Abstract
Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca²⁺ binding sites have been characterized in cytosolic proteins. However, little is known about Ca²⁺ binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca²⁺. However, the D454/fou2 mutation in TPC1 eliminates vacuolar calcium sensitivity and increases store calcium content. In a search for the luminal calcium binding site, structure modeling indicated a possible coordination site formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 on the luminal side of TPC1. Each Glu residue was replaced by Gln, the modified genes were transiently expressed in loss-of-TPC1-function protoplasts, and SV channel responses to luminal calcium were recorded by patch clamp. SV channels lacking any of the four negatively charged residues appeared altered in calcium sensitivity of channel gating. Our results indicate that Glu-450 and Asp-454 are directly involved in Ca²⁺ binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca²⁺ binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants.