Endophilin‐A regulates presynaptic Ca 2+ influx and synaptic vesicle recycling in auditory hair cells
2019; Springer Nature; Volume: 38; Issue: 5 Linguagem: Inglês
10.15252/embj.2018100116
ISSN1460-2075
AutoresJana Kroll, Lina María Jaime Tobón, Christian Vogl, Jakob Neef, Ilona Kondratiuk, Melanie König, Nicola Strenzke, Carolin Wichmann, Ira Milošević, Tobias Moser,
Tópico(s)Ion channel regulation and function
ResumoArticle7 February 2019free access Transparent processSource Data Endophilin-A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells Jana Kroll orcid.org/0000-0003-4243-4088 Synaptic Vesicle Dynamics Group, European Neuroscience Institute (ENI), University Medical Center Göttingen, Göttingen, Germany Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience, InnerEarLab and Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Göttingen Graduate School for Neuroscience and Molecular Biosciences, University of Göttingen, Göttingen, Germany Search for more papers by this author Lina M Jaime Tobón orcid.org/0000-0002-6752-7750 Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Göttingen Graduate School for Neuroscience and Molecular Biosciences, University of Göttingen, Göttingen, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Christian Vogl orcid.org/0000-0003-4432-2733 Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Presynaptogenesis and Intracellular Transport in Hair Cells Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Jakob Neef Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Ilona Kondratiuk orcid.org/0000-0002-7740-8923 Synaptic Vesicle Dynamics Group, European Neuroscience Institute (ENI), University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Melanie König Synaptic Vesicle Dynamics Group, European Neuroscience Institute (ENI), University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Search for more papers by this author Nicola Strenzke orcid.org/0000-0003-1673-1046 Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Auditory Systems Physiology Group and InnerEarLab, Department of Otolaryngology, University of Göttingen Medical Center, Göttingen, Germany Search for more papers by this author Carolin Wichmann Corresponding Author [email protected] orcid.org/0000-0001-8868-8716 Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience, InnerEarLab and Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Ira Milosevic Corresponding Author [email protected] orcid.org/0000-0001-6440-3763 Synaptic Vesicle Dynamics Group, European Neuroscience Institute (ENI), University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Search for more papers by this author Tobias Moser Corresponding Author [email protected] orcid.org/0000-0001-7145-0533 Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Jana Kroll orcid.org/0000-0003-4243-4088 Synaptic Vesicle Dynamics Group, European Neuroscience Institute (ENI), University Medical Center Göttingen, Göttingen, Germany Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience, InnerEarLab and Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Göttingen Graduate School for Neuroscience and Molecular Biosciences, University of Göttingen, Göttingen, Germany Search for more papers by this author Lina M Jaime Tobón orcid.org/0000-0002-6752-7750 Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Göttingen Graduate School for Neuroscience and Molecular Biosciences, University of Göttingen, Göttingen, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Christian Vogl orcid.org/0000-0003-4432-2733 Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Presynaptogenesis and Intracellular Transport in Hair Cells Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Jakob Neef Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Ilona Kondratiuk orcid.org/0000-0002-7740-8923 Synaptic Vesicle Dynamics Group, European Neuroscience Institute (ENI), University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Melanie König Synaptic Vesicle Dynamics Group, European