Role of endolysosomal channels in calcium homeostasis and trafficking

内溶酶体通道在钙稳态和运输中的作用

• 批准号: 8149585
• 负责人: rosa puertollano
• 金额: $ 32.35万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149585
• 关键词: Calcium; Homeostasis; Role; trafficking

Mucolipin-3 (MCOLN3) is a cation channel that belongs to the superfamily of transient receptor potential (TRP) channels. A gain-of-function mutation in MCOLN3 results in the varitint-waddler (Va) phenotype in mice, which is characterized by hearing loss, vestibular dysfunction (circling behavior, head-bobbing, waddling), and coat color dilution. Whole-cell patch-clamp techniques in cells heterologously expressing MCOLN3 revealed that is an inwardly rectifying Ca2+-permeable cation channel which activity is inhibited by acidic extracellular (or luminal) pH and increased by incubation of cells in low Na+ medium. The Va phenotype is caused by a point mutation (V419P) in the pore region that locks the channel in an open conformation. It has been suggested that the Va mutant causes massive entry of Ca2+ inside cells leading to apoptosis and cell death. Endogenous MCOLN3 mainly localizes to intracellular vesicles in hair cells, while lower levels of the protein are also observed at the plasma membrane of the stereocilia. In HeLa and human fibroblasts, endogenous MCOLN3 was found to be distributed along the endocytic pathway. In agreement with these studies, heterologously expressed MCOLN3 co-localizes with early and late endosomes/lysosomes markers in HeLa and ARPE-19 cells. Over-expression of MCOLN3 causes severe alterations in the endosomal pathway, including enlargement and clustering of endosomes, delayed Epidermal Growth Factor Receptor (EGFR) degradation, and impaired autophagosome maturation, thus suggesting that MCOLN3 plays an important role in the regulation of endosomal function. To better understand 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. We found that impairment of MCOLN3 activity caused a significant accumulation of luminal Ca2+ 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 Ca2+ homeostasis at the endosomal pathway and confirm the importance of luminal Ca2+ for proper acidification and membrane trafficking.

MCOLN-3（Mucolipin-3，MCOLN 3）是一种阳离子通道，属于瞬时受体电位（transmittance receptor potential，TRP）通道超家族。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（转圈行为、摇头、蹒跚）和毛色淡化。全细胞膜片钳技术在异源表达MCOLN 3的细胞中显示，MCOLN 3是一种向内整流的Ca 2+渗透性阳离子通道，其活性被酸性细胞外（或管腔）pH抑制，并通过在低Na+培养基中孵育细胞而增加。Va表型是由孔区域中的点突变（V419 P）引起的，该点突变将通道锁定在开放构象。已经表明Va突变体导致大量Ca 2+进入细胞内，导致细胞凋亡和细胞死亡。 内源性MCOLN 3主要定位于毛细胞的细胞内囊泡，而在静纤毛的质膜上也观察到较低水平的蛋白质。在HeLa和人成纤维细胞中，发现内源性MCOLN 3沿着内吞途径分布。与这些研究一致，异源表达的MCOLN 3与HeLa和ARPE-19细胞中的早期和晚期内体/溶酶体标志物共定位。MCOLN 3的过表达导致内体途径的严重改变，包括内体的扩大和聚集、表皮生长因子受体（EGFR）降解延迟和自噬体成熟受损，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。我们发现，MCOLN 3活性的损害引起了显着积累的内腔Ca 2+在内涵体。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Ca 2+稳态中的突出作用，并证实了腔Ca 2+对于适当酸化和膜运输的重要性。