Plant Cyclic Nucleotide Gated Channels: Functional Characterization Using Cloned Channels and Native Plant Membranes

University Of Connecticut, Storrs CT

Investigators

Abstract

This project focuses on the molecular characterization of plant cyclic nucleotide gated nonselective cation channels (CNGCs). CNGCs act in signal transduction cascades, and also are involved in cation uptake into plants. Prior NSF-funded work on this project led to the first electrophysiological analyses of plant CNGCs, focusing on how their molecular architecture is related to differences in their ion conductance properties. Animal CNGCs (six genes in humans) all have similar pore architectures (in the ion selectivity filter region) and nonselectively conduct Ca, Na, as well as K. The situation with plant CNGCs is more complex; the 20 Arabidopsis CNGCs were modeled to have different ion selectivity filters. CNGCs were identified as the first cloned plant channels that conduct Ca (an important cytosolic signaling molecule) and Na (uptake into plants is a major limitation to crop growth). One of the channels was shown to have a pore architecture representing a heretofore unknown molecular paradigm allowing for discrimination between K/Na conduction. Prior work involved the application of heterologous expression systems (oocytes, cell cultures, and yeast mutants) for these characterizations. The new project continues this work, but expands the analysis of plant CNGCs so as to generate new information about how the cation conduction properties of plant CNGCs are related to nutrient uptake and movement within plants. Work underlying the project has led to the first voltage clamp analyses of CNGC currents in native plant membranes. This accomplishment provides a basis for the project to include studies using native membranes to characterize the electrophysiological properties of native CNGC protein complexes in plants, as well as structure/function studies of cloned plant CNGCs expressed in heterologous systems. Comparisons will be made between native CNGC currents in membranes of wild type, and mutant plants that lack specific CNGC genes. These electrophysiological analyses of native CNGCs will be undertaken along with studies of CNGC-dependent cation uptake and movement within plants, and analysis of CNGC mutant plant phenotypes when grown under various nutrient solution regimes. CNGC isoform-specific antibodies will be employed to generate new information about the subunit composition of native plant CNGCs and, along with expression profiling, allow for the dissection of how individual CNGCs contribute to cation fluxes in plants. Insight into the structure/function relationships of these proteins has been aided by three-dimensional modeling and site-directed mutagenesis of functional domains. Understanding protein structure/function through modeling is an important basis for students to understand biological systems at the molecular level. As part of a continuing outreach program to expose high school biology teachers to these insights, a teaching practicum will be developed and offered during summer semester breaks to undergraduate students who are preparing to be secondary school biology teachers. For each training session, ten of these students will be recruited and provided with stipends to participate in the two-week summer practicum that will train students to access web-based protein modeling software, and learn to work with simple biological systems (i.e. growth of yeast mutants to test protein function) so as to bring this information into the high school biology classroom. The broader impact of this project is the focus on a process of 'teaching the teachers' so that undergraduate students training as high school biology teachers can participate in the next horizon of biology in this post-genomic era; i.e. to gain an understanding of how protein structure is related to biological function.