Role of endolysosomal channels in calcium homeostasis and trafficking

粘脂（或TRPML）构成与瞬时受体电位超家族具有同源性的内体阳离子通道家族。在哺乳动物中，粘脂家族包括三个成员，粘脂-1、粘脂-2和粘脂-3（MCOLN 1 -3）。MCOLN 1是该家族中最具特征的成员，因为这种蛋白质的突变与称为IV型粘脂沉积症（MLIV）的人类疾病相关。我们和其他人提出，MCOLN 1在细胞中的主要作用是介导晚期内体和溶酶体的钙外流，从而促进细胞器融合和调节内体运输。 MCOLN 3中的功能获得性突变导致小鼠中的varitint-waddler（Va）表型，其特征在于听力损失、前庭功能障碍和毛色稀释。Va表型由MCOLN 3的孔区域中的点状突变（A419 P）引起，所述点状突变将通道锁定在开放构象中，导致钙大量进入细胞内并通过凋亡诱导细胞死亡。野生型MCOLN 3的过表达导致内体途径的严重改变，包括内体的增大和聚集、延迟的EGF受体降解和受损的自噬体成熟，从而表明MCOLN 3在内体功能的调节中起重要作用。为了更好地理解MCOLN 3的生理作用，我们通过表达通道死亡显性负突变体（458 DD/KK）或通过敲低内源性MCOLN 3来抑制MCOLN 3功能，并测量几个内体参数，包括管腔钙、pH和内体融合。我们发现MCOLN 3活性的损伤导致了内腔钙在内体的显著积累。这种积累导致内体酸化的严重缺陷以及增加的内体融合。我们的研究结果揭示了MCOLN 3在调节内体途径的钙稳态中的突出作用，并证实了腔钙对于适当酸化和膜运输的重要性。 虽然MCOLN 1和MCOLN 3已经得到了很好的表征，但MCOLN 2的细胞功能仍然难以捉摸。我们以前已经描述过，在HeLa细胞中，MCOLN 2分布在Arf 6调节途径的管状再循环内体，并调节特定糖基磷脂酰肌醇锚定蛋白（GPIs）的再循环。为了解决MCOLN 2在生理相关细胞类型中的功能，我们首先分析了MCOLN 2在不同小鼠组织和器官中的表达，发现MCOLN 2主要在淋巴器官和肾脏中表达。定量RT-PCR显示MCOLN 2在转录水平上受到严格调控。虽然MCOLN 2表达在静息巨噬细胞中可以忽略不计，但MCOLN 2 mRNA和蛋白水平在体外和体内均响应于Toll样受体（TLR）激活而显著增加。免疫荧光分析表明，内源性MCOLN 2主要定位于回收内体在培养和原代细胞，与MCOLN 1和MCOLN 3，分布到晚期和早期内体途径，分别。为了更好地理解MCOLN 2的体内功能，我们产生了MCOLN 2敲除小鼠。我们的初步表征表明，MCOLN 2通过内吞途径调节关键免疫调节剂的运输，在先天免疫应答中起重要作用。

Mucolipins (or TRPMLs) constitute a family of endosomal cation channels with homology to the transient receptor potential superfamily. In mammals, the mucolipin family includes three members, mucolipin-1, -2, and -3 (MCOLN1-3). MCOLN1 is the best-characterized member of the family due to the fact that mutations in this protein are associated with a human disease known as mucolipidosis type IV (MLIV). We and others have proposed that the primary role of MCOLN1 in cells is to mediate calcium efflux from late endosomes and lysosomes, thus promoting organelle fusion and regulating endosomal trafficking. 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. 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. While MCOLN1 and MCOLN3 have been well characterized, the cellular function of MCOLN2 has remained elusive. We have previously described that, in HeLa cells, MCOLN2 distributes at the tubular recycling endosomes of the Arf6-regulated pathway and regulates recycling of specific glycosylphosphatidylinositol-anchored proteins (GPIs). To address MCOLN2 function in a physiologically relevant cell type, we first analyzed MCOLN2 expression in different mouse tissues and organs and found that MCOLN2 was predominantly expressed in lymphoid organs and kidney. Quantitative RT-PCR revealed tight regulation of MCOLN2 at the transcriptional level. While MCOLN2 expression was negligible in resting macrophages, MCOLN2 mRNA and protein levels dramatically increased in response to toll-like receptor (TLR) activation both in vitro and in vivo. Immunofluorescence analysis demonstrated that endogenous MCOLN2 primarily localized to recycling endosomes both in culture and primary cells, in contrast with MCOLN1 and MCOLN3 that distribute to the late and early endosomal pathway, respectively. To better understand the in vivo function of MCOLN2 we generated a MCOLN2 knockout mouse. Our initial characterization suggests that MCOLN2 plays an important role in innate immune response by regulating trafficking of key immune modulators through the endocytic pathway.