A highly Ca2+-permeable channelrhodopsin from the ancyromonad Nutomonas longa enables optogenetic control of Ca2+ signaling

Ca{superscript 2} is a ubiquitous regulator of cellular function, linking electrical activity to gene expression, secretion, metabolism, and synaptic plasticity. Yet, tools for its direct, time-resolved optical manipulation remain limited. Here, we report that NlCCR, a channelrhodopsin from Nutomonas longa, possesses high Ca{superscript 2} permeability, enabling precise optical control of Ca{superscript 2} signaling. Compared with CapChR2, the most potent engineered Ca{superscript 2}-conducting channelrhodopsin, NlCCR combines larger and faster photocurrents, higher Ca{superscript 2} permeability, weaker desensitization, and reduced inward rectification. Mutational analysis identified determinants of Ca{superscript 2} selectivity and further enhanced it by introducing carboxylate residues at the channels central gate. NlCCRs blue-shifted absorption (445 nm) minimized optical crosstalk with a red-shifted Ca{superscript 2} indicator, laying the groundwork for all-optical experiments. In mouse cortical pyramidal neurons, NlCCR enabled synaptic transmission independently of endogenous voltage-gated Ca{superscript 2} channels. These findings establish NlCCR as a broadly applicable tool for direct, temporally precise manipulation of Ca{superscript 2}-dependent signaling in living systems.