Mechanisms of voltage regulation of membrane transport

膜转运的电压调节机制 SLC9家族膜转运蛋白结合了钠离子的输入和质子的输出。 它们对细胞质和内体pH的调节至关重要，而胞质和内体的pH反过来又影响几个 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。SLC9C1是SLC9系列中独一无二的成员。与其他SLC9不同的是 具有膜分隔的钠氢交换(NHE)结构域和通常较短的 相对无结构的C-末端可溶域，SLC9C1结合了NHE，一种电压敏感 结构域(VSD)和环核苷酸结合结构域(CNBD)，通过长的、结构化的方式相互连接 连接物，在单个多肽中。最近的基础实验表明，膜 环核苷酸的超极化和结合增强了通过SLC9C1的离子传输。它的 独特的设计使得人们无法预测这种蛋白质的电压和配体调节是如何 表现在结构层面上。SLC9C1表现出精子特异性表达，并已被证明 对小鼠和人类的精子活力至关重要。精子的运动能力由多种因素调节 膜电压、细胞内cAMP水平和pH的变化及所有这些刺激的影响 SLC9C1直接介导离子交换，使理解分子变得至关重要 这种多样化监管的基础。为此，在这项建议中，我们将整合单一- 粒子低温电子显微镜及其生化和重建技术 电生理学方法探索SLC9C1的关键生物物理机制。在目标1中，我们 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的结构重排 通过环核苷酸结合触发，并使用电生理测试一个关键接口的作用 在调解CNBD的监管效果方面。在目标3中，我们将确定pH和Per 离子影响SLC9C1的结构和功能。这项建议有很强的科学基础。 建立在我们严谨的初步研究基础上。它具有创新性，因为它将提供 不同构象的新膜蛋白及其在细胞上的挑衅假说 转运蛋白的电压和环核苷酸调节机制。所获得的见解 我们的研究将有助于以结构为基础的药物设计来治疗男性不育。它还将 对SLC9的结构和功能机制有广泛的影响 细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.