13002:

Cloning and functional characterization of a new class of plant ion channels activated by cyclic nucleotide.

Authors:	Leng, Qiang(A)Mercier, Richard(A)Berkowitz, Gerald, A.(A)
Affiliations:	(A): Department of Plant Science, University of Connecticut
Presenter:	Berkowitz, Gerald A., gberkowi@canr1.can.uconn.edu

Cyclic nucleotides (cNMP) act in animal cell signal transduction pathways by activating cyclic nucleotide-gated cation channels (cngc's) in sensory cells such as olfactory and taste receptors, and retinal rod and cone cells. Cngc's act in signal transduction cascades by facilitating Ca entry into animal cells, which can transduce signal perception by altering membrane potential and/or cytosolic [Ca]. We have cloned an Arabidopsis cDNA ('Atcngc1') which shows sequence homology to, and shares some structural features with animal cngc's. Atcngc1 was shown to function as a cNMP-activated cation transporter in two independent heterologous expression systems. Atcngc1 was found to complement growth of a K uptake-deficient yeast mutant only in the presence of (lipophilic) dibutyryl-cAMP or cGMP. Atcngc1 cRNA expression in Xenopus laevis oocytes induced inward rectified K currents at hyperpolarizing step voltages. Addition of 10 mM cAMP or cGMP dramatically increased whole cell currents, and shifted the activation threshold to less negative (< -150 mV) voltages; in a plant cell with a typical resting potential of ~ -120 to -150 mV, generation of cytosolic cAMP should effectively activate currents through this channel. Atcngc1 was found to conduct K; and to a much less extent Ca, Li, Rb, and Cs, but not Na. Animal cngc's are typically nonselective cation channels which do conduct Na. Like animal cngc's, Atcngc1 has cNMP and calmodulin binding sites. However, conversely to animal cngc's, these binding sites are located in tandem and are downstream from the transmembrane regions in Atcngc1. The capacity for Ca conductance, cAMP activation, and calmodulin binding suggests that these cytosolic secondary messengers may mediate signal transduction cascades by acting in concert to modulate Atcngc1 currents. Ca/calmodulin binding to Atcngc1 may restrict cAMP activation of the channel, and provide a mechanism for inward Ca currents facilitated by Atcngc1 to feedback inhibit activation. We are currently initiating cell-detached patch clamp analysis to evaluate the interplay between cAMP, Ca, and calmodulin in the gating of Atcngc1 currents. In addition, we are cloning two Arabidopsis cDNAs with sequence homology to Atcngc1 and may comprise a newly characterized family of plant ion channels capable of acting in signal transduction pathways. Supported by NSF grants MCB-9513921 and BIR-9512977 and DOE grant DE-FG02-95ER20202.