Role of endolysosomal channels in calcium homeostasis and trafficking

粘膜脂-3(MCOLN3)是一种pH调节的钙通道，定位于内体途径。MCOLN3的功能获得突变导致小鼠的Va表型，其特征是听力损失、前庭功能障碍和毛色变淡。Va表型是由MCOLN3孔区的一个点突变(A419P)引起的，该突变将通道锁定在开放的构象中，导致钙大量进入细胞内，并通过凋亡诱导细胞死亡。野生型MCOLN3的过表达导致内体途径的严重改变，包括内小体的增大和聚集，EGF受体降解延迟，自噬小体成熟受阻，因此MCOLN3在内体功能的调节中起重要作用。为了更好地了解MCOLN3的生理作用，我们通过表达通道死亡的显性负性突变体(458DD/KK)或通过敲除内源MCOLN3来抑制MCOLN3的功能，并测量了几个内体参数，包括管腔钙、pH和内体融合。 用荧光比值成像法测定管腔内钙离子浓度和pH值。为了监测细胞内的钙离子浓度，同时用Oregon Green 488BAPTA-5N(钙指示剂)和Alexa Fluor 555结合的葡聚糖(对钙不敏感)负载细胞，同时用FITC结合的葡聚糖(对pH敏感)和Alexa Fluor 555结合的葡聚糖(对pH不敏感)同时负载细胞来测量细胞内膜的pH。绿色荧光与红色荧光的比值指示了胞内囊泡内的钙离子浓度和pH值。最后，我们开发了一种体外实验来测量内小体的同型融合。简单地说，一组细胞与Alexa-Fluor-488 EGF孵育，另一组细胞与Alexa-Fluor-555 EGF孵育15分钟以标记早期内膜室。然后纯化内容体，并进行体外融合试验。孵育结束后，将内小体固定在玻片上，用共聚焦显微镜进行分析。通过测量黄色内小体的比例来量化内小体融合。 我们发现，MCOLN3活性的受损导致了腔内钙在内吞体内的显著积累。这种堆积导致内体酸化的严重缺陷以及内体融合的增加。我们的发现揭示了MCOLN3在调节内体途径的钙稳态方面的显著作用，并证实了腔钙对适当的酸化和膜转运的重要性。未来的实验将解决MCOLN3在动物模型中的作用。

Mucolipin-3 (MCOLN3) is a pH-regulated calcium channel that localizes to the endosomal pathway. Gain-of-function mutation in MCOLN3 causes the varitint-waddler (Va) phenotype in mice, which is characterized by hearing loss, vestibular dysfunction, and coat color dilution. The Va phenotype results from a punctual mutation (A419P) in the pore region of MCOLN3 that locks the channel in an open conformation causing massive entry of calcium inside cells and inducing cell death by apoptosis. Overexpression of wild-type MCOLN3 produces severe alterations of the endosomal pathway, including enlargement and clustering of endosomes, delayed EGF receptor degradation, and impaired autophagosome maturation, thus suggesting that MCOLN3 plays an important role in the regulation of endosomal function. To understand better the physiological role of MCOLN3, we inhibited MCOLN3 function by expression of a channel-dead dominant negative mutant (458DD/KK) or by knockdown of endogenous MCOLN3 and measure several endosomal parameters including luminal calcium, pH, and endosomal fusion. Luminal calcium concentration and pH were measured by fluorescence ratio imaging. To monitor calcium, cells were simultaneously loaded cell-impermeant Oregon Green 488 BAPTA-5N (calcium indicator) and Alexa Fluor 555-conjugated dextran (nonsensitive to calcium), while measurement of endosomal pH was performed by simultaneous loading of cells with FITC-conjugated dextran (sensitive to pH) and Alexa Fluor 555-conjugated dextran (not sensitive to pH). The ratio of green to red fluorescence is indicative of the luminal calcium concentration and pH in endocytic vesicles. Finally, we developed an in vitro assay to measure homotypic fusion of endosomes. Briefly, one population of cells was incubated with Alexa-Fluor-488 EGF, and the other cells were incubated with Alexa-Fluor-555 EGF for 15 min to label the early endosomal compartment. Endosomes were then purified and subjected to an in vitro fusion assay. At the end of the incubation, endosomes were fixed on glass coverslips and analyzed by confocal microscopy. Endosomal fusion was quantified by measuring the proportion of yellow endosomes. We found impairment of MCOLN3 activity caused a significant accumulation of luminal calcium at endosomes. This accumulation led to severe defects in endosomal acidification as well as to increased endosomal fusion. Our findings reveal a prominent role for MCOLN3 in regulating calcium homeostasis at the endosomal pathway and confirm the importance of luminal calcium for proper acidification and membrane trafficking. Future experiments will address the role of MCOLN3 in animal models.