Molecular Basis of K+ Current During Secretion

唾液腺功能减退会增加患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。唾液的分泌通过润滑和滋润粘膜表面来帮助维持口腔健康，同时保护口腔免受机械和化学压力以及微生物入侵。分泌过程耦合了多种离子通道、共转运体和交换体的作用，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活化钾通道与顶端膜上的钙活化氯通道偶联，这些通道共同作用，允许毒蕈碱激动剂刺激分泌。这些通道的分子特性仍然未知。直到最近，大电导BK （slo）通道被认为在刺激分泌过程中携带大部分钙活化钾电流。然而，药理学证据表明，下颌骨腺的分泌主要是通过中导钙激活钾通道的作用发生的。一个候选cDNA，命名为SK4/IK1，最近克隆基于同源性的小电导钙活化钾通道的SK家族。在检查的组织范围内，人类IK亚型hIK1的最高表达水平发生在唾液腺中。为了更好地确定IK1在刺激分泌中的作用，我们建议使用分子和免疫学工具在小鼠模型系统的腮腺中定位IK1转录物和蛋白。我们将通过研究mIK1活性丧失的生理后果来进一步利用这一系统的效用。利用我们目前正在产生的含有靶向mIK1基因破坏的转基因菌株，我们将研究mIK1介导的唾液流速和离子组成的变化。由于重组mIK1被认为在非洲爪蟾卵母细胞中传递了一种新的经历调节性体积减少（RVD）的能力，我们将进一步分析mIK1是否在哺乳动物表达系统和腮腺中参与了RVD。最后，我们将研究在转基因菌株中发生的功能相关离子转运蛋白表达的代偿性变化。

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.