Poster: Membrane transport

Abs # 178: Use of yeast as a heterologous expression system for electrophysiological analysis of plant CNGC channels

Presenter:	Lemtiri-Chllieh, Fouad , flemtiri@canr.uconn.edu
Authors	Lemtiri-Chllieh, Fouad  (A)   Ali, Rashid  (A)   Berkowitz, Gerald  (A)
Affiliations:	(A): University of Connecticut

Plant cyclic nucleotide gated cation channels are inhibited by calmodulin (CaM) and activated by cAMP. Functional study of these channels has been limited by the recalcitrance of their expression in heterologous systems amenable to patch clamp analysis. Here, we report the expression of a deletion mutant of the Arabidopsis CNGC AtCNGC1 in yeast for electrophysiological analysis. Our results indicate the following. AtCNGC1 mRNA is generated in yeast. GFP-fusion protein expression studies indicate the recombinant plant protein is targeted to the yeast cell membrane. Suppression of a K-uptake mutation phenotype by expression of AtCNGC1 in the trk1,2 yeast mutant suggests the channel is functional in yeast. We increase AtCNGC1 function in this yeast mutant by deletion of the CaM binding domain; perhaps reducing yeast CaM inhibition of the channel. In preliminary patch clamp studies, we observe a change in channel activity upon addition of cAMP in a tetraploid wild type yeast cell transfected with this mutant AtCNGC1 construct. Further work is ongoing to characterize this inward current in the trk1,2 mutant transformed with deletion mutant of AtCNGC1. These results suggest that this system may provide a new approach to studying these plant channels. Supported by NSF award 0344141.