Plant Cyclic Nucleotide Gated Ion Channels; Structure: Function Analysis of a Newly Identified and Unique Family of Proteins

University Of Connecticut, Storrs CT

Investigators

Abstract

This laboratory has recently cloned and undertaken the first functional characterization of Arabidopsis thaliana cyclic nucleotide (cNMP) gated K-conducting ion channel ('AtCNGC2'). Cyclic nucleotide gated (nonselective) cation channels (cngc's) (in animals) are ligand-gated, conduct K, Na, and Ca, and are regulated by cytosolic levels of Ca, calmodulin, and cyclic nucleotides. Cngc's typically function in signal transduction pathways, providing a mechanism by which external signal perception invokes signaling cascades that in turn alter cellular functions. The role that cAMP and cGMP play in specific signal transduction systems in animals is well known. However, their involvement in specific signal transduction systems in plants is not well understood. AtCNGC2 is apparently a member of a large gene family in plants: Database searches have identified 10 sequences in the A. thaliana genome which encode putative cngc's. The deduced amino acid sequences of these plant cngc's differ from the animal cngc sequences in regions that are critical for function. The electrophysiological properties of this plant class of ion channels have not been elucidated. AtCNGC2 and other plant cngc's will be expressed in heterologous systems amenable to patch/voltage clamp analysis (Xenopus laevis oocytes, and/or HEK293 cell cultures). Some of the AtCNGC2 homologs will be expressed in oocytes for functional characterization. The primary objective of this work will be an electrophysiological structure-function analysis of AtCNGC2 and homologs, focusing firstly on the selectivity filter of the pore and secondly on the cyclic nucleotide binding domains of these channels. These studies will include site-directed mutagenesis of AtCNGC2, followed by voltage clamp analysis of oocytes expressing the mutated channels. A detailed analysis will be undertaken of differences in ion selectivity profiles of these channels, and also differences in affinity for cAMP and cGMP. This research objective should allow for a comparison of how differences in the primary protein structure of members of this plant channel family correlate with differences in function and regulation. As other members of this large gene family are cloned, Northern analyses using coding sequences as a probe will be undertaken to monitor expression patterns of different members of this gene family in Arabidopsis. This research project will generate new information about one of the most intensively studied areas of ion channel characterization: The molecular basis of ion selectivity. A landmark study by others led to the first X-ray crystallographic analysis of a K-selective ion channel. This pioneering work confirmed that a triplet of amino acids ('GYG') in the pore selectivity filter of K channels is absolutely required for channel conductance to favor K over Na. Modeling of the AtCNGC2 pore selectivity filter indicates that the amino acid triplet 'AND' is associated with this unique channel's ability to conduct K and specifically select against Na, indicating that AtCNGC2 selects for K over Na conductance in a manner heretofore unknown in biology! This finding suggests that current understanding of the molecular basis for ion selectivity profiles of channels should be reappraised. The significance of this project is supported by the following: a) the primary sequence differences amongst this channel family in functional domains suggests different channel properties; b) the availability of a large family of cloned plant channels for study at the molecular level is unprecedented; c) the channel properties (e.g. K and Ca conductance, but not Na) may be a unique biological paradigm that may provide valuable insights into how protein architecture affects channel properties; and d) this family of proteins requires cAMP and cGMP for activation and these studies may provide insights into how cyclic nucleotides regulate ion channels in plant cells.