Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany.
Molecular Neuroscience and Biophysics, Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany.
Nat Commun. 2022 Dec 21;13(1):7844. doi: 10.1038/s41467-022-35373-4.
Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H, Na, K and anions have been discovered or engineered, Ca-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca permeability and improve their Ca affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for calcium-permeable channelrhodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca permeation at negative voltage and low extracellular Ca concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca concentrations. Moreover, CapChR2 can robustly trigger Ca signalling in hippocampal neurons. When expressed together with genetically encoded Ca indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca entry in brain explants.
通道视紫红质是光门控离子通道，用于以高时空分辨率控制大网络中指定细胞的兴奋性。虽然已经发现或工程改造了对 H、Na、K 和阴离子有选择性的 ChR，但迄今为止尚未报道对 Ca 有选择性的 ChR。在这里，我们分析了 ChR 和突变衍生物，以了解它们的 Ca 通透性，并通过在中央选择性过滤器处进行靶向诱变来提高它们的 Ca 亲和力。这些工程通道被称为 CapChR1 和 CapChR2，用于钙渗透性通道视紫红质，它们在负电压和低细胞外 Ca 浓度下表现出降低的钠和质子传导能力，同时强烈改善了 Ca 通透性。在培养的细胞和神经元中，CapChR2 可靠地增加细胞内 Ca 浓度。此外，CapChR2 可以在海马神经元中强有力地触发 Ca 信号转导。当在果蝇黑腹果蝇蘑菇体输出神经元中与遗传编码的 Ca 指示剂一起表达时，CapChRs 介导脑外植体中的光诱导 Ca 内流。
