Chuang J Z, Milner T A, Zhu M, Sung C H
Department of Ophthalmology, The Margaret M. Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, New York 10021, USA.
J Neurosci. 1999 Apr 15;19(8):2919-28. doi: 10.1523/JNEUROSCI.19-08-02919.1999.
A novel class of intracellular chloride channels, the p64 family, has been found on several types of vesicles. These channels, acting in concert with the electrogenic proton pump, regulate the pH of the vesicle interior, which is critical for vesicular function. Here we describe the molecular cloning of p64H1, a p64 homolog, from both human and cow. Northern blot analysis showed that p64H1 is expressed abundantly in brain and retina, whereas the other members of this family (e.g., p64 and NCC27) are expressed only at low levels in these tissues. Immunohistochemical analysis of p64H1 in rat brain, using an affinity-purified antibody, revealed a high level of expression in the limbic system-the hippocampal formation, the amygdala, the hypothalamus, and the septum. Immunoelectron microscopic analysis of p64H1 in hippocampal neurons demonstrated a striking association between p64H1 and large dense-core vesicles (LDCVs) and microtubules. In contrast, very low p64H1 labeling was found in perikarya or associated with small synaptic vesicles (SSVs) in axonal profiles. Immunoblot analysis confirmed that p64H1 is colocalized with heavy membrane fractions containing LDCVs rather than the fractions containing SSVs. These results suggest that p64H1-mediated Cl- permeability may be involved in the maintenance of low internal pH in LDCVs and in the maturation of LDCVs and the biogenesis of functional neuropeptides.
一类新型的细胞内氯离子通道，即p64家族，已在几种类型的囊泡上被发现。这些通道与生电质子泵协同作用，调节囊泡内部的pH值，这对囊泡功能至关重要。在此，我们描述了来自人和牛的p64同源物p64H1的分子克隆。Northern印迹分析表明，p64H1在脑和视网膜中大量表达，而该家族的其他成员（如p64和NCC27）在这些组织中仅低水平表达。使用亲和纯化抗体对大鼠脑内的p64H1进行免疫组织化学分析，结果显示在边缘系统——海马结构、杏仁核、下丘脑和隔膜中表达水平很高。对海马神经元中的p64H1进行免疫电子显微镜分析，结果表明p64H1与大致密核心囊泡（LDCV）和微管之间存在显著关联。相比之下，在胞体中或轴突轮廓中与小突触囊泡（SSV）相关的p64H1标记非常低。免疫印迹分析证实，p64H1与含有LDCV的重膜组分共定位，而不是与含有SSV的组分共定位。这些结果表明，p64H1介导的Cl-通透性可能参与维持LDCV内的低内部pH值以及LDCV的成熟和功能性神经肽的生物合成。
