Chloride fluxes in organellar membranes

这是该项目的第二年，该项目使用多种方法来分析溶酶体膜的离子运输特性。溶酶体是细胞内的细胞器，在大多数细胞中作为消化细胞器，尽管在某些组织中它们被用于其他功能。溶酶体功能紊乱可导致多种疾病，包括神经功能障碍（溶酶体贮积病）和骨质疏松症（骨过度钙化）。溶酶体利用atp驱动的质子泵来维持酸性的腔内pH值并促进其消化功能。这样的泵只有在伴随着额外的离子传输来消散由atp酶建立的跨膜电压时才能有效。遗传证据表明，这一额外的离子途径是clc型阴离子转运体，但功能实验尚未证实这一途径。我们试图确定溶酶体中的离子运输途径，表征它们的性质，并确定负责的蛋白质。今年我们详细描述了去年发现的氯离子渗透率。我们证明了这种渗透性源于Cl-/H+反转运子而不是离子通道，并揭示了其化学计量为2Cl-/1H+。我们进一步表明，该反转运蛋白是溶酶体的主要Cl-通透性。通过siRNA敲低实验，我们进一步证明了阴离子转运蛋白家族的成员ClC-7负责溶酶体Cl-传导。这一结果对细胞器pH调节以及人类疾病具有启示意义，因为ClC-7阻滞剂可能作为骨质疏松症的治疗方法。

This was the second year for this project which is using a combination of methods to analyze the ion transport properties of lysosomal membranes. Lysosomes are intracellular organelles that serve in most cells as digestive organelles although in some tissues they are used for other functions. Disorders of lysosome function lead to a variety of diseases including neurological dysfunction (lysosomal storage diseases) and osteopetrosis (overcalcification of bone). Lysosomes utilize an ATP-driven proton pump to maintain an acidic luminal pH and facilitate their digestive function. Such a pump can only be effective if accompanied by additional ion transport to dissipate the transmembrane voltage built up by the ATPase. Genetic evidence suggests that this additional ion pathway a ClC-type anion transporter, but functional experiments have not yet demonstrated such a pathway. We seek to identify the ion transport pathways in lysosomes, to characterize their properties, and to identify the responsible proteins. This year we characterized in detail the Cl permeability that we discovered last year. We demonstrated that this permeability stems from a Cl-/H+ antirporter rather than an ion channel and revealed its stoichiometry as 2Cl-/1H+. We further showed that this antiporter is the major Cl- permeability of lysosomes. Using siRNA knockdown experiments we further demonstrated that ClC-7, a member of a family of anion transport proteins, is responsible for the lysosomal Cl- conductance. This results have implications for organellar pH regulation as well as for human disease, as blockers of ClC-7 might act as therapies for osteoporosis.