Physical and Functional Interaction of the Arabidopsis K⁺ Channel AKT2 and Phosphatase AtPP2CA

Abstract

The AKT2 K ؉ channel is endowed with unique functional properties, being the only weak inward rectifier characterized to date in Arabidopsis. The gene is expressed widely, mainly in the phloem but also at lower levels in leaf epiderm, mesophyll, and guard cells. The AKT2 mRNA level is upregulated by abscisic acid. By screening a two-hybrid cDNA library, we isolated a protein phosphatase 2C (AtPP2CA) involved in abscisic acid signaling as a putative partner of AKT2. We further confirmed the interaction by in vitro binding studies. The expression of AtPP2CA ( ␤ -glucuronidase reporter gene) displayed a pattern largely overlapping that of AKT2 and was upregulated by abscisic acid. Coexpression of AtPP2CA with AKT2 in COS cells and Xenopus laevis oocytes was found to induce both an inhibition of the AKT2 current and an increase of the channel inward rectification. Site-directed mutagenesis and pharmacological analysis revealed that this functional interaction involves AtPP2CA phosphatase activity. Regulation of AKT2 activity by AtPP2CA in planta could allow the control of K ؉ transport and membrane polarization during stress situations. INTRODUCTIONPotassium is the most abundant cation in the cytoplasm of the living cell, where it is involved in the regulation of ionic strength, osmotic potential, and membrane polarization. K ϩ channels of the so-called Shaker family (nine genes in Arabidopsis) have been shown to play a role in K ϩ uptake by the root periphery (AKT1: Lagarde et al., 1996; Hirsch et al., 1998), K ϩ secretion into the root xylem sap (SKOR: Gaymard et al., 1998), K ϩ transport in the phloem tissues (AKT2: Marten et al., 1999;Lacombe et al., 2000), or K ϩ inward (KAT1: Ichida et al., 1997;Szyroki et al., 2001; KAT2: Pilot et al., 2001) and outward (GORK: Ache et al., 2000) fluxes in guard cells, leading to stomatal opening/closing.To adapt to fluctuating K ϩ availability in the environment and to cope with other stresses, plants need to tightly regulate K ϩ transport at both the whole plant and the cell level (Kochian and Lucas, 1988;Schroeder et al., 1994). Studies aimed at revealing the molecular determinants of these regulations have highlighted mechanisms likely to target Shaker K ϩ channels at both the transcriptional and posttranslational levels. Expression studies have revealed that transcript levels of Shaker channels are sensitive to hormones (Gaymard et al., 1998;Philippar et al., 1999;Lacombe et al., 2000), sugar synthesis and accumulation, and environmental signals (Deeken et al., 2000). At the post-translational level, indications have been found for regulation by ATP and cyclic GMP (Hoshi, 1995), phosphorylation events (Li et al., 1994; Armstrong et al., 1995; Tang and Hoshi, 1999), functional interactions with the cytoskeleton (Hwang et al., 1997), 14-3-3 proteins (Saalbach et al., 1997 Booij et al., 1999), sulfonylurea receptors (Leonhardt et al., 1997), syntaxins (Leyman et al., 1999), and G proteins (Wu andWang et al., 2001).Searches for interacting proteins have been focused on t...

Increased Functional Diversity of Plant K+ Channels by Preferential Heteromerization of the Shaker-like Subunits AKT2 and KAT2

“…Yeast Two-hybrid Assays-The C-terminal region of AKT2 was previously cloned in pGBT9 (48), yielding the (bait) construct named pGBT9-AKT2. The plasmids used for the expression of AKT2 and KAT2 C-terminal regions fused to the GAL4 activation domain (prey constructs) are the previously described plasmids pACT2-AKT2 and pACT2-KAT2 (36,37).…”

“…These constructs were used to screen an Arabidopsis thaliana cDNA library prepared with the GAL4 activation domain plasmid pGAD10 (MATCHMAKER, Clontech), using for transformation the lithium acetate method, as described in Chérel et al (48). Transformed cells were spread directly on minimal medium lacking histidine, leucine, and tryptophane (plating medium described in Ref.…”

Potassium channel α-subunit AtKC1 negatively regulates AKT1-mediated K+ uptake in Arabidopsis roots under low-K+ stress

“…Previous studies have shown that posttranslational regulation is an important mechanism for regulation of K + channel activities in plant cells [13,[38][39][40]. One of the examples is the regulation of AKT1 activity by a protein kinase AtCIPK23.…”

The Nuclear Interactor PYL8/RCAR3 ofFagus sylvaticaFsPP2C1 Is a Positive Regulator of Abscisic Acid Signaling in Seeds and Stress

“…These substrates include transcription factors involved in the drought/stress response (Himmelbach et al, 2002), protein kinases such as OST1/SnRK2.6 (Yoshida et al, 2006a), K + channels (Cherel et al, 2002), calcineurin B-like calcium sensors (Guo et al, 2002), a glutathione peroxidase (Miao et al, 2006), and a stress-induced photoprotective plastid protein .…”