Structural and biophysical basis of Connexin26 channel mediated disease

Connexin26 通道介导疾病的结构和生物物理基础

• 批准号: MR/P010393/1
• 负责人: Nicholas Dale
• 金额: $ 141.79万
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FP010393%2F1
• 关键词: Structural; biophysical; basis; Connexin26; channel

CO2 is the unavoidable by-product of metabolism and its concentration controls the acidity of blood. Because only a small increase in the acidity of blood can prove fatal, the regulated excretion of CO2 via breathing is an extremely important life-preserving process. We discovered that CO2 binds to and opens membrane channels formed from Connexin26 (Cx26), allowing them to release ATP, which then activates the neural circuits that control breathing. This is a key mechanism for the CO2-dependent regulation of breathing.Cx26 is one of 20 human connexin genes. It encodes a membrane channel that can dock to identical membrane channels in adjacent cells, to form a "gap junction". Gap junctions allow direct passage of ions and small molecules between cells. In addition, undocked connexin membrane channels, "hemichannels", can permit release of signalling substances such as the neurotransmitter ATP. Both gap junctions and hemichannels provide important but distinctive mechanisms for cell-to-cell communication.Cx26 is critical for human physiology -over 100 different Cx26 mutations have been linked to human pathologies. Cx26 mutations are the commonest genetic cause of hearing loss. Other Cx26 mutations cause potentially fatal syndromes that involve serious disorders of skin, vision and hearing. Unexpectedly, some of the Cx26 mutations that cause hearing loss and syndromes also alter the CO2-sensitivity of Cx26 hemichannels. CO2-dependent signalling via Cx26 may therefore have further vital, yet currently unrecognised, roles in human physiology. Surprisingly, we have now found that CO2 closes Cx26 gap junctions in contrast to its opening action on hemichannels. This closing action of CO2 on gap junctions may occur as a result of binding to the same location in the protein that causes the opening of the hemichannel. This is extremely important, as both Cx26 gap junctions and hemichannels co-exist in the same tissues, such as those involved in the control of breathing and hearing. Understanding the differential modulation of gap junctions and hemichannels by CO2 is thus fundamentally important and will provide new insight into the aetiology of pathologies linked to mutations of Cx26.We shall analyze whether CO2 does indeed bind to the same site on gap junctions and hemichannels, by mutating the key amino acids that comprise the CO2-binding site in hemichannels to test whether this also alters the CO2-sensitivity of the gap junction. We shall then test whether the pathology-causing mutations of Cx26, which alter the sensitivity of hemichannels to CO2, also change the sensitivity of the gap junction to CO2.To understand exactly how CO2 binds to Cx26 and opens the hemichannel, we need atomic level structures of the Cx26 in various states. We shall purify Cx26, grow crystals (with and without CO2 bound) and use X-ray methods to determine the atomic structures. As the human mutations that alter the CO2 sensitivity of Cx26 do not affect the CO2 binding site, it is unclear why they should have this effect. Therefore we shall crystallize mutant variants of Cx26, with and without CO2 bound, to see how the structure has been altered and whether this can explain the altered CO2 sensitivity. We shall also explore whether a complementary method, which does not require protein crystals, can provide structural information at sufficient resolution.Our research will show how CO2 binds to Cx26 and how the channels open and close. This will provide the structural underpinnings to one of the most important life preserving reflexes -the CO2-dependent regulation of breathing. Additionally, we will transform mechanistic understanding of how certain Cx26 mutations linked to human pathology alter CO2 binding. This may suggest therapies to lessen pathology, and management strategies to enhance patients' quality of life. This new structural information may aid development of drugs to rescue the CO2-sensitivity of the mutated Cx26 protein.

