Poster: Membrane Transport
Abs #
1213: Functional characterization of inwardly K-conducting plant cyclic nucleotide gated channels using a trk1, trk2 yeast mutant
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Presenter: |
Rabinowitz, Natasha M, nrabinow@canr.cag.uconn.edu |
Authors | Rabinowitz, Natasha M (A) Mercier, Richard W (A) Gaxiola, Roberto A (A) Berkowitz, Gerald A (A) | | Affiliations: |
(A): University of Connecticut, Plant Science Department
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The Arabidopsis genome encodes 20 cyclic nucleotide gated channels (CNGCs). Here we evaluate inward K conduction of three of these channels (AtCNGC1,2 and 4) by heterologous expression in the trk1,2, K-uptake deficient yeast mutant. Channel function was demonstrated by complementing hygromycin B (Hyg) sensitivity of trk1,2 yeast growth around a filter disk containing 3 M KCl on solid YPD medium. trk1,2 yeast is hypersensitive to the toxic cation Hyg due to the hyperpolarized state of the cell cytosol; prior work has shown that K influx depolarizes the yeast cell cytosol, reducing Hyg uptake. At low (50 mg/L) [Hyg] in YPD, trk1,2 transformed with empty plasmid (pYES) grew only around the KCl filter disk (growth was dependent on [KCl] on the filter); higher [Hyg] (100 mg/L) was lethal at all [KCl] tested. The plant inward rectified K channel KAT1 has been shown previously to increase K uptake into trk1,2. We show trk1,2 transformed with pYES-KAT1 grew at 100 mg/L Hyg, demonstrating utility of this assay to identify function of inward K-conducting channels. Transformation of trk1,2 with AtCNGC1,2, and 4 also resulted in growth around the KCl filter disk with 100 mg/L Hyg in YPD. This growth complementation was enhanced by addition of 100 mM lipophilic dibutyryl-cAMP to the YPD medium. In contrast, growth of trk1,2 with empty plasmid on 50 mg/L Hyg, and trk1,2 with pYES-KAT1 was inhibited by addition of cAMP. These results demonstrate AtCNGCs as encoding cyclic nucleotide gated, inwardly K-conducting channels, and further, the utility of this assay for characterization of these plant channels. Functional expression of AtCNGCs in other heterologous systems (oocytes and HEK cells) is recalcitrant.
Supported by NSF award 0090675.