Neuroscience Institute (ENI), University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Search for more papers by this author Nicola Strenzke orcid.org/0000-0003-1673-1046 Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Auditory Systems Physiology Group and InnerEarLab, Department of Otolaryngology, University of Göttingen Medical Center, Göttingen, Germany Search for more papers by this author Carolin Wichmann Corresponding Author [email protected]ettingen.de orcid.org/0000-0001-8868-8716 Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience, InnerEarLab and Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Ira Milosevic Corresponding Author [email protected] orcid.org/0000-0001-6440-3763 Synaptic Vesicle Dynamics Group, European Neuroscience Institute (ENI), University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Search for more papers by this author Tobias Moser Corresponding Author [email protected] orcid.org/0000-0001-7145-0533 Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Author Information Jana Kroll1,2,3,4,‡, Lina M Jaime Tobón3,4,5,6,7,‡, Christian Vogl3,5,7,8,‡, Jakob Neef3,5,6,7, Ilona Kondratiuk1, Melanie König1,3, Nicola Strenzke3,7,9, Carolin Wichmann *,2,3,7, Ira Milosevic *,1,3 and Tobias Moser *,3,5,6,7 1Synaptic Vesicle Dynamics Group, European Neuroscience Institute (ENI), University Medical Center Göttingen, Göttingen, Germany 2Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience, InnerEarLab and Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany 3Collaborative Research Center 889, University of Göttingen, Göttingen, Germany 4Göttingen Graduate School for Neuroscience and Molecular Biosciences, University of Göttingen, Göttingen, Germany 5Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany 6Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany 7Auditory Neuroscience Group, Max Planck Institute for Experimental Medicine, Göttingen, Germany 8Presynaptogenesis and Intracellular Transport in Hair Cells Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany 9Auditory Systems Physiology Group and InnerEarLab, Department of Otolaryngology, University of Göttingen Medical Center, Göttingen, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 551 39 61128; Fax: +49 551 3912950; E-mail: [email protected] *Corresponding author. Tel: +49 551 3912379; Fax: +49 551 3912346; E-mail: [email protected] *Corresponding author. Tel: +49 551 39 63070; Fax: +49 551 3912950; E-mail: [email protected] EMBO J (2019)38:e100116https://doi.org/10.15252/embj.2018100116 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT–PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis–endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation. Synopsis Using a multidisciplinary approach, we show that endophilin-A positively regulates presynaptic Ca2+ influx and interacts with otoferlin. Moreover, endophilin-A supports vesicle endocytosis and clathrin-dependent vesicle reformation at ribbon synapses of murine inner hair cells. Absence of endophilin-A leads to reduced presynaptic Ca2+channel cluster size and attenuated Ca2+ influx at inner hair cell ribbon synapses. Otoferlin physically interacts with endophilin; loss of endophilin-A1 and -A3 leads to a reduction in otoferlin levels of ˜25%. The number of cytosolic and ribbon-associated SVs is decreased in mutants missing several endophilin-A genes; consequently, IHC sustained exocytosis was found to be reduced. Absence of endophilin-A leads to accumulations of endosome-like vacuoles and clathrin-coated organelles; the severity of the phenotype depends on the number of missing endophilin alleles. Introduction Ribbon synapses of auditory IHCs faithfully convert acoustic signals into an action potential code in spiral ganglion neurons (SGNs). Individual presynaptic active zones (AZs) of IHCs are thought to drive firing in a single SGN at rates of up to hundreds of Hz for as long as the sound continues (Safieddine et al, 2012; Wichmann & Moser, 2015; Rutherford & Moser, 2016). Exocytosis of synaptic vesicles (SVs) at IHC AZs is both, fast and indefatigable. It seems to operate independently of classical neuronal SNARE proteins, Munc13-like priming factors, or complexins (Strenzke et al, 2009; Nouvian et al, 2011; Vogl et al, 2015), but, instead, involves the deafness gene product otoferlin (Roux et al, 2006; Pangrsic et al, 2010). Probably the most important coordinator of synaptic transmission is Ca2+ that enters IHCs primarily through presynaptic voltage-gated CaV1.3 Ca2+ channels and mediates excitation–secretion coupling (Platzer et al, 2000; Brandt et al, 2003, 2005; Weiler et al, 2014; Wong et al, 2014). At IHC AZs, Ca2+ channels are present in defined numbers, organized in a stripe-like manner, and show little inactivation, which enables reliable information transfer during sustained stimulation (Brandt et al, 2005; Frank et al, 2009, 2010; Ohn et al, 2016; Neef et al, 2018). Interestingly, Ca2+ channels have been reported to interact with otoferlin (Ramakrishnan et al, 2009) and with endophilins (Chen et al, 2003), the protein family under study here. To sustain high transmission rates, IHCs need to balance exocytosis by equally efficient SV recycling (Siegel & Brownell, 1986; Parsons et al, 1994; Moser & Beutner, 2000; Beutner et al, 2001; Lenzi et al, 2002; Trapani et al, 2009; Neef et al, 2014; Revelo et al, 2014; Jung et al, 2015). Here, at least three kinetically distinct forms of endocytic membrane retrieval—rapid (300 ms), fast (4 s), and slow (20 s half-time recovery)—have been described for IHCs (Moser & Beutner, 2000; Beutner et al, 2001; Neef et al, 2014). However, to date, knowledge of the molecular entities mediating these kinetically distinct forms of endocytosis in IHCs remains scarce. In line with findings at conventional synapses (Ferguson & De Camilli, 2012; Kononenko & Haucke, 2015), work on endocytosis in IHCs has indicated a role of dynamins (Boumil et al, 2010; Neef et al, 2014), synaptojanin-1 (Trapani et al, 2009), and clathrin (Siegel & Brownell, 1986; Neef et al, 2014; Jung et al, 2015) in slow endocytosis that most likely represents clathrin-mediated endocytosis [CME, recently reviewed in Pangrsic and Vogl (2018)]. Surprisingly, genetic disruption of the clathrin adaptor AP-2 did not noticeably affect endocytic membrane retrieval in IHCs (Jung et al, 2015). However, in AP-2μ mutants, the abundance of clathrin-coated structures near the presynaptic AZs was reduced and large membranous organelles (endosome-like vacuoles, ELVs) accumulated after stimulation (Jung et al, 2015) similar to findings at conventional synapses (Kononenko et al, 2014). This supports the notion that, next to CME, bulk retrieval (reviewed in Kokotos and Cousin (2015)) may play a prominent role in hair cells (Lenzi et al, 2002; Neef et al, 2014; Revelo et al, 2014; Jung et al, 2015). SVs are then rapidly reformed from endocytosed membranes (Kamin et al, 2014; Revelo et al, 2014), which seems to employ clathrin-dependent and clathrin-independent mechanisms (Jung et al, 2015). Importantly, the processes of exocytosis and endocytosis are intimately coupled and tightly coordinated—both at classical neuronal and IHC ribbon synapses—and the proper function of both types of synapses depends on this coupling (Haucke et al, 2011; Wichmann & Moser, 2015; Milosevic, 2018). In IHCs, AP-2, which interacts with otoferlin (Duncker et al, 2013; Jung et al, 2015), has been implicated in exocytosis and endocytosis coupling (Jung et al, 2015). In neurons, a range of molecular key players have been identified that orchestrate endocytic membrane retrieval and SV reformation (Kononenko & Haucke, 2015; Milosevic, 2018); yet, their respective relevance for these processes in IHCs remains unclear. In this context, one interesting molecular target is the evolutionary conserved family of endophilin-A proteins (henceforth “endophilin”), which are involved in endocytic membrane retrieval