A Novel Calcium Binding Site in the Slow Vacuolar Cation Channel TPC1 Senses Luminal Calcium Levels |
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Authors: | Beata Dadacz-Narloch Diana Beyhl Christina Larisch Enrique J López-Sanjurjo Ralf Reski Kazuyuki Kuchitsu Thomas D Müller Dirk Becker Gerald Sch?nknecht Rainer Hedrich |
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Institution: | aUniversity of Wuerzburg, Institute for Molecular Plant Physiology and Biophysics, D-97082 Wuerzburg, Germany;bPlant Biotechnology, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany;cDepartment of Applied Biological Science, Tokyo University of Science, Noda 278-8510 Japan;dOklahoma State University, Department of Botany, Stillwater, Oklahoma 74078;eKing Saud University, Riyadh 11451, Saudi Arabia |
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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. |
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