Recovery of transduction after EGTA treatment
We began by examining the recovery of transduction following disruption of tip links using low calcium solutions that contained 5 mM EGTA. Previously, it has been shown that calcium chelation breaks tip links in frog (Assad et al., 1991
; Marquis and Hudspeth, 1997
) and chicken (Zhao et al., 1996
) hair cells. Gale et al. (2001)
showed that FM1-43 uptake is reduced in mouse cochlear hair cells exposed to EGTA. To extend these observations we harvested the cochlear basal turn from neonatal mice at postnatal day 0 (P0). After one day in culture, explants where treated for 15 minutes with a low calcium solution supplemented with 5 mM EGTA. Mechanoelectrical transduction currents were recorded from outer hair cells using the whole-cell, tight-seal technique. Hair bundles were deflected with a stiff glass probe mounted on a fast piezo actuator (Stauffer and Holt, 2007
). We were unable to detect transduction currents immediately following EGTA treatment (n = 5; , EGTA) whereas untreated control hair cells had robust transduction currents (, Control). These data are consistent with the notion that tip links are severed by solutions in which the calcium concentration is <1 μM (Assad et al., 1991
; Zhao et al., 1996
; Marquis and Hudspeth, 1997
; Furness et al., 2008
Figure 1 Recovery of transduction current after EGTA treatment. (A) Representative families of transduction currents recorded from outer hair cells in control conditions (left), immediately following EGTA treatment (center) and twelve hours after EGTA treatment (more ...)
In the chicken basilar papilla, recovery of transduction currents following EGTA treatment has been reported after calcium was restored to the culture medium (Zhao et al., 1996
). Gale et al. (2001)
demonstrated recovery of FM1-43 uptake after 12–24 hours in mouse cochlear hair cells and Jia et al. (2009)
reported recovery of transduction currents in gerbil hair cell cultures after 24 hours. In each case the authors suggested that reformation of tip links and the transduction apparatus had occurred. In our experiments when the EGTA-treated cultures were incubated in standard culture media (MEM with 1.3 mM Ca2+
) for 12 hours we found that transduction currents returned and that the current had maximal amplitudes similar to controls (, Recovery). We measured recovered transduction currents from 17 outer hair cells: the mean maximal transduction current was −401 ± 33 pA. To characterize the time course of transduction current recovery we performed similar experiments at 3, 6, 12 and 24 hours after EGTA treatment. Representative currents are shown for each time point (). We also plotted the mean maximal current amplitudes as a function of recovery time and fit the data with an exponential curve (). After 12 hours the amplitude of the transduction currents was 86% of that reported for P2 basal turn outer hair cells (, circle; Lelli et al., 2009
) and the recovery was complete 24 hours post EGTA treatment. The exponential fit revealed a time constant of 10.6 hours. Interestingly, the time course of transduction current recovery closely paralleled the time course of recovery from noise induced temporary threshold shifts in hearing threshold (Zhao et al., 1996
; Husbands et al., 1999
), suggesting that regeneration of tip links and the transduction apparatus might be an important cellular mechanism that underlies the recovery of auditory function following noise exposure.
We wondered whether the recovery in current amplitude was paralleled by a recovery of other biophysical aspects of hair cell transduction. In contrast to chick hair cells (Zhao et al., 1996
) but consistent with gerbil hair cells (Jia et al., 2009
), mouse cochlear hair cells had prominent adaptation (the decline of transduction current in the presence of a constant stimulus) at the earliest time point we examined: three hours post EGTA treatment. We examined the time course of hair cell adaptation and the shape of the stimulus-response relationship at several points during the recovery. Current traces were selected that had peak currents approximately equal to the half maximal response, the point at which the activation curve is most linear and the time course of the current decline roughly parallels the underlying shift of the activation curve (Shepherd and Corey, 1994
; Stauffer and Holt, 2007
). The selected traces were fit with a double exponential equation (Vollrath and Eatock, 2003
). We found both fast and slow components of adaptation were present three hours post EGTA (), which suggested that the mechanisms of fast and slow adaptation are tightly linked with the transduction recovery process. However, the time constants were slower than in control cells and became faster as the recovery proceeded (). The trend toward faster adaptation is illustrated by the mean time constants measured from 37 outer hair cells shown as a function of recovery time ().
Figure 2 Properties of transduction currents during recovery. (A) Representative transduction currents at 3, 12 and 24 hours after EGTA treatment (black line). Current traces that represented ~50% activation were fitted with a double exponential equation (green (more ...)
We also noted that as the recovery proceeded, the sensitivity of the cells became sharper. The greater sensitivity is illustrated in the representative stimulus-response curves which became progressively steeper at 3, 12 and 24 hours after EGTA exposure (). To quantify the change in sensitivity we plotted the 10–90% operating range for 37 cells as a function of recovery time ().
In a previous study we characterized the development of hair cell adaptation and sensitivity and found a similar trend toward faster adaptation and greater sensitivity as a function of development (Lelli et al., 2009
). We suggest that the parallel changes in these properties of hair cell transduction indicate that similar molecular mechanisms may contribute to both development and regeneration of hair cell transduction.
Next we asked whether regeneration of hair cell transduction in mice requires new protein synthesis. We added 1μg/ml of the protein synthesis inhibitor cycloheximide (CXM) to the medium and recorded transduction currents 24 hours later. We found no significant difference between cultures exposed to both EGTA and CXM (−239 ± 47 pA, n = 12) and CXM-treated controls (−212 ± 48 pA, n = 10). The similar amplitude between EGTA-treated and untreated cells suggests that regeneration of transduction does not require new protein synthesis, but rather takes advantage of a readily available pool of transduction molecules. This result is consistent with a previous report that showed a similar concentration of CXM was sufficient to block protein synthesis in hair cells but did not block regeneration of tip links (Zhao et al., 1996
The mean amplitude of the transduction currents recorded from CXM-exposed cultures, regardless of whether or not the cells had been exposed to EGTA, was about half of that recorded from untreated outer hair cells of the equivalent developmental stage (P2) and region but about the same as control currents at P1 (Lelli et al., 2009
). Because CXM is a broad spectrum protein synthesis inhibitor, we suspect the relative transduction current reduction was due to the 24-hour halt in global protein synthesis and development rather than a specific effect on tip link molecules.
Exogenous CDH23 and PCDH15 fragments inhibit transduction recovery
A recent study (Kazmierczak et al., 2007
) presented immunolocalization evidence that placed PCDH15 at the tips of shorter stereocilia and CDH23 along the side of the adjacent taller stereocilia. Kazmierczak et al. (2007)
also showed that the two molecules interact at their N-terminal tips to form filaments of sufficient length to span the distance between adjacent stereocilia. Lastly, they found that the interaction was calcium-dependent. Although these data are consistent with the hypothesis that tip links are formed by PCDH15 and CDH23, the Kazmierczak et al. (2007)
study did not address whether the proteins are required for mechanotransduction. To test the hypothesis that a functional interaction between the PCDH15 and CDH23 is required for hair cell mechanotransduction we used recovery of transduction current following chemical disruption of tip links as an assay. Since newly synthesized protein is not necessary for the recovery of transduction, we reasoned that an abundance of exogenous protein fragments might interact specifically with native cadherins to prevent the reformation of tip links by outcompeting the endogenous binding partner. Specifically, we predicted that exogenous extracellular CDH23 fragments would outcompete native full-length CDH23 and thus prevent formation of CDH23/PCDH15 filaments (, cartoon). Likewise, extracellular addition of PCDH15 fragments was predicted to outcompete endogenous PCDH15 and block formation of full-length filaments (, cartoon).
CDH23 and PCDH15 fragments interfere with transduction current recovery.
To test these predictions we first treated mouse cochlear hair cells with 5 mM EGTA for 15 minutes. The cells were then incubated for 12 hours in a solution that contained 1.3 mM calcium and purified CDH23 fragments (CDH23-His) which included the full length CDH23 ectodomain (Supplemental Figure 1
). We recorded from 19 hair cells and found that the mean maximal transduction current was −156 ± 36 pA, a significant (p<0.0001) reduction relative to the currents in hair cells that were not exposed to CDH23-His ( and ). We found a similar significant (p<0.0001) reduction in the maximal transduction current amplitude (−178 ± 38 pA, n = 17) when the cells were exposed to purified PCDH15 ectodomain fragments (PCDH15-His; Supplemental Figure 1
) for 12 hours ( and ). As a control for the specificity of the interaction between the exogenous fragments and endogenous proteins we used a purified integrin fragment (INTG-His) that was generated and purified in the same manner. We found no significant (p = 0.6) reduction of the transduction current recovery relative to untreated controls: the mean maximal transduction current following exposure to INTG-His was −375 ± 29 pA (n = 28; & ). These data suggest that the recovery of transduction can be inhibited by extracellular application of CDH23 or PCDH15 fragments but not integrin, consistent with the notion that a functional interaction between endogenous CDH23 and PCDH15 is required for the recovery of hair cell transduction following chemical disruption of tip links.
