Mechanisms of voltage regulation of membrane transport

膜转运的电压调节机制 膜转运蛋白的SLC9家族将钠离子的输入与质子的输出偶联。 它们对于调节细胞质和内体pH至关重要，这反过来又影响几种细胞因子。 生理过程。它们的功能障碍与许多疾病有关，如糖尿病， 高血压、心力衰竭和癌症。特定SLC9成员中的基因突变也 与Angelman综合征样障碍、ADHD、家族性自闭症、癫痫和 男性不育症SLC9C1是SLC9家族的独特成员。与其他SLC9不同， 其特征在于膜界定的钠-氢交换（NHE）结构域和通常短且 相对非结构化的C-末端可溶性结构域，SLC9C1结合了NHE，电压敏感性结构域， 结构域（VSD）和环核苷酸结合结构域（CNBD），通过长的，结构化的 连接子，在单个多肽中。最近的基础实验表明，膜 超极化和环核苷酸的结合增强了通过SLC9C1的离子转运。其 独特的设计使得不可能预测这种蛋白质的电压和配体调节是如何进行的。 表现在结构层面。SLC9C1表现出精子特异性表达， 对小鼠和人类的精子活力至关重要。精子活力受到以下因素的强烈调节： 膜电压、细胞内cAMP水平和pH的变化以及所有这些刺激影响 SLC9C1直接介导离子交换，这使得了解SLC9C1的分子机制至关重要。 这种多样性的监管基础。为此，在本提案中，我们将整合单一- 粒子冷冻电子显微镜和重建技术与生化和 电生理学方法探索SLC9C1的关键生物物理机制。目标1： 将确定SLC9C1的第一个高分辨率结构，并确定关键的相互作用 管理其组织。在目标2中，我们将阐明SLC9C1中的结构重排 触发环核苷酸结合，并使用电生理学测试的关键接口的作用 在调节CNBD的调节作用中的作用。在目标3中，我们将确定pH值和渗透率 离子影响SLC 9C1的结构和功能。这个建议有很强的科学基础 建立在我们严格的初步研究之上它是创新的，因为它将提供一个 新的膜蛋白在不同的构象和测试挑衅性的假设， 转运蛋白的电压和环核苷酸调节机制。获得的见解 将有助于基于结构的药物设计用于治疗男性不育症。它还将 具有广泛的影响的结构和功能机制的SLC9调控，其 胞质结构域进一步强调了其对人类健康的重要性。

Mechanisms of voltage regulation of membrane transport SLC9 family of membrane transporters couple the import of sodium ions to export of protons. They are vital for regulation of cytoplasmic and endosomal pH, which in turn affect several physiological processes. Their disfunction has been linked to many diseases such as diabetes, hypertension, heart failure and cancer. Genetic mutations in specific SLC9 members have also been associated with Angelman-syndrome like disorders, ADHD, familial autism, epilepsies and male infertility. The SLC9C1 is a unique member of the SLC9 family. Unlike other SLC9s which feature a membrane delimited sodium-hydrogen exchange (NHE) domain and a usually short and relatively unstructured C-terminal soluble domain, SLC9C1 combines an NHE, a voltage-sensing domain (VSD) and a cyclic nucleotide binding domain (CNBD), interconnected via long, structured linkers, in a single polypeptide. Recent foundational experiments have revealed that membrane hyperpolarization and binding of cyclic nucleotides potentiates ion transport via SLC9C1. Its unique design makes it impossible to predict how voltage and ligand regulation of this protein is manifested at a structural level. SLC9C1 exhibits sperm-specific expression and has been shown to be critical for sperm motility in mouse and humans. Sperm motility is robustly modulated by changes in membrane voltage, intracellular cAMP levels and pH and all these stimuli influence SLC9C1 mediated ion exchange directly, making it vital to understand the molecular underpinnings of such diverse regulation. To this end, in this proposal we will integrate single- particle cryo-electron microscopy and reconstruction techniques with biochemical and electrophysiological methods to explore key biophysical mechanisms of SLC9C1. In Aim 1, we will determine the first high-resolution structure of SLC9C1 and identify the key interactions governing its organization. In Aim 2, we will elucidate the structural rearrangements in SLC9C1 triggered by cyclic nucleotide binding and use electrophysiology to test the role of a key interface in mediating the regulatory effects of the CNBD. In Aim 3, we will determine how pH and permeant ions affect the structure and function of SLC9C1. The proposal has a strong scientific foundation built on our rigorous preliminary studies. It is innovative as it will provide the first snapshots of a novel membrane protein in different conformations and test provocative hypotheses on the mechanisms of voltage and cyclic nucleotide regulation of a transporter. The insights obtained from our studies will aid structure-based drug design for treatment of male infertility. It will also have broad implications on the structural and functional mechanisms of SLC9 regulation by their cytoplasmic domains further underscoring its importance for human health.