Functional interaction of calmodulin with a plant cyclic nucleotide gated cation channel

A family of plant ligand gated nonselective cation channels (cngcs) can be activated by direct, and reversible binding of cyclic nucleotide. These proteins have a cytoplasm-localized cyclic nucleotide binding domain (CNBD) at the carboxy-terminus of the polypeptide. A portion of the cngc CNBD also acts as a calmodulin (CaM) binding domain (CaMBD). The objective of this work is to further characterize interaction of cyclic nucleotide and CaM in gating plant cngc currents. The three-dimensional structure of an Arabidopsis thaliana cngc (Atcngc2) CNBD was modeled, indicating cAMP binding to the Atcngc2 CNBD in a pocket formed by a beta barrel structure appressing a shortened (relative to animal cngc CNBDs) alphaC helix. The Atcngc2 CaMBD was expressed as a fusion peptide linking blue and green fluorescent proteins, and used to quantify CaM (A. thaliana CaM isoform 4) binding. CaM bound the fusion protein in a Ca2+-dependent manner with a K-d of 7.6 nM and a Ca2+ binding K-d of 200 nM. Functional characterization (voltage clamp analysis) of Atcngc2 was undertaken by expression in human embryonic kidney cells. CaM reversed cAMP activation of Atcngc2 currents. This functional interaction was dependent on free cytosolic Ca2+. Increasing cytosolic Ca2+ was found to inhibit cAMP activation of the channel in the absence of added CaM. We conclude that the physical interaction of Ca2+/CaM with plant cngcs blocks cyclic nucleotide activation of these channels. Thus, the cytosolic secondary messengers CaM, cAMP, and Ca2+ can act in an integrated fashion to gate currents through these plant ion channels. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.