Abstract
<jats:p> Ca <jats:sup>2+</jats:sup> 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 Ca <jats:sup>2+</jats:sup> permeability, enabling precise optical control of Ca <jats:sup>2+</jats:sup> signaling. Compared with CapChR2, the most potent engineered Ca <jats:sup>2+</jats:sup> -conducting channelrhodopsin, <jats:italic>Nl</jats:italic> CCR combines larger and faster photocurrents, higher Ca <jats:sup>2+</jats:sup> permeability, weaker desensitization, and reduced inward rectification. Mutational analysis identified determinants of Ca <jats:sup>2+</jats:sup> 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 Ca <jats:sup>2+</jats:sup> 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 Ca <jats:sup>2+</jats:sup> channels. These findings establish <jats:italic>Nl</jats:italic> CCR as a broadly applicable tool for direct, temporally precise manipulation of Ca <jats:sup>2+</jats:sup> -dependent signaling in living systems. </jats:p>