title: AKT2/3 subunits render guard cell K+ channels Ca2+ sensitive.
abstract: Inward-rectifying K+ channels serve as a major pathway for Ca2+-sensitive K+ influx into guard cells. Arabidopsis thaliana guard cell inward-rectifying K+ channels are assembled from multiple K+ channel subunits. Following the recent isolation and characterization of an akt2/3-1 knockout mutant, we examined whether the AKT2/3 subunit carries the Ca2+ sensitivity of the guard cell inward rectifier. Quantification of RT-PCR products showed that despite the absence of AKT2 transcripts in guard cells of the knockout plant, expression levels of the other K+ channel subunits (KAT1, KAT2, AKT1, and AtKC1) remained largely unaffected. Patch-clamp experiments with guard cell protoplasts from wild type and akt2/3-1 mutant, however, revealed pronounced differences in Ca2+ sensitivity of the K+ inward rectifier. Wild-type channels were blocked by extracellular Ca2+ in a concentration- and voltage-dependent manner. Akt2/3-1 mutants lacked the voltage-dependent Ca2+ block, characteristic for the K+ inward rectifier. To confirm the akt2/3-1 phenotype, two independent knockout mutants, akt2-1 and akt2::En-1 were tested, demonstrating that the loss of AKT2/3 indeed affects the Ca2+ dependence of guard cell inward rectifier. In contrast to AKT2 knockout plants, AKT1, AtKC1, and KAT1 loss-of-function mutants retained Ca2+ block of the guard cell inward rectifier. When expressed in HEK293 cells, AKT2 channel displayed a pronounced susceptibility toward extracellular Ca2+, while the dominant guard cell K+ channel KAT2 was Ca2+ insensitive. Thus, we conclude that the AKT2/3 subunit constitutes the Ca2+ sensitivity of the guard cell K+ uptake channel.
authors.affiliation: Julius-von-Sachs Institute of Biosciences, Biocenter, Department of Molecular Plant Physiology and Biophysics, University of Würzburg, Germany.
authors.forename: Natalya
authors.initials: N
authors.lastname: Ivashikina
authors.forename: Rosalia
authors.initials: R
authors.lastname: Deeken
authors.forename: Susanne
authors.initials: S
authors.lastname: Fischer
authors.forename: Peter
authors.initials: P
authors.lastname: Ache
authors.forename: Rainer
authors.initials: R
authors.lastname: Hedrich
mesh_terms: D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000235:genetics / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials / Q000502:physiology; D016296:Mutagenesis; D018515:Plant Leaves / Q000502:physiology*; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000235:genetics / Q000502:physiology*
chemical_list: C097485:AKT2 protein, Arabidopsis; C431716:AKT3 protein, Arabidopsis; D029681:Arabidopsis Proteins; D015221:Potassium Channels; D011188:Potassium; D002118:Calcium