and uncoating in neurons of invertebrates (Verstreken et al, 2002, 2003; Schuske et al, 2003) and mammals (Milosevic et al, 2011; Watanabe et al, 2018). The current view on mammalian endophilins (A1-A3) pictures them as hubs of a protein network that coordinates cargo packing, bud constriction, actin assembly, and recruitment of factors needed for fission and uncoating (Saheki & Camilli, 2012). Structurally, endophilins contain a BAR domain that senses and induces membrane curvature, as well as a SH3 domain that recruits the GTPase dynamin and the PI(4,5)P2 phosphatase synaptojanin-1 to clathrin-coated pits (Verstreken et al, 2002, 2003; Schuske et al, 2003; Perera et al, 2006; Ferguson et al, 2009; Simunovic et al, 2017). Upon fission, PI(4,5)P2 degradation initiates the shedding of clathrin adaptor proteins from the endocytosed membranes, ultimately leading to the uncoating of SVs (Schuske et al, 2003; Verstreken et al, 2003; Milosevic et al, 2011; Pechstein et al, 2015; Watanabe et al, 2018). To clarify a potential contribution of endophilins in IHC presynaptic physiology, we performed a comprehensive functional and morphological analysis encompassing single-cell RT–PCR, immunoblotting, electron microscopy, immunohistochemistry, patch-clamp recordings, biochemical interaction studies, and auditory systems physiology using constitutive endophilin knockout mice. Results All three endophilins are expressed in the cochlea To investigate the expression of endophilin genes in the organ of Corti and, more specifically, in IHCs, we collected mRNA from IHCs of the apical cochlear coil of Wt mice (C57BL/6J, 2 weeks old, i.e., right after hearing onset). After reverse transcription, we performed single-cell multiplex-nested real-time PCR. In these experiments, all three endophilin-A transcripts (i.e., A1, A2, and A3; Fig 1A and B) could be detected in all tested single IHC samples that were also positive for the housekeeping gene HPRT (see Appendix Table S1 for primer sequences). Importantly, we could not detect endophilin mRNAs in our negative control samples, i.e., a small volume of bath solution that was collected in close proximity to the IHC row prior to and directly after the extraction of the IHC cytoplasm. We note that, while this approach does not provide a quantitative assessment of expression levels due to the nature of the amplification procedure with nested primer pairs, it reliably indicates the presence of endophilin-A1-3 mRNAs in IHCs. Figure 1. Endophilin-A expression in the cochlea A. Schematic domain overview of endophilins-A1-3, highlighting the BAR and SH3 domains. B–B′. Schematic overview of the sample collection procedure for single-cell RT-PCR (scPCR). Single IHC cytoplasms from acutely dissected organs of Corti of C57BL/6J (Wt) mice after hearing onset were aspirated and processed for scPCR as depicted. (B′) Expression analysis of endophilins-A1-3 from individually isolated IHC cytoplasms using RT–PCR from a representative experimental run. Please note that for these experiments, negative bath control samples from before and after the isolation procedure were an essential requirement to ensure lack of contamination from cellular debris in the bath solution. HPRT was used as a housekeeping gene. C–C′. Immunoblotting of tissue lysates from postnatal day (p)15 Wt (C57BL/6J), 1-SKO, 1/2-DKO, and 1/3-DKO revealed protein expression of endophilin-A1 and endophilin-A2 in the murine cochlea and ensured antibody specificity. Unfortunately, none of the commercially available endophilin-A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts, but rather appeared to (also) detect A1. γ-Adaptin was used as loading control. All antibody epitopes localize to the distinct C-terminal regions of the different endophilin-A family members. Hi, hippocampus; Ce, cerebellum; Co, pooled cochleae from a single individual of the indicated genotype; Md, modioli (micro-dissected and pooled from 10 Wt animals); OC, organs of Corti (micro-dissected and pooled from 10 Wt animals). Source data are available online for this figure. Source Data for Figure 1 [embj2018100116-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint To further investigate endophilin protein expression in situ, we have tested several commercially available as well as custom-made anti-endophilin-A antibodies (see Materials and Methods) in various fixation and permeabilization conditions; yet, we did not obtain specific immunolabeling in the organ of Corti. Therefore, we proceeded to perform immunoblotting with KO-verified antibodies (Milosevic et al, 2011) on cochlear samples of all genotypes using hippocampal and cerebellar tissue extracts as positive controls. Here, we detected bands with the expected molecular weight of endophilin-A1 (~39 kD) and endophilin-A2 (~42 kD) in Wt cochleae. These bands were absent in cochlear lysates of the respective KO genotypes, hence strongly suggesting target specificity (Fig 1C–C′; γ-adaptin was used as an independent loading control). Taken together, these data indicate the expression of endophilins-A1-3 in IHCs and show the presence of endophilin-A1 and endophilin-A2 protein in the murine cochlea. Endophilin promotes Ca2+ influx and efficient SV replenishment in IHCs Next, we employed perforated patch-clamp recordings to assess the role of endophilins in presynaptic IHC function. Since the cumulative loss of all three endophilin genes is perinatally lethal, we first prepared organotypic cultures of organs of Corti harvested from endophilin-A1/2/3 triple KO (TKO; see Materials and Methods for exact genetic descriptions and breeding schemes of endophilin mutants) and endophilin-A1/3 double KO (1/3-DKO) mice, as well as C57BL/6J pups within 3–8 h after birth. Thereafter, organs of Corti were maintained in culture for 1 week to enable synaptic maturation and the otoferlin-dependence of exocytosis to be established (Sobkowicz et al, 1982; Vogl et al, 2015) prior to detailed electrophysiological analysis (Fig 2A′). In order to boost depolarization-induced exocytosis, whole-cell Ca2+ currents (ICa) and the ensuing exocytic membrane capacitance changes (ΔCm) were recorded at an elevated extracellular Ca2+ concentration of 10 mM ([Ca2+]e; 1.3 mM is considered physiological). In these experiments, IHCs of 1/3-DKOs exhibited a 25% reduction of the presynaptic ICa and TKO IHCs showed a non-significant trend toward smaller ICa (Fig 2A and A″, Imax: −317 ± 27.3 pA for TKO IHCs, −298 ± 22.9 pA for 1/3-DKO, and −403 ± 32.2 pA for Wt; one-way ANOVA, F(2, 26) = 3.89, P = 0.0334; post hoc Tukey's test: P = 0.046 Wt versus 1/3-DKO; P = 0.103 Wt versus TKO for the maximal ICa elicited by depolarization to –17 mV). Interestingly, ICa of TKO IHCs showed enhanced inactivation, as evident from a significantly reduced fraction of ICa remaining at 100 ms of depolarization, which was not found in 1/3-DKO IHCs (Fig 2B–B′, Inorm, res 100 ms: 0.65 ± 0.02 for TKO IHCs, 0.73 ± 0.02 for 1/3-DKO, and 0.71 ± 0.02 for Wt; one-way ANOVA, F(2, 26) = 4.89, P = 0.0158; post hoc Tukey's test: P = 0.046 for TKO versus Wt; P = 0.794 for 1/3-DKO versus Wt). Such ICa reduction and enhanced ICa inactivation suggest a functional interaction of endophilins and Ca2+ channel complexes, which is in line with previous biochemical interaction studies (Chen et al, 2003). Figure 2. Reduced presynaptic Ca2+ currents and exocytosis in endophilin-deficient IHCs maintained in organotypic culture A–A″. Ca2+ current–voltage relationships evoked by incremental 15-ms step depolarizations from −87 mV to +58 mV revealed a ˜25% reduction of ICa in 1/3 DKO and TKO mice. (A′) Due to the perinatal lethality of TKO mice, detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically cultured organs of Corti after 7 days in vitro (DIV). C57BL/6J (Wt) and 1/3-DKO served as controls. Please note that all recordings from cultured IHCs were performed at [Ca2+]e of 10 mM to maximize IHC exocytic performance. (A″) Quantification