Molecular Mechanisms of Ion Transport by the SMG

SMG 离子传输的分子机制

• 批准号: 6870173
• 负责人: Shmuel Muallem
• 金额: $ 39万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6870173
• 关键词: acinar cell; bicarbonates; chloride channels; chloride ion; confocal scanning microscopy; cystic fibrosis; electrophysiology; homeostasis; ion exchange chromatography; ion transport; laboratory mouse; northern blottings; oral health; oral mucosa; polymerase chain reaction; potassium ion; saliva; salivary glands; secretion; single cell analysis; sodium ion; submandibular gland; voltage /patch clamp

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.

唾液腺功能异常可发生于放射治疗、药物治疗、干燥综合征和囊性纤维化。 唾液由腺泡细胞分泌蛋白质和液体形成。导管系统吸收Na+和Cl-并分泌K+和HCO 3-以形成最终的唾液。 在过去的几年里，我们开始认识到CFTR调节的HCO 3转运在上皮细胞液体和电解质分泌中的核心作用，包括唾液腺分泌。 CFTR支持Cl-依赖性HCO 3-转运，具有正常Cl-通道活性的CF-引起突变具有异常的HCO 3-转运。 在初步的数据中，我们表明SMG表达新的SLC 26家族的几个成员的Cl-/HCO 3-交换器-PDS-SLC 26，PDS，SMCBT-和BTR 1，在HCO 3-分泌的功能。 SMG还表达NBCn 1家族上的剪接变体，其在HCO 3补救中起作用。 这两个家族的蛋白质都受到CFTR的调控。 基于这些发现，我们提出了一个新的假设，提出跨细胞HCO 3运输是中央唾液腺功能和CFTR协调HCO 3运输在休息和刺激。 该假设将通过以下四个目标进行检验。 在目的1中，我们将研究CFTR和mrna之间的相互调节相互作用。初步数据表明，CFTR显着刺激mCl 3活性，mCl 3可能会影响CFTR的阴离子选择性。 我们将使用几种CFTR和Δ F突变体来a）表征CFTR激活Δ F的机制，B）研究Δ F如何影响CFTR通道特性，以及c）研究WT和Δ F小鼠体内的相互调节。 在目标2中，我们将探测CFTR与SLC 6家族成员Pendrin和SLC 26 A6之间的相互作用，这些家族成员在SMG导管中以高水平表达。 在SLC 26 A6 Cl-/HCO 3-交换活性的基本表征之后，我们将探索CFTR是否在体外和体内调节PDS和SMCBT，就像它控制线粒体一样。 在目的3中，我们将研究BTR 1在SMG功能中的作用。 BTR 1是一个新的HCO 3转运蛋白家族的第一个成员，在SMG导管和腺泡细胞中表达。 我们建议表征Cl-和HCO 3-运输BTR 1作为潜在的HCO 3-转运蛋白或通道中的LM的SMG，产生最终的140 mM HCO 3-在唾液中。 在目标4中，我们将表征CFTR对NBCn 1剪接变体的调节及其在SMG的HCO 3补救中的作用。 我们将使用deltaF小鼠在体外和体内表征NBCn 1同种型的个体和组合的活性以及CFTR抑制它们的机制。 实验的成功完成应大大澄清CFTR在调节HCO 3-稳态在休息和刺激状态的作用。 这些研究也可能将重点从纠正Cl-转运转移到纠正分泌性上皮疾病（如CF和Sjogren综合征）中Cl-和HCO 3-转运。