The potassium channel KAT1 is activated by plant and animal 14-3-3 proteins.
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.
Sottocornola
Barbara
B
Dipartimento di Biologia and Istituto di Biofisica-Consiglio Nazionale delle Ricerche, Università degli Studi di Milano, 20133 Milano, Italy.
Visconti
Sabina
S
Orsi
Sara
S
Gazzarrini
Sabrina
S
Giacometti
Sonia
S
Olivari
Claudio
C
Camoni
Lorenzo
L
Aducci
Patrizia
P
Marra
Mauro
M
Abenavoli
Alessandra
A
Thiel
Gerhard
G
Moroni
Anna
A
D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D029681:Arabidopsis Proteins / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D002586:Cesium / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D015640:Ion Channel Gating; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry; D014981:Xenopus; D048490:raf Kinases / Q000737:chemistry
D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; C501210:KAT1 protein, Arabidopsis; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D002586:Cesium; D048490:raf Kinases