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.

CO2是代谢不可避免的副产品，其浓度控制着血液的酸度。因为只有少量增加血液的酸度才能证明是致命的，所以通过呼吸的调节二氧化碳的排泄是一个极为重要的保留生命的过程。我们发现CO2与Connexin26（CX26）形成的膜通道结合并打开，使它们可以释放ATP，然后激活控制呼吸的神经回路。这是呼吸的二氧化碳依赖性调节的关键机制。CX26是20种人连接基因之一。它编码一个可以停靠在相邻细胞中相同的膜通道的膜通道，形成“间隙连接”。间隙连接允许直接通过离子在细胞之间传递小分子。此外，未锁定的连接膜膜通道“半通道”可以允许释放信号物质，例如神经递质ATP。间隙连接和半通道都为细胞到细胞通信提供了重要但独特的机制。CX26对于人类生理至关重要 - 100种不同的CX26突变与人类病理有关。 CX26突变是最常见的听力丧失遗传原因。其他CX26突变会引起潜在的致命综合征，涉及严重的皮肤，视力和听力。出乎意料的是，一些引起听力损失和综合征的CX26突变也会改变CX26半通道的二氧化碳敏感性。因此，通过CX26通过CO2依赖性信号传导可能在人类生理学中具有进一步的至关重要但目前未被认可的作用。出乎意料的是，我们现在发现CO2与对半通道的开场作用相比，关闭了CX26差距。二氧化碳对间隙连接的闭合作用可能是由于与引起半通道开放的蛋白质中的同一位置结合而发生的。这是非常重要的，因为CX26间隙连接和半通道共存在相同的组织中，例如参与呼吸和听力的涉及的组织。因此，了解与CX26突变相关的病理学的病因，了解与CX26突变有关的病因的差异连接和半通道的差异调节。我们应分析二氧化碳是否确实在间隙连接和半chan中与同一位点结合，该二氧化碳是否能够通过二氧化碳酸突变二氧化碳元素来实现二氧化碳的分析。间隙交界处。然后，我们将测试CX26引起病理的突变（会改变半通道对CO2的敏感性）是否也改变了间隙连接对CO2的敏感性。要确切地了解CO2与CX26的结合并打开Hemichannel，我们需要各个州CX26的原子水平结构。我们将纯化CX26，生长晶体（有或没有CO2结合），并使用X射线方法来确定原子结构。由于改变CX26的CO2敏感性的人类突变不会影响CO2结合位点，因此尚不清楚为什么它们应该具有这种作用。因此，我们将结晶的CX26的突变体变体有或没有二氧化碳结合，以查看结构如何改变以及这是否可以解释二氧化碳敏感性的改变。我们还将探讨不需要蛋白质晶体的互补方法是否可以在足够的分辨率下提供结构信息。我们的研究将显示二氧化碳如何与CX26结合以及通道如何打开和关闭。这将为保存反射的最重要的寿命之一 - 依赖于CO2的呼吸调节，为结构上的基础提供了基础。此外，我们将改变对某些CX26突变与人类病理学如何改变CO2结合的机理理解。这可能暗示了减少病理学的疗法，并提高了提高患者生活质量的管理策略。这种新的结构信息可能有助于开发药物以挽救突变的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