Cholesterol Crystalline Domain Function in Eye Lens: EPR Spin-Labeling Studies

摘要 人眼晶状体的纤维细胞膜充满了胆固醇(显示出胆固醇与 磷脂摩尔比从皮质的1到细胞核的高达4)，这导致了 这些膜中含有不相容的胆固醇结晶结构域(CCDs)。胆固醇含量最高时， 高达50%的细胞膜表面可被CCDs占据。这些条件存在于晶状体核中， 其中胆固醇含量可能显著超过膜的溶解度阈值。这个 CCDs的出现通常是一种病理征象；然而，只有在眼睛的晶状体中，CCDs才能发挥积极的作用 生理功能，保持晶状体透明度，并可能预防白内障 队形。 这项建议的长期目标是更好地了解胆固醇在 纤维细胞膜。在短期内，我们将(I)研究高胆固醇含量如何影响侧向 磷脂晶状体膜的组织，特别注意ccd的形成，以及(Ii)试验 CCDs的存在决定了周围主体磷脂的性质的假设- 胆固醇膜。(Iii)此外，实验将揭示晶状体的磷脂成分 膜影响CCDs的形成、大小和稳定性。(4)最后，制定的方法和 在模型膜系统中测试将用于检测和表征共存的胆固醇结晶 和晶状体纤维细胞膜中大量的磷脂-胆固醇结构域在成熟、衰老和白内障过程中 队形。用于研究从眼晶状体分离的膜中的共存结构域，以及在 纤维细胞膜，将使用氧传输(DOT)辨别法。DOT方法， 它基于电子顺磁共振(EPR)自旋标记技术，允许区分 不同的膜结构域，并提供关于结构和分子动力学的信息 共存区域的膜深度，而不需要分离它们。它还允许提供信息 关于氧在膜结构域内和跨膜结构域的传输。

ABSTRACT Fiber-cell membranes of the human eye lens are saturated with cholesterol (showing cholesterol-to- phospholipid mole ratios from 1 in the cortex to as high as 4 in the nucleus), which leads to the formation of immiscible cholesterol crystalline domains (CCDs) within these membranes. At the highest cholesterol content, up to 50% of the cell-membrane surface can be occupied by CCDs. These conditions exist in the lens nucleus, where the cholesterol content can significantly exceed the solubility threshold of the membrane. The appearance of CCDs is usually a sign of pathology; however, only in the eye lens can CCDs play a positive physiological function, maintaining lens transparency and possibly protecting against cataract formation. The long-term objective of this proposal is to achieve a greater understanding of cholesterol's function in fiber-cell membranes. In the short-term, we will (i) examine how high cholesterol content affects the lateral organization of phospholipid lens membranes, with special attention paid to the formation of CCDs, and (ii) test the hypothesis that the presence of CCDs determines the properties of the surrounding bulk phospholipid- cholesterol membranes. (iii) Additionally, experiments will reveal if the phospholipid composition of the lens membrane affects the formation, size, and stability of CCDs. (iv) Finally, the methodology developed and tested in model-membrane systems will be applied to detect and characterize coexisting cholesterol crystalline and bulk phospholipid-cholesterol domains in lens fiber-cell membranes during maturation, aging, and cataract formation. For studies of coexisting domains in membranes isolated from the eye lens, as well as in models of fiber-cell membranes, the discrimination by oxygen transport (DOT) method will be used. The DOT method, which is based on electron paramagnetic resonance (EPR) spin-labeling techniques, permits discrimination of different membrane domains and gives information about structure and molecular dynamics as a function of the membrane depth in coexisting domains without the need for their separation. It also allows information about oxygen transport within and across membrane domains to be obtained.