Abstract
14-3-3 proteins modulate the plant inward rectifier K+ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by +11 mV and increased the tau(on). KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (approximately 70% at -150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.
Highlights
Voltage-dependent (Kv) potassium channels mediate an inward Kϩ current (IKin) at the plasma membrane of plant cells
Recombinant 14-3-3 Proteins Increase the Activity of KAT1 Channels Expressed in Xenopus Oocytes—Fig. 1A shows the current traces recorded from KAT1 channels expressed in a Xenopus oocyte
Recombinant 14-3-3 proteins from maize activate KAT1 channels expressed in Xenopus oocytes
Summary
Expression of KAT1 in Oocytes—KAT1 cDNA was cloned into pSGEM vector (a modified version of pGEM-HE). cRNA was transcribed in vitro using T7 RNA polymerase (Promega) and injected (25 ng/oocyte) into Xenopus laevis oocytes prepared according to standard methods [22]. 40 control or KAT1-expressing oocytes were homogenized in 800 l of 20 mM Tris-HCl, pH 7.4, containing 5 mM MgCl2, 5 mM NaH2PO4, 1 mM EDTA, 80 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, and 5 g/ml pepstatin and centrifuged two times for 10 min at 3000 ϫ g at 4 °C to remove yolk proteins. The membrane was blocked with 5% fatty acid-free milk in 25 mM Hepes-OH, 75 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, 0.05% Tween 20, pH 7.5 (buffer H) and incubated overnight at 4 °C in the same buffer containing 2% fatty acid-free milk, 3 g of 32P-labeled GF14-6 The overlay experiment was performed three times, and similar results were obtained
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