Role of endolysosomal channels in calcium homeostasis and trafficking

粘膜脂(或TRPML)构成了一个与瞬时受体电位超家族同源的内体阳离子通道家族。在哺乳动物中，粘蛋白家族包括三个成员，即粘蛋白-1、-2和-3(MCOLN1-3)。MCOLN1是该家族中特征最好的成员，因为这种蛋白的突变与一种称为IV型粘脂沉积症(MLIV)的人类疾病有关。我们和其他人提出，MCOLN1在细胞中的主要作用是介导晚期内小体和溶酶体的钙外流，从而促进细胞器融合和调节内小体运输。 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。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。 虽然MCOLN1和MCOLN3已经被很好地描述，但MCOLN2的细胞功能仍然难以捉摸。我们之前已经描述过，在HeLa细胞中，MCOLN2分布在Arf6调控途径的管状循环内小体，并调控特定糖基磷脂酰肌醇锚定蛋白(GGIs)的循环。为了研究MCOLN2在生理相关细胞类型中的功能，我们首先分析了MCOLN2在小鼠不同组织和器官中的表达，发现MCOLN2主要在淋巴器官和肾脏中表达。定量RT-PCR显示MCOLN2在转录水平上有严格的调控。在静息的巨噬细胞中，MCOLN2mRNA和蛋白的表达水平可以忽略不计，但在体外和体内，Toll样受体(TLR)激活后，MCOLN2mRNA和蛋白的表达水平显著增加。免疫荧光分析表明，在培养细胞和原代细胞中，内源性MCOLN2主要定位于循环内体，而MCOLN1和MCOLN3分别分布于晚期和早期的内体途径。为了更好地了解MCOLN2在体内的功能，我们产生了一只MCOLN2基因敲除小鼠。我们的初步鉴定表明，MCOLN2通过内吞途径调节关键免疫调节剂的运输，在先天性免疫反应中发挥重要作用。

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.