A highly Ca 2+ -permeable channelrhodopsin from the ancyromonad Nutomonas longa enables optogenetic control of Ca 2+ signaling.

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 Nl CCR, a channelrhodopsin from Nutomonas longa, possesses high Ca²⁺ permeability, enabling precise optical control of Ca²⁺ signaling. Compared with CapChR2, the most potent engineered Ca²⁺-conducting channelrhodopsin, Nl 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. Nl 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, Nl CCR enabled synaptic transmission independently of endogenous voltage-gated Ca²⁺ channels. These findings establish Nl CCR as a broadly applicable tool for direct, temporally precise manipulation of Ca²⁺-dependent signaling in living systems.