Minisymposium 12: Intracellular Signaling

Abs # 24004: CNGC2-CaM interaction, a paradigm for Ca²⁺ and cNMP-modulated signaling in plants

Presenter:	Zielinski, Ray , zielinsk@life.uiuc.edu
Authors	Zielinski, Ray  (A)   Berkowitz, Gerry  (B)   Lemtiri-Chlieh, Fouad  (B)   Mercier, Richard  (B)   Ditzler, Mark  (A)   Harish, Ajith  (A)
Affiliations:	(A): Dept. of Plant Biology, University of Illinois (B): Dept. of Plant Sciences, University of Connecticut

Arabidopsis cyclic nucleotide (cNMP)-gated channel, AtCNGC2/dnd1, conducts cations and mediates hypersensitive cell death in response to pathogens. However, the mechanism by which it acts is not understood. Calmodulin (CaM) and cNMP bind AtCNGC2 and regulate its activity. All CNGCs have cytoplasmic cNMP-binding domains (CNBD) at their carboxy-termini. Unlike neuronal CNGCs, a portion of the plant CNGC CNBD also acts as a CaM binding domain (CaM-BD). Functional analysis of plant CNGCs was undertaken in AtCNGC2-transfected HEK cells and in native Arabidopsis cell membranes. An inward rectified Ca²⁺ current in native mesophyll and guard cells was activated by cAMP. Activation of inward Ca²⁺ current by cAMP was also demonstrated in membrane patches and was reversed upon removal of cAMP, results consistent with the presence of functional CNGCs in planta. Ca²⁺ entry into guard cells is a critical component of signaling stomatal closure. GdCl₃ blocks inward Ca²⁺ currents in guard cells; we found this inhibitor also blocks these cAMP-activated currents. CaM reversed cAMP-activated AtCNGC2 (whole cell) currents in HEK cells in a Ca²⁺-dependent manner. These results are consistent with competitive binding of CaM and cNMP modulating the activity of AtCNGC2. To better understand AtCNGC2's regulation and its role in the hypersensitive response we measured CaM binding to the channel's CaM-BD, with the goal of decoupling cNMP and CaM regulation of the channel by altering its affinity for CaM. Fluorescent indicator proteins comprising GFP, the CNGC2 CaM-BD and BFP as well as split cameleons consisting of GFP-CNGC2 CaM-BD and BFP-CaM were used to measure CNGC2-CaM interaction. Site directed mutations in the CaM-BD were constructed and expressed that enabled us to examine the roles of strongly hydrophobic residues and regions that influence the domain's propensity for a-helix formation and identify mutations that increase CaM's affinity for the CNGC2 CaM-BD and those that decrease its affinity. Current studies are aimed at introducing mutations into the intact channel coding sequence in order to assess their effect on cNMP regulation and channel activity in yeast and Arabidopsis. We are also introducing the fluorescent protein reporters into transgenic plants to determine whether they can be used to image CaM interaction with AtCNGC2 CaM-BD in vivo. Supported by USDA-NRI 2001-353-10927 and NSF 0090675.