Minisymposium 24: Membrane transport

Abs # 45005: Plant CNGCs: functional characterization of plant cation channels using native membranes, mutant plants, and heterologous expression systems

Presenter:	Berkowitz, Gerald A, Gerald.Berkowitz@uconn.edu
Authors	Berkowitz, Gerald A (A)   Loder, Laura  (A)   Ali, Rashid  (A)   Lemtiri-Chlieh, Fouad  (A)   Salt, David E (B)
Affiliations:	(A): University of Connecticut (B): Purdue University

Plant cyclic nucleotide gated nonselective cation channels (CNGCs) conduct external Ca, Na, and/or K into cells. Plant CNGCs are candidates for involvement in plant physiological functions related to cytosolic Ca signaling. Some CNGC isoforms may also contribute to Na uptake into plants; impacting growth under saline conditions. Here, we characterize some functional properties of two of the twenty Arabidopsis CNGC isoforms (AtCNGC1 and 2). Patch clamp analysis demonstrated direct activation by cAMP of an inward Ca current in Arabidopsis leaf mesophyll and guard cell protoplasts, results consistent with the presence of functional CNGC channel complexes in plant cell membranes. AtCNGC2 is highly expressed in the leaf mesophyll. An Arabidopsis AtCNGC2 mutant accumulated less Ca in the leaf than wild type (WT) plants; this difference was accentuated at high growth medium Ca. In contrast to AtCNGC2, AtCNGC1 is highly expressed in Arabidopsis roots. AtCNGC1 mutant plants accumulated slightly less Na in leaves than WT plants. AtCNGC1 mutant seed germination and seedling growth was less sensitive than WT plants to increasing growth medium Na. The above CNGC mutant plant phenotypes are consistent with AtCNGC2 involvement in Ca movement into plant cells, and AtCNGC1 as contributing to Na uptake into plant roots. Preliminary studies suggest root-expressed AtCNGC1 may also be involved in the gravitropic signal transduction pathway. In prior work from this lab, plant CNGCs were functionally characterized by expression in animal cell cultures and frog oocytes. Here, we employed other heterologous systems for channel characterization; K uptake deficient yeast and Escherichia coli mutants. Green fluorescent protein (GFP):AtCNGC fusion protein constructs demonstrated expression and insertion into the cell membrane of yeast. A number of AtCNGC isoforms were found to enhance growth at low K of a K uptake deficient yeast mutant. In some cases, removal of the calmodulin binding domain from the carboxy-terminus of the AtCNGC coding sequence enhanced growth. This result suggests that endogenous calmodulin in heterologous systems may be binding to CNGCs and preventing cyclic nucleotide activation of these ligand gated channels. Consistent with this, removal of the calmodulin binding domain of AtCNGC1 did not enhance growth of a K uptake-deficient E. coli mutant; do not express any endogenous calmodulin protein. Supported by NSF award 0344141.