EDNOGENOUS ION CHANELS OF THE GOLGI COMPLEX

高尔基复合体的内源性离子通道

• 批准号: 6387009
• 负责人: JOHN H CALDWELL
• 金额: $ 37.2万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387009
• 关键词: Golgi apparatus; acidity /alkalinity; adenosinetriphosphatase; carbohydrate receptor; complementary DNA; electrophysiology; endoplasmic reticulum; gel electrophoresis; immunofluorescence technique; inositol phosphates; intracellular membranes; ion channel blocker; laboratory rat; lipid bilayer membrane; liposomes; mass spectrometry; membrane channels; membrane reconstitution /synthesis; membrane transport proteins; organelles; protein sequence; protein structure function; solubility; tissue /cell culture

The Golgi complex is present in every eukaryotic cell, from yeast to humans, and functions in posttranslational protein modifications and sorting of these molecules to post-Golgi destinations. Both processes require an acidic lumenal pH and transport of substrate and reactants into and out of the Golgi lumen. Endogenous ion channels are expected to be important for regulating the ionic environment within the Golgi lumen. This proposal is to isolate and characterize these endogenous ion channels of the Golgi complex and is a collaboration between two laboratories, one that studies ion channels and one that studies Golgi function. Single ion channel studies are now feasible because we have recently improved the isolation of a Golgi fraction from rat liver. We eliminated proteins transiting the Golgi and achieved a 400-700 fold enrichment of endogenous Golgi proteins. Ion channels in the enriched fraction have been incorporated into planar lipid bilayers. We named the most prevalent ion channel GOLAC1 (Golgi Anion Channel 1). This channel has novel properties and is modulated by pH on the lumenal surface. Two hypotheses are proposed for the function of the GOLAC 1: first, it provides counterions necessary for acidification of the Golgi lumen by an electrogenic H+ATPase and second, it removes phosphate (generated by glycosylation and sulfation) from the Golgi lumen. There are three specific aims. (1) Electrophysiologically characterize anion and cation channels of the Golgi. (2) Obtain peptide and cDNA sequence by enriching for channel activity with subfractionation of detergent- solubilized Golgi proteins. Proteins that enrich in parallel with channel activity will be identified using 2D-gel electrophoresis, mass spectrometry, and peptide sequencing. The cDNA identified will be expressed, purified, and confirmed to be a GOLAC in bilayer studies and a Golgi protein by immunofluorescence. (3) Study modulation of Golgi channels by candidate molecules and cell factors. From a clinical viewpoint, there are an increasing number of diseases classified as ion channelopathies. It is likely that some human diseases will be due to mutations of endogenous Golgi channels.