Figure 5 Summary bar graph and dose response relation for CDH23 fragments. (A) Bar graph that summarizes mean maximal transduction current amplitudes in the various conditions presented in –, indicated as follows: CTRL: control (no fragments (more ...)
To control for the possibility that the His tag may have affected transduction recovery we also applied CDH23 or PCDH15 fragments that lacked a His tag. In this case the fragments were tagged with the Fc domain of Human IgG (Supplemental Fig. 1
). Both Fc-tagged fragments induced significant inhibition in transduction current recovery (CDH23F11Fc: −229 ± 53 pA, n = 10, p = 0.016; PCDH15-Fc: −221 ± 25pA, n = 25, p = 0.00015) similar to the His-tagged fragments, which minimized the concern that the inhibition was the result of the tag itself.
The transduction currents that recovered during incubation with CDH23 or PCDH15 fragments had normal activation curves and normal adaptation. Since a reduction in the whole-cell current amplitude was the only effect observed, we interpret these data to suggest that the total number of functional transduction units per cell was reduced. The alternate explanation - that each hair cell had the same number of functional transduction units before and after application of CDH23 or PCDH15 fragments, but that there was a reduction in current per transduction unit - seems less likely, since the biophysical properties were otherwise unaltered.
We also found that the decrease in transduction current recovery depended on the concentration of the protein fragments applied to the cultures. At higher concentrations of CDH23-His, the inhibition of transduction current recovery was more pronounced. A dose response curve fitted with an exponential indicated a half maximal inhibition dose of 13.6 ng/μl which corresponds to a concentration of ~40 nM ().
Next we wondered whether the interaction between CDH23 and PCDH15 was dynamic or stable. To investigate this question we performed the same experiments except that hair bundles were not exposed to EGTA. In other words, tip links remained intact prior to the 12-hour incubation with the protein fragments. In this case we observed no reduction on the mean maximal transduction current amplitudes in any of the three conditions: CDH23-His: −425 ± 28 pA, (n = 21); PCDH15-His: −486 ± 24 pA, (n = 13); INTG-His: −477 ± 24 pA, (n = 5). These data suggest that, once formed, the interaction between endogenous CDH23 and PCDH15 is fairly stable and is not vulnerable to competitive inhibition by external application of exogenous protein fragments. Furthermore, the lack of effect without prior EGTA treatment shows that neither CDH23-His nor PCHD15-His had a non-specific, blocking effect on transduction channels directly.
Washout of exogenous protein fragments
Since the inhibition of transduction recovery was robust and repeatable we wondered whether the block could be washed out. That is, could a second treatment, with the low-calcium EGTA solution followed by a vigorous wash, break the interaction between endogenous PCDH15 and exogenous CDH23-His or between endogenous CDH23 and exogenous PCDH15-His? If so, the endogenous proteins might be available to reassemble and reform full-length functional interactions. Indeed, 24 hours after a second treatment with the EGTA solution and vigorous wash we found that the transduction currents recovered ( and ). The mean maximal transduction current amplitudes following washout of CDH23-His (−372 ± 24 pA, n = 16) and PCDH15-His (−371 ± 33 pA, n = 10) were similar and were not significantly (p>0.05) different from washout of INTG controls (−423 ± 12 pA, n = 3).
