Expression, function and regulation of renal claudins

描述（由申请人提供）：紧密连接蛋白是决定细胞旁通透性的紧密连接蛋白，可能形成细胞旁孔的衬里。在肾小管中，它们在电解质和酸碱处理中起重要作用。这个建议的长期目标是确定在细胞旁孔的离子选择性的结构和功能的决定因素。我们建议使用claudin-2作为模型阳离子选择性孔。我们的总体假设是，细胞旁阳离子孔是一个相对较宽的充满水的圆柱体，其壁由细胞外结构域形成。细胞外结构域内的酸性残基的侧链贡献了排列在孔中的带负电荷的位点。我们假设一价阳离子通过部分脱水渗透到孔隙中，然后通过离散的静电结合位点或简单地通过孔隙内改变的介电环境来稳定。具体目标是：（1）表征密蛋白-2细胞旁孔的生物物理性质并模拟阳离子通过孔的渗透;（2）鉴定密蛋白-2的细胞外结构域中的酸性残基，其决定细胞旁阳离子选择性并表征其作用;（3）鉴定claudin中的其他孔衬残留物-2通过半胱氨酸诱变，并确定它们的位置和功能，所用方法包括用封闭蛋白-2稳定转染MDCK I细胞，扩散电位和电导的Ussing室研究，通过pH和羧基修饰试剂干扰带负电荷的残基，细胞外残基的定点诱变，以及通过甲硫基磺酸盐试剂探测半胱氨酸取代的残基。所获得的数据将导致一个结构模型来解释基于密蛋白的孔的功能。这可能有助于阐明高血压和肾性盐耗等疾病，并导致开发可调节肾小管细胞旁通透性和纠正电解质紊乱的药物。

DESCRIPTION (provided by applicant): Claudins are tight junction proteins that determine paracellular permeability and probably form the lining of the paracellular pore. In the renal tubule, they play important roles in electrolyte and acid-base handling. The long-term goal of this proposal is to determine the structural and functional determinants of ionic selectivity in the paracellular pore. We propose to use claudin-2 as a model cation-selective pore. Our overall HYPOTHESIS is that the paracellular cation pore is a relatively wide, water-filled cylinder, the wall of which is formed from the extracellular domains. The side chains of acidic residues within the extracellular domain contribute negatively charged sites that line the pore. We hypothesize that monovalent cations permeate the pore by partially dehydrating and are then stabilized either by discrete electrostatic binding sites, or simply by the altered dielectric environment within the pore. The SPECIFIC AIMS are to: (1) Characterize the biophysical properties of the claudin-2 paracellular pore and model the permeation of cations through the pore; (2) Identify acidic residues in the extracellular domains of claudin-2 that determine paracellular cation selectivity and characterize their role; (3) Identify other pore-lining residues in claudin-2 by cysteine mutagenesis and determine their location and function METHODS to be used include stable transfection of MDCK I cells with claudin-2, Ussing chamber studies of diffusion potential and conductance, interference with negatively charged residues by pH and carboxyl modifying reagents, site-directed mutagenesis of extracellular residues, and probing of cysteine-substituted residues by methanethiosulfonate reagents. The data obtained will lead to a structural model to explain the function of claudin-based pores. This may shed light on disorders such as hypertension and renal salt-wasting and lead to the development of drugs that could regulate paracellular permeability of the renal tubule and correct electrolyte disorders.