A Novel Calcium Binding Site in the Slow Vacuolar Cation Channel TPC1 Senses Luminal Calcium Levels

Abstract

Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca 2+ binding sites have been characterized in cytosolic proteins. However, little is known about Ca 2+ binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca 2+ . 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 Ca 2+ binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca 2+ binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants.

Intracellular localization and physiological function of a rice Ca2+-permeable channel OsTPC1

“…[17][18][19] In contrast, TPCs in monocots including OsTPC1 have been suggested to be localized at the PM and confirmed by several groups independently. [13][14][15]20 To resolve this discrepancy, and confirm the intracellular localization of TPC family proteins, we introduced a GFP construct fused to the coding sequence of the C-terminus of OsTPC1 (OsTPC1-GFP) -d, f-h) and differential interference contrast (DIC) images (a, e) of tobacco BY-2 cells expressing GFP (a-d) or OsTPC1-GFP (e-h) stained with the fluorescent styryl membrane probe FM4-64 (Molecular Probes, USA).…”

A Single-Pore Residue Renders the Arabidopsis Root Anion Channel SLAH2 Highly Nitrate Selective

“…Site-directed mutations were introduced by means of a modified USER fusion method as described by Dadacz-Narloch et al (2011). Primer sequences are provided in Supplemental Table 1.…”

Reduced Tonoplast Fast-Activating and Slow-Activating Channel Activity Is Essential for Conferring Salinity Tolerance in a Facultative Halophyte, Quinoa

“…One key factor is luminal Ca 2+ , which shifts the SV voltage dependence, decreasing the SV activity at physiologically attainable transtonoplast potentials (Pottosin et al, 2004). The respective binding site in the TPC (SV) channel, sensitive to luminal Ca 2+ changes between 0 and 10 mM, was recently characterized at the molecular level (Dadacz-Narloch et al, 2011). Under physiological conditions (relevant membrane voltage, cytosolic and vacuolar pH, and Ca 2+ , Mg 2+ , and other cations), the time-averaged SV channel activity yields from less than one to only a few open SV channels per vacuole.…”