Mutations in CDH23 and PCDH15 that cause deafness
Next we examined the effects of two missense mutations. E737V occurs in the seventh EC domain of CDH23 and causes deafness in salsa
mice (Schwander et al., 2009
), a model of the non-syndromic, recessive deafness in humans known as DFNB12. The second mutation we examined, R139G, occurs in the putative interaction domain (EC1) of PCDH15 and causes the non-syndromic recessive deafness DFNB23 in humans (Ahmed et al., 2003
mice have normal hair bundle morphology and normal transduction current amplitudes at early postnatal stages (Schwander et al., 2009
). Interestingly, the CDH23 mutation in the seventh cadherin domain affects calcium binding and is thought to render the molecule susceptible to mechanical damage, accumulation of which may be the cause of the deafness that occurs at later stages (Schwander et al., 2009
). The presence of normal transduction current amplitudes at early postnatal stages suggests that CDH23 is functional at these stages and that the E737V mutation does not affect its ability to bind PCDH15. To examine the ability of mutant CDH23 to interact with PCDH15 we used our standard assay and applied CDH23 fragments that carried the salsa
mutation (CDH23-E737V). The fragments were applied for 12 hours following treatment with the low-calcium, EGTA solution. We found that the CDH23-E737V fragments blocked the recovery of transduction in a manner similar to the wild-type CDH23-His fragments. The mean maximal transduction currents were significantly reduced (−178 ± 49 pA, n = 11, p<0.005; and ) relative to controls. Since application of the exogenous CDH23-E737V fragments inhibited the recovery of transduction, we conclude that the E737V mutation does not disrupt the functional interaction with endogenous cadherin molecules in hair cells, which is consistent with available biochemical data (Schwander et al., 2009
). As such, our data help explain the presence of normal transduction current amplitudes in salsa
mice as reported by Schwander et al. (2009)
. Furthermore, these data are consistent with the suggestion that the mutation, in the seventh cadherin domain, affects the mechanical properties of molecule but not its ability to bind PCDH15.
Figure 4 Effects of mutations in CDH23 and PCDH15 fragments. (A) A form of CDH23-His that carried the salsa mutation (E737V) was applied to hair bundles following the EGTA treatment. A representative family of transduction currents showed reduction in current (more ...)
In contrast, mutations in PCDH15 that cause DFNB23 can occur in either the first or second cadherin domain but only those that occur in the first cadherin domain abolish the interaction with CDH23 in vitro
(Kazmierczak et al., 2007
). As a control for non-specific effects and to gain insight into the etiology of DFNB23, we applied exogenous PCDH15 fragments that carried the R139G mutation in the first cadherin domain (PCDH15-R139G). Twelve hours after exposure to the low-calcium EGTA solution and application of the PCDH15-R139G fragments we observed no reduction in the mean maximal transduction currents. The currents recovered to control levels (−444 ± 21 pA, n = 12; ). This finding suggests that the inhibition of transduction current recovery observed following incubation with the wild-type PCDH15-Fc fragments was not due to a non-specific interaction since, except for the mutation, the experiments were identical. Furthermore, the lack of inhibition suggests that R139G mutation renders PCDH15 unable to bind endogenous CDH23, consistent with the conclusions of Kazmierczak et al. (2007)
Development of transduction
Since recovery of transduction was inhibited by external application of exogenous CDH23 and PCDH15 fragments, we wondered whether development of transduction might also be vulnerable to inhibition. To test whether a functional interaction between CDH23 and PCDH15 is required for the development of transduction we examined four conditions. In this case, the apical region of the mouse cochlea was excised at P0, prior to the developmental onset of mechanotransduction in mouse outer hair cells (Lelli et al., 2009
). The organs of Corti were placed in culture for three days and were exposed to CDH23-His, PCDH15-His, a control solution that contained neither, or PCDH15-R139G fragments. Transduction currents were recorded at the equivalent of P3 after application of the various protein fragments. We found that hair cells exposed to either CDH23-His or PCDH15-His had maximal transduction currents that were significantly reduced (p<0.0005) relative to untreated controls and relative to cells exposed to either the control solution or the PCDH15-R139G fragments (). We conclude that a functional interaction between CDH23 and PCDH15 is required for the normal development of transduction currents and that the interaction is established extracellularly.
Figure 6 Transduction current development is impaired by CDH23 and PCDH15 fragments. (A) Representative families of transduction currents recorded from apical outer hair cells. Cochleas were harvested before the onset of mechanotransduction (P0) and cultured with (more ...)
Localization of CDH23 and PCDH15 fragments
Data presented in the previous sections suggest that a functional interaction between CDH23 and PCDH15 is required for both development and regeneration of hair cell transduction. Other recent work has suggested that these molecules are localized at the tips of stereocilia and that they are thought to interact to form tip links (Kazmierczak et al., 2007
) which are essential for transduction (Assad et al., 1991
). However, CDH23 and PCDH15 are also localized elsewhere in the hair bundle and may play a key role in the formation of proper hair bundle morphology during development (Michel et al., 2005
; Di Palma et al., 2001
; Bolz et al., 2001
; Lagziel et al., 2005
). Therefore, we wondered whether the inhibition we observed was the result of interactions between CDH23 or PCDH15 fragments and endogenous PCDH15 and CDH23 located at the tips of stereocilia or elsewhere in the bundle. To localize the exogenous protein fragments we fixed cultured organs of Corti after the tip links had been cut by EGTA and tissue had been exposed to CDH23F11-Fc, PCDH15-Fc, or PCDH15-R139G-Fc fragments for 12 hours. Since we observed similar transduction current inhibition between His-tagged fragments and Fc-tagged fragments, we opted to use fragments fused to the Fc domain of human IgG (Kazmierczak et al., 2007
) and an antibody against the human Fc domain conjugated to FITC to localize the exogenous fragments. The tissue was counterstained with phalloidin-Alexa 546 to illuminate the hair bundles. shows immunoreactivity for CDH23F11-Fc at the tips of the stereocilia and in the kinocilium, revealing the localization pattern of its endogenous binding partner, presumably PCDH15. We observed punctuate staining at the tips of the outer hair cell stereocilia and in the kinocilium but not elsewhere in the bundle. The pattern was consistent with the data from Ahmed et al. (2006)
who showed immunolocalization of PCDH15 at the tips of stereocilia and, after treatment with BAPTA, in the kinocilium.
Figure 7 Binding of CDH23 and PCDH15 fragments to hair bundles. Confocal images of cultures fixed 12 hours after EGTA treatment and exposure to CDH23F11-Fc, PCDH15-Fc or PCDH15-R139G. To increase the specificity for immunolocalization of the exogenous proteins, (more ...)
To localize the binding partner of PCDH15-Fc we performed essentially the same experiment using the human Fc domain antibody conjugated to FITC. The distribution of PCDH15-Fc (), which presumably reflected the localization of endogenous CDH23, was also at the stereociliary tips. A noticeable difference was the lack of prominent immunofluorescence in the kinocilium. Lastly, we were unable to detect the binding of mutant PCDH15-R139G fragments (), consistent with its inability to inhibit development and recovery of transduction currents. No immunofluorescence was detected in bundles incubated the human Fc domain conjugated to FITC by itself or with a control solution that did not contain protein fragments.
Presence of tip links during transduction recovery
To determine whether inhibition of transduction corresponded to inhibition of tip link recovery we used scanning electron microscopy to collect images of hair bundles under control conditions; immediately following exposure to the low calcium EGTA solution; 12 hours after exposure to the EGTA solution; and 12 hours post EGTA in the presence of CDH23-His or PCDH15-Fc (). Importantly, incubation with CDH23-His or PCDH15-Fc did not disrupt normal hair bundle morphology (). At high magnification a myriad of linkages between adjacent stereocilia in many different orientations were apparent, consistent with previous reports of developing cochlear hair bundles (Goodyear et al., 2005
). Thus, we were unable to conclusively distinguish true tip links from the numerous other linkages present at this developmental stage. However, at higher magnification it was clear that control bundles contained numerous links oriented parallel to the bundles’ morphological axis of sensitivity (, arrows). Immediately following EGTA treatment the number of linkages with such orientation was greatly reduced and sites between adjacent stereocilia where tip links might be found (arrows) were mostly vacant. Twelve hours after exposure to the EGTA solution linkages oriented along the bundle’s morphological axis were again apparent (arrows). Consistent with our physiological data we found that bundles exposed to PCDH15-Fc had fewer linkages aligned along the bundle’s morphological axis. A few pairs of stereocilia did contain links which was consistent with the partial transduction currents that remained following CDH23-His and PCDH15-Fc treatment.
Fragments prevent recovery of tip links after EGTA treatment.
To quantify apparent tip links, we examined pairs of stereocilia in the tallest three rows. We counted the proportion of potential tip link sites that were occupied by links oriented parallel to the sensitive axis. Hair bundles were scored by two independent observers and the counts for each bundle were averaged. We examined 37 bundles in five conditions and present the data as a bar graph (). Consistent with the data from chick hair bundles, (Zhao et al., 1996
) we found few occupied tip link sites in bundles just after exposure to EGTA but significant recovery 12 hours later. When cells were exposed to CDH23-His or PCDH15-Fc, the proportion of occupied sites was significantly reduced (p<0.005). Based on the general preservation of the hair bundle morphology and the lack of occupied tip link sites we conclude that the inhibition in the development and recovery of transduction currents was the result of an acute and specific inhibition of tip-link formation rather than the result of chronic structural disruption of hair bundle morphology.