New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency
1997; Springer Nature; Volume: 16; Issue: 10 Linguagem: Inglês
10.1093/emboj/16.10.2565
ISSN1460-2075
Autores Tópico(s)Ion Channels and Receptors
ResumoArticle15 May 1997free access New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency Katrin Czempinski Katrin Czempinski Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl–Liebknecht-Str. 25, Haus 20, D-14476 Golm, GermanyK.Czempinski and S.Zimmermann contributed equally to this work Search for more papers by this author Sabine Zimmermann Sabine Zimmermann Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl–Liebknecht-Str. 25, Haus 20, D-14476 Golm, GermanyK.Czempinski and S.Zimmermann contributed equally to this work Search for more papers by this author Thomas Ehrhardt Thomas Ehrhardt Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl–Liebknecht-Str. 25, Haus 20, D-14476 Golm, Germany Search for more papers by this author Bernd Müller-Röber Corresponding Author Bernd Müller-Röber Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl–Liebknecht-Str. 25, Haus 20, D-14476 Golm, Germany Search for more papers by this author Katrin Czempinski Katrin Czempinski Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl–Liebknecht-Str. 25, Haus 20, D-14476 Golm, GermanyK.Czempinski and S.Zimmermann contributed equally to this work Search for more papers by this author Sabine Zimmermann Sabine Zimmermann Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl–Liebknecht-Str. 25, Haus 20, D-14476 Golm, GermanyK.Czempinski and S.Zimmermann contributed equally to this work Search for more papers by this author Thomas Ehrhardt Thomas Ehrhardt Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl–Liebknecht-Str. 25, Haus 20, D-14476 Golm, Germany Search for more papers by this author Bernd Müller-Röber Corresponding Author Bernd Müller-Röber Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl–Liebknecht-Str. 25, Haus 20, D-14476 Golm, Germany Search for more papers by this author Author Information Katrin Czempinski1, Sabine Zimmermann1, Thomas Ehrhardt1 and Bernd Müller-Röber 1 1Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl–Liebknecht-Str. 25, Haus 20, D-14476 Golm, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:2565-2575https://doi.org/10.1093/emboj/16.10.2565 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Potassium (K+) channels mediating important physiological functions are characterized by a common pore–forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of 'two-pore' K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium-binding EF-hand motifs. Heterologous expression of KCO1 in baculovirus-infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+-selective currents elicited by depolarizing voltage pulses in whole-cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to 28 000 ESTs deposited; September 1996), using the complete kco1 sequence as bait, did not result in the identification of additional ESTs (besides H76771, see above). Taken together, kco1 appears to represent only >Cl−). The replacement of K+ by other monovalent cations and tail current analysis resulted in the selectivity sequence: K+ (1)>NH4+ (0.3)>Na+(0.16)>Li+ (0.1) (n = 3–6). Conductance–voltage plots revealed a shift of the activation potential with external K+, saturating at a concentration range of 10–30 mM K+ (Figure 4E). In the absence of external divalent cations (i.e. in Ca2+- and Mg2+-free bath solution), KCO1-expressing insect cells still displayed outward rectifying K+ currents (n = 4; data not shown), indicating that the rectification properties were intrinsic to the KCO1 channel protein (see Discussion). Ba2+, described as an inhibitor of K+ outward channels in a series of studies on plant protoplasts (see, e.g. Schroeder et al., 1987; Thomine et al., 1994), was tested on Sf9 and Sf21 cells displaying stable KCO1 currents (see Figure 5A for a representative Sf21 cell). Inhibition by ∼90% of the positive current amplitudes was observed rapidly after addition of 5 mM Ba2+ (n = 6; Figure 5B and C). A dose–response curve (0.5–10 mM Ba2+) revealed a half-maximal inhibition (IC50) of 3.8 mM (Figure 5D). The Ba2+ effect was not reversible after inhibition of KCO1 currents with 5 mM Ba2+ and perfusion with up to 15 volumes of bath solution (n = 4), indicating strong binding of the ion to the pore. Figure 5.Inhibition of kco1-induced currents by external Ba2+. (A) Control currents of a representative Sf21 cell expressing KCO1 in standard solution. (B) Reduced current amplitudes in response to depolarizing voltage pulses after application of 5 mM Ba2+ (representative of six cells). (C) Current–voltage relationship before and 15 s and 1 min after addition of Ba2+, respectively. (D) Dose–response curve of inhibition of KCO1 currents by external Ba2+. Experimental data (n = 3–6 for each Ba2+ concentration) were fitted with a Michaelis–Menten equation. Download figure Download PowerPoint Taken together, these data identified KCO1 as the first outward rectifying K+ channel cloned from higher plants. KCO1 requires nanomolar concentrations of cytosolic free Ca2+ for activity and shows a very steep Ca2+ dependency The structural analysis performed on KCO1 indicated the presence of tandem EF-hands within the protein's C-terminus (see above). We were interested, therefore, in seeing whether KCO1 shows Ca2+-dependent activation kinetics. All results described above for KCO1 expressed in insect cells were obtained using a pipet solution buffered to 100 μM Ca2+ (see Materials and methods). Reduction of cytosolic free calcium to 1 μM (not shown) or 500 nM still allowed the detection of positive activating currents (Figure 6A, lower panel). Further reduction of [Ca2+]cyt to 100 nM (Figure 6A, upper panel) or a pipet solution without Ca2+ prevented voltage activation of KCO1 (Figure 6B and 6C), indicating Ca2+ dependency of this outward rectifier. To characterize KCO1 further, we determined channel activity at intermediate Ca2+ concentrations, i.e. with [Ca2+]cyt buffered to 150, 175, 200 and 300 nM, respectively. The results of these experiments are summarized in Figure 6B and 6C. As is evident, KCO1 shows a very steep Ca2+ dependency with maximal channel activity already at ∼300 nM [Ca2+]cyt. Fitting these data with a sigmoidal function revealed a half-maximal activation concentration of ∼200 nM [Ca2+]cyt. Figure 6.Ca2+-dependent activation of outward rectifying K+ currents in insect cells expressing KCO1. (A) Current responses of representative Sf21 cells with 100 nM (upper panel) and 500 nM [Ca2+]cyt (lower panel), respectively. Currents were elicited by 300 ms depolarizing pulses from −90 to +90 mV in 10 mV steps from a holding potential of −20 mV. (B) Current–voltage relationships in the presence of different concentrations of intracellular free Ca2+ (n = 4–7). (C) Mean current amplitudes (±SE) at a membrane potential of +10 mV, extracted from data presented in (B), dependent on [Ca2+]cyt. Experimental data were fitted by a logistic function using SigmaPlot software (Jandel Corp.). Note that no currents were observed when [Ca2+]cyt was buffered to <150 nM. (D) Normalized conductance–voltage plot indicates a shift of the activation potential dependent on [Ca2+]cyt. Data were fitted as described in the legend to Figure 4E. ○, 500 nM Ca2+; ▵, 175 nM Ca2+. Download figure Download PowerPoint Rather large variations of maximal current amplitudes were observed in individual insect cells expressing KCO1 under elevated [Ca2+]cyt (see e.g. Figure 6C). However, no current above background was detected in any of the insect cells when [Ca2+]cyt was buffered to 10 min. Download figure Download PowerPoint Membrane patches from uninfected control cells did not display single channel activities in the absence or presence of Ca2+ (500 nM) under the conditions applied (n = 5; data not shown). In KCO1-expressing cells, no single channel activities could be resolved when Ca2+ was omitted from the bath solution (n = 11), whereas a rise in the Ca2+ concentration towards 200–500
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