI of Ca²⁺ channels (Belles et al., 1988; von Gersdorff and Matthews, 1996).

Based on the present work using potent kinase inhibitors, a modulation of the IHC Ca_V1.3 channel by phosphorylation via CaMKII is unlikely. The majority of protein kinases (including CaMKII and PKA) can use ATP-γ-S, although it is a poorer substrate than ATP (Palvimo et al., 1985; Ishida et al., 1996), further arguing against an implication of CaMKII- and PKA-mediated phosphorylation in the ATP-γ-S-induced I_Ca reduction. Interestingly, it has been suggested that ATP-γ-S supports the normal function of Ca_V1.4 channels in synaptic terminals of bipolar cells by serving kinases as a substrate for thiophosphorylation (Heidelberger et al., 2002). In this context, the better maintained I_Ca in recordings with ATP-γ-S compared with those without exogenous ATP together with the mild effect on I_Ca of the PKA inhibitor KT5720 may indicate a modest positive effect of PKA-mediated phosphorylation of Ca_V1.3 on IHC I_Ca.

Steady-state CDI of Ca_V1.3 channels by elevated resting [Ca²⁺] in IHCs

Parallel measurements of IHC [Ca²⁺] and I_Ca revealed a coincident and correlated increase of resting [Ca²⁺] and I_Ca inactivation in the absence of hydrolyzable ATP or pharmacological block of PMCAs in IHCs. PMCAs are the major source of Ca²⁺ extrusion from IHCs (Kennedy, 2002) and other cells with ribbon synapses (Zenisek and Matthews, 2000). Lack of ATP or its hydrolysis (ATP-γ-S) (Eckstein, 1985) disables their pumping activity; indeed, the ATP-γ-S-mediated global Ca²⁺ increase observed in IHCs could be mimicked by the PMCA inhibitor CE. These findings emphasize the essential role of PMCA-mediated Ca²⁺ clearance for synaptic transmission. In addition to PMCA endogenous immobile and mobile Ca²⁺ buffers, the latter estimated at 0.5–1 mm Ca²⁺ binding sites (Hackney et al., 2005; Johnson and Marcotti, 2008) have been proposed to shape synaptic Ca²⁺ signals in IHCs (Frank et al., 2009). Our results suggest that, in conditions of metabolic stress that lowers the cytosolic ATP levels (likely 1–2 mm in hair cells) (Puschner and Schacht, 1997; Shin et al., 2007), IHCs may fail to maintain low resting [Ca]_i and normal I_Ca, which would then impede sensory signaling during prolonged stimulation.

Steady-state CDI driven by enhanced basal cytosolic Ca²⁺ has been documented for Ca_V channels of cardiomyocytes and retinal bipolar cells (Belles et al., 1988; von Gersdorff and Matthews, 1996). In the latter, the dialysis with elevated Ca²⁺ led to a block of I_Ca. In the present study, we corroborated our hypothesis that the absence of hydrolyzable ATP triggers steady-state CDI in IHCs via increased basal cytosolic Ca²⁺ by showing that EGTA, BAPTA, and the calmodulin inhibitors antagonize the I_Ca rundown. Work on the molecular mechanism of Ca²⁺/calmodulin modulation of the C terminus of the Ca_V channels indicates that the N-terminal lobe of CaM might respond preferentially to the global accumulation of Ca²⁺ (Dick et al., 2008). IHCs use several mechanisms to counteract CDI. An increase of steady-state inactivation of Ca_V1.3 channels in IHCs, as found here upon manipulation of the ATP supply, is expected to reduce the rate of transmitter release and, consequently, of spiking in the postsynaptic spiral ganglion neurons. Interestingly, a human mutation in the gene coding for Ca²⁺ binding protein 2 that antagonizes CDI impairs hearing (Schrauwen et al., 2012) potentially because of increased steady-state inactivation. It is conceivable that IHC Ca²⁺ influx, synaptic sound coding, and hearing can be compromised also by other mechanisms that lead to enhanced CDI, and the metabolic state of the IHC may couple to sound encoding via the mechanism described in this study.