Characterization of Ca²⁺ Permeable Channel AtTPC1 Using a Ca²⁺ Reporter Chimeric GFP-Aequorin Protein

Abstract

Aequorin is widely utilized for analyzing calcium signaling in cells. We constructed chimeric proteins (GFP-AQ) consisted of GFP (S65T) and aequorin with various length linker peptides, according to Baubet et al (Proc Natl Acad Sci U S A.; 97(13):7260-5). Using GFP-AQ, calcium fluxes are converted to strong fluorescence of GFP without exitation light.
In this study, we characterized Arabidopsis Ca²⁺ permeable channel AtTPC1 using GFP-AQ in cch1 mutant yeast cells. Glucose addition to the sugar-starved yeast cells expressing AtTPC1 caused a transient increase of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt). This glucose-induced [Ca²⁺]_cyt increase was clearly inhibited by BAPTA, an extracellular Ca²⁺ chelator and La³⁺, a Ca²⁺ channel blocker. This [Ca²⁺]_cyt increase was also specifically inhibited by K⁺, but not by other mono- and divalent cations. Moreover, the expression of AtTPC1 accelerated ⁸⁶Rb⁺ uptake into the cells, indicating that AtTPC1 is a Ca²⁺ and K⁺ selective cation channel.