Department of Chemistry & Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts, United States of America.
Sandia National Laboratories, Albuquerque, New Mexico, United States of America.
PLoS One. 2024 Sep 6;19(9):e0309553. doi: 10.1371/journal.pone.0309553. eCollection 2024.
Cation conducting channelrhodopsins (ChRs) are a popular tool used in optogenetics to control the activity of excitable cells and tissues using light. ChRs with altered ion selectivity are in high demand for use in different cell types and for other specialized applications. However, a detailed mechanism of ion permeation in ChRs is not fully resolved. Here, we use complementary experimental and computational methods to uncover the mechanisms of cation transport and valence selectivity through the channelrhodopsin chimera, C1C2, in the high- and low-conducting open states. Electrophysiology measurements identified a single-residue substitution within the central gate, N297D, that increased Ca2+ permeability vs. Na+ by nearly two-fold at peak current, but less so at stationary current. We then developed molecular models of dimeric wild-type C1C2 and N297D mutant channels in both open states and calculated the PMF profiles for Na+ and Ca2+ permeation through each protein using well-tempered/multiple-walker metadynamics. Results of these studies agree well with experimental measurements and demonstrate that the pore entrance on the extracellular side differs from original predictions and is actually located in a gap between helices I and II. Cation transport occurs via a relay mechanism where cations are passed between flexible carboxylate sidechains lining the full length of the pore by sidechain swinging, like a monkey swinging on vines. In the mutant channel, residue D297 enhances Ca2+ permeability by mediating the handoff between the central and cytosolic binding sites via direct coordination and sidechain swinging. We also found that altered cation binding affinities at both the extracellular entrance and central binding sites underly the distinct transport properties of the low-conducting open state. This work significantly advances our understanding of ion selectivity and permeation in cation channelrhodopsins and provides the insights needed for successful development of new ion-selective optogenetic tools.
阳离子传导通道型视紫红质（ChR）是光遗传学中常用的工具，用于控制可兴奋细胞和组织的活性。具有改变的离子选择性的 ChR 对于不同的细胞类型和其他专门应用具有很高的需求。然而，ChR 中离子渗透的详细机制尚未完全解决。在这里，我们使用互补的实验和计算方法来揭示通道型视紫红质嵌合体 C1C2 在高导和低导开放状态下阳离子运输和价态选择性的机制。电生理学测量确定了中央门内的单个残基取代，N297D，在峰值电流时使 Ca2+通透性相对于 Na+增加近两倍，但在稳态电流时则不然。然后，我们在开放状态下为二聚野生型 C1C2 和 N297D 突变体通道开发了分子模型，并使用调整良好/多步元动力学计算了每种蛋白质中 Na+和 Ca2+渗透的 PMF 曲线。这些研究的结果与实验测量结果非常吻合，并表明细胞外侧的孔入口与原始预测不同，实际上位于 I 螺旋和 II 螺旋之间的间隙中。阳离子运输是通过一种接力机制发生的，其中阳离子通过沿孔全长排列的柔性羧酸盐侧链进行侧链摆动，就像猴子在藤上摆动一样，在中央和胞质结合位点之间传递。在突变体通道中，残基 D297 通过直接配位和侧链摆动介导中央和胞质结合位点之间的交接来增强 Ca2+通透性。我们还发现，细胞外入口和中央结合位点处改变的阳离子结合亲和力是低导开放状态下独特传输特性的基础。这项工作显著提高了我们对阳离子通道型视紫红质中离子选择性和渗透的理解，并为成功开发新的离子选择性光遗传学工具提供了必要的见解。
