Poster: Intracellular Signaling

Abs # 846: Molecular Identification and Characterization of AtTPC1, a Voltage-gated Ca²⁺-permeable Channel in Arabidopsis thaliana

Presenter:	Furuichi, Takuya , takuya-f@jf6.so-net.ne.jp
Authors	Furuichi, Takuya  (A)   Kawaguchi, Masakazu  (B)   Sokabe, Masahiro  (A)   Muto, Shoshi  (B)
Affiliations:	(A): Graduate School of Medicine, Nagoya University (B): Nagoya University Bioscience Center, Nagoya University

Calcium plays an essential role as an intracellular second messenger in plants. Rapid increases in [Ca²⁺]_cyt are mediated by calcium channels located on the plasma membrane and endomembranes. Recently, we isolated a full-length cDNA encoding a voltage-gated Ca²⁺-permeable channel (AtTPC1) from A. thaliana (Furuichi et al., Plant Cell Pyisiol.42, 900-905 [2001a]). AtTPC1 has two conserved homologous domains, both of which contain six transmembrane segments (S1-S6) and a pore loop (P) between S5 and S6 in each domain. The overall structure is similar to the half of the general structure of a-subunits of voltage-activated Ca²⁺ channels in animal cells. From searching the last available release of the DNA databases, AtTPC1 homologues appear to be well conserved in planta. In the present study, AtTPC1 was expressed in Saccharomyces cerevisiae mutant lacking ScCCH1, a putative plasma membrane Ca²⁺ channel, which expressing an engineered Ca²⁺-sensitive reporter gene to monitor AtTPC-mediated Ca²⁺ influx. The expression of AtTPC1 significantly promoted a glucose-induced [Ca²⁺]_cyt increase in yeast cells. This glucose-induced [Ca²⁺]_cyt increase was clearly inhibited by BAPTA, an extracellular Ca²⁺ chelator and by La³⁺, a Ca²⁺ channel blocker. Using these cells, ion selectivity and voltage-dependency of AtTPC1 expressing in yeast were tested. A desired mutation (Asp335 to Ala) in EF-hand motif in a long hydrophilic segment between domain-I and -II disrupted Ca²⁺-binding activity and promoted the glucose-induced [Ca²⁺]_cyt increase in the yeast cells. This suggests that intracellular Ca²⁺-binding to the EF-hand controls the gating of AtTPC1.