and statistical analysis of individual maximum ICa amplitudes (Imax) of the respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes (*P = 0.046, one-way ANOVA with post hoc Tukey's test). B–B′. Ca2+ current inactivation was probed by test pulses of 100 ms to the Imax potential and revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt and 1/3-DKO cells. (B′) Quantification and statistical analysis of the residual current (Ires 100 ms) at the end of the test pulse (*P = 0.046, one-way ANOVA with post hoc Tukey's test). C–C″. Representative ICa (upper panel) and Cm (lower panel) in response to a 50 ms depolarizing step to the potential eliciting Imax. (C′) Exocytic ΔCm and corresponding QCa elicited by depolarizations of stimulus durations from 2 to 100 ms for all respective genotypes and at [Ca2+]e = 6 mM for a second set of recordings from wild-type IHCs to experimentally approximate the decreased ICa observed in the endophilin mutants. (C″) Magnification of the initial, short depolarizing steps (2–20 ms) for clarity. Exocytic ΔCm of cultured endophilin-deficient IHCs was strongly reduced (*P < 0.05; **P < 0.01; one-way ANOVA with post hoc Tukey's or non-parametric K–W with post hoc Dunn's test; please also refer to Appendix Tables S2 and S3 for detailed statistical analysis). D. The reduced Ca2+ efficiency of exocytosis (ΔCm/QCa) in endophilin-deficient IHCs indicates that diminished Ca2+ influx cannot fully account for the reduction of exocytosis (*P < 0.05; **P < 0.01; one-way ANOVA with post hoc Tukey's or non-parametric K–W with post hoc Dunn's test; please also refer to Appendix Table S4 for detailed statistical analysis). Data information: For panels (A–D), the following numbers of replicates were used: Wt 10 mM [Ca2+]e number of cells (n) = 12, number of animals (N) = 9, number of organotypic cultures (C) = 5; Wt 6 mM [Ca2+]e n = 8/N = 5/C = 4; 1/3-DKO 10 mM [Ca2+]e n = 8/N = 7/C = 3; TKO 10 mM [Ca2+]e n = 9/N = 7/C = 4. Error bars in (C′–D) indicate the SEM; box plots in (A″) and (B′) illustrate the median with the interquartile range, whiskers indicate 10–90% of data points, and the squares present the respective mean value. Download figure Download PowerPoint Recordings of exocytic changes in membrane capacitance (ΔCm) showed impaired exocytosis. Exocytosis of the readily releasable pool (RRP), as approximated by ΔCm responses to 20-ms depolarizations, was significantly attenuated in 1/3 DKO IHCs and tended to be reduced in TKO IHCs (one-way ANOVA, F(3, 33) = 5.35, P = 0.0041; post hoc Tukey's test: P = 0.075 for Wt versus TKO; P = 0.006 for Wt versus 1/3-DKO). Similarly, sustained exocytosis, probed by 100-ms-long depolarizations, tended to be attenuated in both genotypes (Fig 2C–C″, Kruskal–Wallis statistic (KWS) = 10.93, P = 0.0121; post hoc Dunn's test: P = 0.220 for Wt versus TKO; P = 0.025 for Wt versus 1/3-DKO). In contrast, no significant difference was found for responses to short stimuli (< 10 ms, also see Appendix Tables S2 and S3), indicating that endophilins are dispensable for SV fusion. In order to disentangle the reduction of exocytosis caused by diminished ICa from a potential impairment of SV replenishment in the absence of endophilin-A1 and endophilin-A3, we attempted to match the decreased ICa amplitudes by performing additional recordings from Wt (C57BL/6J) IHCs at lower [Ca2+]e (i.e., 6 mM instead of 10 mM; Fig 2C′–D). Under these conditions, ICa of Wt IHCs closely resembled the ones of endophilin-deficient mutant IHCs. However, the extent of exocytosis from Wt IHCs still exceeded that of cultured 1/3-DKO and TKO IHCs for depolarizations ≥ 10 ms and remained comparable to the data acquired at 10 mM [Ca2+]e (Fig 2C′–C″). Hence, the reduction in ICa in the endophilin mutants cannot fully account for the observed impairment of exocytosis, suggesting an additional requirement for endophilin-A1 and endophilin-A3 in exocytosis, e.g., in vesi
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