Molecular Mechanisms of Ion Transport by the SMG

唾液腺功能异常可见于放射治疗、药物治疗、干燥综合征和囊性纤维化。唾液是由腺泡细胞分泌蛋白质和液体形成的。导管系统吸收Na和Cl-，并分泌K和HCO3-形成最终唾液。在过去的几年里，我们逐渐认识到CFTR调节的HCO3-转运在上皮液和电解质分泌中的核心作用，包括唾液腺的分泌。CFTR支持依赖氯离子的HCO3-转运，而具有正常C1通道活性的致病突变具有异常的HCO3-转运。在初步的数据中，我们发现SMG表达新的SLC26家族中的几个成员-DRA、PDS、SMCBT-和BTR1，它们在HCO3-的分泌中起作用。SMG还表达NBCn1家族的剪接变异体，这些剪接变异体在HCO3-SAVERAGE中发挥作用。这两个家族的蛋白质都受cftr调控。基于这些发现，我们提出了一个新的假说，提出跨细胞的HCO3运输是唾液腺功能的中心，CFTR协调静止和刺激期间的HCO3运输。这一假设将通过以下四个目标进行检验。在目标1中，我们将研究CFTR和MDRA之间的相互调控相互作用。初步数据表明，CFTR显著刺激MDRA活性，MDRA可能影响CFTR的阴离子选择性。我们将使用几个cftr和dra突变体来a)表征cftr激活mdra的机制b)研究mdra如何影响cftr通道特性以及c)研究在WT和deltaF小鼠体内的相互调节。在目标2中，我们将探讨CFTR与SLC6家族成员Pendrin和SLC26A6之间的相互作用，这两个成员在SMG导管中高水平表达。在对SLC26A6的C1-/HCO3-交换活性进行基本表征后，我们将探讨CFTR在控制MDRA的同时，是否在体外和体内调节PDS和SMCBT。在目标3中，我们将研究BTR1在SMG功能中的作用。BTR1是新的HCO3转运蛋白家族的第一个成员，该转运蛋白在SMG导管和腺泡细胞中表达。我们建议将BTR1的Cl-和HCO3-转运描述为SMG的LM中潜在的HCO3-转运体或通道，最终产生唾液中140 mM的HCO3-。在目标4中，我们将表征CFTR对NBCn1剪接变体的调节以及它们在SMG对HCO3-挽救中的作用。我们将利用deltaF小鼠在体外和体内研究NBCn1亚型的单个和组合的活性以及CFTR抑制它们的机制。这些实验的成功完成将在很大程度上阐明CFTR在调节静息和刺激状态下的HCO3动态平衡方面的作用。这些研究还可能将重点从纠正氯离子转运的努力转向纠正分泌上皮疾病(如CF和干燥综合征)中的氯离子和HCO3-转运的努力。

Abnormal function of salivary gland occurs in radiation, drug therapies, Sjogren's syndrome and Cystic Fibrosis. Saliva is formed by secretion of proteins and fluid by acinar cells. The ductal system absorbs the Na+ and Cl- and secretes K+ and HCO3- to form the final saliva. Over the last few years, we came to appreciate the central role of CFTR-regulated HCO3- transport in epithelial fluid and electrolyte secretion, including that by salivary glands. CFTR supports Cl--dependent HCO3- transport, and CF-causing mutations with normal C1- channel activity have aberrant HCO3- transport. In the preliminary data we show that SMG express several members of the new SLC26 family of Cl-/HCO3- exchangers-DRA, PDS, SMCBT-and BTR1, that function in HCO3- secretion. SMG also express splice variants on the NBCn1 family that function in HCO3- salvage. The proteins of both families are regulated by CFTR. Based on these findings, we developed a new hypothesis to propose that transcellular HCO3- transport is central to salivary gland function and that CFTR coordinates HCO3- transport at rest and during stimulation. The hypothesis will be tested by following four aims. In aim 1 we will study the reciprocal regulatory interaction between CFTR and mDRA. Preliminary data shows that CFTR markedly stimulates mDRA activity and that mDRA may affect anion selectivity of CFTR. We will use several CFTR and DRA mutants to a) characterize the mechanism by which CFTR activates mDRA b) study how mDRA affects CFTR channel properties and c) study the reciprocal regulation in vivo in WT and deltaF mice. In aim 2 we will probe interactions between CFTR and the SLC6 family members Pendrin and SLC26A6 that are expressed at high levels in SMG ducts. After basic characterization of SLC26A6 C1-/HCO3- exchange activity, we will probe whether CFTR regulates PDS and SMCBT in vitro and in vivo as it controls mDRA. In aim 3 we will study the role of BTR1 in SMG function. BTR1 is the first member of a new family of HCO3- transporters that is expressed in SMG duct and acinar cells. We propose to characterize Cl- and HCO3- transport by BTR1 as the potential HCO3- transporter or channel in the LM of the SMG that generates the final 140 mM HCO3- in saliva. In aim 4 we will characterize regulation of the NBCn1 splice variants by CFTR and their role in HCO3- salvage by the SMG. We will characterize the activity of individual and combinations of NBCn1 isoforms and the mechanism of their inhibition by CFTR in vitro and in vivo using the deltaF mouse. Successful completion of the experiments should considerably clarify the role of CFTR in regulating HCO3- homeostasis in the resting and stimulated states. The studies may also shift the emphasis from efforts to correct Cl- transport to efforts to correct Cl- and HCO3- transport in diseases of secretory epithelia such as CF and Sjogren's syndrome.