A highly Ca ²⁺ -permeable channelrhodopsin from the ancyromonad <i>Nutomonas longa</i> enables optogenetic control of Ca ²⁺ signaling

<jats:title>Abstract</jats:title> <jats:p> Ca²⁺ 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 <jats:italic>Nl</jats:italic> CCR, a channelrhodopsin from <jats:italic>Nutomonas longa,</jats:italic> possesses high Ca²⁺ permeability, enabling precise optical control of Ca²⁺ signaling. Compared with CapChR2, the most potent engineered Ca²⁺-conducting channelrhodopsin, <jats:italic>Nl</jats:italic> CCR combines larger and faster photocurrents, higher Ca²⁺ permeability, weaker desensitization, and reduced inward rectification. Mutational analysis identified determinants of Ca²⁺ selectivity and further enhanced it by introducing carboxylate residues at the channel’s central gate. <jats:italic>Nl</jats:italic> CCR’s blue-shifted absorption (445 nm) minimized optical crosstalk with a red-shifted Ca²⁺ indicator, laying the groundwork for all-optical experiments. In mouse cortical pyramidal neurons, <jats:italic>Nl</jats:italic> CCR enabled synaptic transmission independently of endogenous voltage-gated Ca²⁺ channels. These findings establish <jats:italic>Nl</jats:italic> CCR as a broadly applicable tool for direct, temporally precise manipulation of Ca²⁺-dependent signaling in living systems. </jats:p>