Abstract:
Two-pore channels are pathophysiologically important Na+- and Ca2+-permeable channels expressed in lysosomes and other acidic organelles. Unlike most other ion channels, their permeability is malleable and ligand-tuned such that when gated by the signaling lipid PI(3,5)P2, they are more Na+-selective than when gated by the Ca2+ mobilizing messenger nicotinic acid adenine dinucleotide phosphate. However, the structural basis that underlies such plasticity and single-channel behavior more generally remains poorly understood. A recent Cryo-electron microscopy (cryo-EM) structure of TPC2 bound to PI(3,5)P2 in a proposed open-channel conformation provided an opportunity to address this via molecular dynamics (MD) simulation. To our surprise, simulations designed to compute conductance through this structure revealed almost no Na+ permeation events even at very high transmembrane voltages. However further MD simulations identified a spontaneous transition to a dramatically different conformation of the selectivity filter that involved expansion and a flip in the orientation of two core asparagine residues. This alternative filter conformation was remarkably stable and allowed Na+ to flow through the channel leading to a conductance estimate that was in very good agreement with direct single-channel measurements. Furthermore, this conformation was more permeable for Na+ over Ca2+. Our results have important ramifications not just for understanding the control of ion selectivity in TPC2 channels but also more broadly in terms of how ion channels discriminate ions.
摘要:
双孔通道是在溶酶体和其他酸性细胞器中表达的病理生理上重要的Na和Ca2渗透通道。与大多数其他离子通道不同,它们的通透性是可延展的和配体调节的,使得当通过信号脂质PI(3,5)P2门控时,它们比通过Ca2+动员信使烟酸腺嘌呤二核苷酸磷酸门控时更具Na+选择性。然而,作为这种可塑性和单通道行为基础的结构基础通常仍然知之甚少。最近提出的开放通道构象中与PI(3,5)P2结合的TPC2的低温电子显微镜(cryo-EM)结构提供了通过分子动力学(MD)模拟解决这一问题的机会。令我们惊讶的是,设计用于计算通过该结构的电导的模拟显示,即使在非常高的跨膜电压下,也几乎没有Na+渗透事件。然而,进一步的MD模拟确定了向选择性过滤器的明显不同构象的自发转变,该选择性过滤器涉及两个核心天冬酰胺残基的膨胀和取向的翻转。这种替代的过滤器构象非常稳定,并且允许Na流过通道,从而导致电导率估算与直接单通道测量非常吻合。此外,这种构象对Na+的渗透性高于Ca2+。我们的结果不仅对于理解TPC2通道中离子选择性的控制,而且在离子通道如何区分离子方面也具有重要意义。