MOLECULAR MECHANISMS OF MECHANOSENSITIVE CHANNEL GATING

机械敏感通道门控的分子机制

• 批准号: 6387116
• 负责人: PAUL BLOUNT
• 金额: $ 27.38万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387116
• 关键词: bacterial proteins; biological signal transduction; cysteine; electrophysiology; intermolecular interaction; membrane channels; molecular genetics; site directed mutagenesis; structural biology; voltage /patch clamp; voltage gated channel

Bacterial mechanosensitive channels are emerging as molecular paradigms for investigation of the mechanosensory transduction that occurs in physiological processes such as touch, proprioception, cardiovascular regulation, hearing and balance. The use of the MscL channel of Escherichia coli has advanced the field considerably by allowing molecular genetic analysis to be combined with electrophysiology and transport assays in the study of a protein with a well-defined physiological role. Hence, MscL serves as a good model for determining the molecular mechanisms of mechanosensitive channel gating as well as the general principles of how proteins detect and respond to membrane tension. Purification of the MscL channel protein led to the identification of the structural gene and ultimately to a 3.5 angstrom X-ray crystallographic structure of the Mycobacterium tuberculosis MscL. This is a closed structure, which unfortunately provides few clues to the process of channel gating or to the structure of the open channel. Because a large pore of 30 to 40 angstrom diameter is generated upon gating, a large conformational change must occur and several residues normally embedded in protein or lipid environments must contribute to the lining of the open pore. The residue interations that are strongest and of greatest importance for keeping the channel closed, the movements of residues and domains that take place upon gating, and the residues that contribute to the lining of the open pore are currently all unknown. The experiments in this application are designed to use molecular analyses based on the solved structure to determine these functional and structural properties of the channel. The approaches include: 1) Cysteine scanning to determine which interactions within the transmembrane domains are of most importance in MS channel gating. 2) Utilizing the "Substituted Cysteine Accessibility Method" (SCAM) to identify the residues that move into an aqueous environment upon channel opening, and thus are likely candidates for lining the open-channel pore. 3) Isolating and characterizing suppressor mutations of gain-of-function MscL mutants to identify pairs of residues that potentially interact in the closed, transition or open states.

细菌机械敏感通道以分子形式出现