Calcium- and otoferlin-dependent exocytosis by immature outer hair cells
Calcium- and otoferlin-dependent exocytosis by immature outer hair cells
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DOI：
10.1523/jneurosci.4653-07.2008
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发表时间：
2008-02-20
影响因子：
5.3
通讯作者：
Dulon, Didier
中科院分区：
文献类型：
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作者：
Beurg, Maryline;Safieddine, Saaid;Dulon, Didier
未成熟的耳蜗外毛细胞 (OHC) 与 I 型传入神经纤维发生短暂的突触接触（带状突触），但突触小泡胞吐作用的直接证据仍然缺失。因此，我们研究了小鼠 OHC 在出生后第 2 天 (P2)-P3 的钙依赖性胞吐作用，这是钙电流最大幅度最高的发育阶段。通过使用时间分辨膜片钳电容测量，我们表明 L 型钙通道的电压阶跃激活会触发膜电容快速增加。电容增加显示出两个动力学成分，这可能反映了两个功能不同的突触小泡池，一个易于释放的池（RRP；tau = 79 ms）和一个缓慢释放的池（tau = 870 ms）。 RRP 大小和最大释放速率估计分别类似于 1200 个囊泡和 15,000 个囊泡/秒。此外，我们发现电容增加和钙流入之间存在线性关系，就像在成熟的内毛细胞 (IHC) 中一样。这些结果有力地支持了未成熟 OHC 中存在有效的钙依赖性神经递质释放。此外，我们发现，未成熟的 OHC，就像未成熟的 IHC 一样，能够产生再生钙依赖性动作电位，从而触发体内突触胞吐作用。最后，尽管钙电流正常，但在 Otof(-/-) 突变小鼠的 P2-P3 OHC 中，诱发的膜电容增加被消除。我们得出的结论是，Otoferlin（IHC 带状突触中假定的主要钙传感器）对于未成熟 OHC 中的突触胞吐作用也至关重要。
Immature cochlear outer hair cells (OHCs) make transient synaptic contacts (ribbon synapses) with type I afferent nerve fibers, but direct evidence of synaptic vesicle exocytosis is still missing. We thus investigated calcium-dependent exocytosis in murine OHCs at postnatal day 2 (P2)-P3, a developmental stage when calcium current maximum amplitude was the highest. By using time-resolved patch-clamp capacitance measurements, we show that voltage step activation of L-type calcium channels triggers fast membrane capacitance increase. Capacitance increase displayed two kinetic components, which are likely to reflect two functionally distinct pools of synaptic vesicles, a readily releasable pool (RRP;tau = 79 ms) and a slowly releasable pool (tau = 870 ms). The RRP size and maximal release rate were estimated at similar to 1200 vesicles and similar to 15,000 vesicles/s, respectively. In addition, we found a linear relationship between capacitance increase and calcium influx, like in mature inner hair cells (IHCs). These results give strong support to the existence of efficient calcium-dependent neurotransmitter release in immature OHCs. Moreover, we show that immature OHCs, just like immature IHCs, are able to produce regenerative calcium-dependent action potentials that could trigger synaptic exocytosis in vivo. Finally, the evoked membrane capacitance increases were abolished in P2-P3 OHCs from mutant Otof(-/-) mice defective for otoferlin, despite normal calcium currents. We conclude that otoferlin, the putative major calcium sensor at IHC ribbon synapses, is essential to synaptic exocytosis in immature OHCs too.