Molecular Basis of K+ Current During Secretion

唾液腺功能减退会增加口腔和全身疾病的风险，并导致咀嚼、吞咽和说话能力的恶化。唾液的分泌有助于通过润滑和水合粘膜表面来维持口腔健康，以及保护口腔免受机械和化学应力以及微生物入侵。分泌过程耦合多个离子通道、共转运蛋白和交换剂的作用，以驱动水的跨上皮运动。特别是，位于腺泡细胞基底外侧膜上的钙激活钾通道与顶膜上的钙激活氯离子通道偶联，这些通道一起工作以允许毒蕈碱激动剂刺激分泌。这些通道中的每一个的分子身份仍然未知。直到最近，大电导BK（BK）通道被认为在刺激分泌期间携带大部分钙激活钾电流。然而，药理学证据表明，下颌下腺的分泌主要通过中电导钙激活钾通道的作用发生。一个候选的cDNA，称为SK 4/IK 1，最近克隆的基础上同源性与SK家族的小电导钙激活钾通道。在检查的组织范围内，人IK亚型hIK 1的最高表达水平发生在唾液腺中。为了更好地定义IK 1参与刺激分泌，我们建议使用分子和免疫学工具本地化的IK 1转录本和蛋白质在腮腺的小鼠模型系统。我们将通过研究mIK 1活性丧失的生理后果来进一步利用该系统的实用性。使用转基因菌株含有一个有针对性的基因破坏mIK 1，我们目前正在产生，我们将检查mIK 1介导的变化，唾液流速和离子组成。由于重组mIK 1已被建议传达一种新的能力，在非洲爪蟾卵母细胞进行调节体积减小（RVD），我们将进一步分析是否mIK 1参与RVD在哺乳动物表达系统和腮腺。最后，我们将研究转基因株中功能相关离子转运蛋白表达的补偿性变化。

Salivary gland hypofunction enhances the risk of oral and systemic disease, and results in a deterioration in the ability to chew, swallow, and speak. The secretion of saliva helps to maintain oral health by lubricating and hydrating mucosal surfaces, as well as protecting the oral cavity from mechanical and chemical stresses and microbial invasion. The process of secretion couples the action of multiple ion channels, co-transporters, and exchangers to drive the transepithelial movement of water. In particular, a calcium-activated potassium channel located on the basolateral membrane of acinar cells is coupled to a calcium-activated chloride channel on the apical membrane, and these channels work together to allow stimulated secretion by muscarinic agonists. The molecular identity of each of these channels remains unknown. Until recently, the large-conductance BK (slo) channel was thought to carry the majority of the calcium-activated potassium current during stimulated secretion. However, pharmacological evidence exists that secretion in the submandibular gland occurs primarily through the action of an intermediate-conductance calcium-activated potassium channel. A candidate cDNA, termed SK4/IK1, was recently cloned based upon homology with the SK family of small-conductance calcium-activated potassium channels. Over the range of tissues examined, the highest level of expression of the human IK isoform, hIK1, occurs in salivary glands. To better define the involvement of IK1 in stimulated secretion, we propose to use molecular and immunological tools to localize the IK1 transcript and protein in the parotid gland of a mouse model system. We will further exploit the utility of this system by studying the physiological consequences of the loss of mIK1 activity. Using a transgenic strain containing a targeted gene disruption of mIK1 which we are generating presently, we will examine mIK1-mediated changes in saliva flow rate and ion composition. Because recombinant mIK1 has been suggested to convey a novel ability to undergo regulatory volume decrease (RVD) in Xenopus oocytes, we will further assay whether mIK1 is involved in RVD in a mammalian expression system and in the parotid gland. Finally, we will look at compensatory changes that occur in the expression of functionally-related ion transport proteins in the transgenic strain.