二氧化碳是新陈代谢不可避免的副产品，它的浓度控制着血液的酸度。因为血液酸度的小幅上升就足以致命，所以通过呼吸排出二氧化碳是一个极其重要的维持生命的过程。我们发现，二氧化碳与Connexin26 （Cx26）形成的膜通道结合并打开，允许它们释放ATP，然后激活控制呼吸的神经回路。这是依赖二氧化碳的呼吸调节的关键机制。Cx26是20个人类连接蛋白基因之一。它编码一个膜通道，可以停靠到相邻细胞的相同膜通道，形成一个“间隙连接”。间隙连接允许离子和小分子在细胞间直接通过。此外，未连接的连接蛋白膜通道，“半通道”，可以允许释放信号物质，如神经递质ATP。间隙连接和半通道都为细胞间的通讯提供了重要而独特的机制。Cx26对人体生理至关重要——超过100种不同的Cx26突变与人类病理有关。Cx26突变是听力损失最常见的遗传原因。其他的Cx26突变会导致潜在的致命综合症，包括严重的皮肤、视力和听力障碍。出乎意料的是，一些导致听力损失和综合征的Cx26突变也改变了Cx26半通道的二氧化碳敏感性。因此，通过Cx26传递的二氧化碳依赖信号可能在人体生理学中具有进一步的重要作用，但目前尚未被认识到。令人惊讶的是，我们现在发现二氧化碳关闭了Cx26缝隙连接，而不是打开半通道。二氧化碳对间隙连接的关闭作用可能是由于与蛋白质中导致半通道打开的相同位置结合而发生的。这是非常重要的，因为Cx26间隙连接和半通道共存于同一组织中，例如那些参与控制呼吸和听力的组织。因此，了解二氧化碳对间隙连接和半通道的差异调节是非常重要的，并将为与Cx26突变相关的病理病因学提供新的见解。我们将通过突变构成半通道中二氧化碳结合位点的关键氨基酸来分析二氧化碳是否确实结合在间隙连接和半通道上的同一位点，以测试这是否也改变了间隙连接对二氧化碳的敏感性。然后，我们将测试致病的Cx26突变是否会改变半通道对CO2的敏感性，也会改变间隙连接对CO2的敏感性。为了准确地了解二氧化碳是如何与Cx26结合并打开半通道的，我们需要不同状态下Cx26的原子水平结构。我们将提纯Cx26，生长晶体（有或没有二氧化碳结合），并使用x射线方法确定原子结构。由于改变Cx26的CO2敏感性的人类突变不影响CO2结合位点，因此尚不清楚它们为什么会产生这种影响。因此，我们将对Cx26的突变体进行结晶，观察其结构是如何改变的，以及这是否可以解释二氧化碳敏感性的改变。我们还将探索一种不需要蛋白质晶体的补充方法是否可以在足够的分辨率下提供结构信息。我们的研究将展示二氧化碳如何与Cx26结合，以及通道如何打开和关闭。这将为最重要的维持生命的反射之一——依赖二氧化碳的呼吸调节——提供结构基础。此外，我们将转变对某些与人类病理相关的Cx26突变如何改变二氧化碳结合的机制理解。这可能建议治疗以减轻病理，和管理策略，以提高患者的生活质量。这一新的结构信息可能有助于开发药物来挽救突变的Cx26蛋白的二氧化碳敏感性。

项目成果

Mechanism of substrate binding and transport in BASS transporters

BASS转运蛋白中底物结合和转运的机制

• DOI: 10.7554/elife.89167.3
• 发表时间: 2023
• 期刊: eLife
• 影响因子: 7.7
• 作者: Becker P

Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO 2

connexin26 野生型和组成型封闭突变体的结构揭示了 CO 2 的通道调节

• DOI: 10.1101/2023.08.22.554292
• 发表时间: 2023
• 作者: Brotherton D

Mechanism of substrate binding and transport in BASS transporters.

• DOI: 10.7554/elife.89167
• 发表时间: 2023-11-14
• 期刊: eLife
• 影响因子: 7.7
• 作者: Becker P;Naughton F;Brotherton D;Pacheco-Gomez R;Beckstein O;Cameron AD
• 通讯作者: Cameron AD