KDC1, a carrot Shaker-like potassium channel, reveals its role as a silent regulatory subunit when expressed in plant cells
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The Shaker potassium channels are tetrameric proteins formed by the assembly of four α-subunits. The oligomerization can occur among both homo- and hetero-α-subunits. KDC1 is a carrot Shaker-like potassium channel expressed in the epidermis of plantlet roots and the protoderm of somatic embryos. KDC1 was previously characterised electrophysiologically in CHO and Xenopus oocytes cells, but the experiments performed in these systems did not provide conclusive evidence that KDC1 forms a functional homomeric channel in plant cells. In this report, we show that KDC1 localizes to the plasma membrane of root cells in transgenic tobacco plants transformed with a KDC1∷GFP fusion construct. In tobacco mesophyll protoplasts, transiently transformed with KDC1∷GFP, KDC1 was present on the endomembrane and the protoplasts did not show any inward potassium current, as demonstrated by patch-clamp experiments. The co-expression of KDC1∷GFP with the Arabidopsis thaliana potassium channel AKT1 in tobacco mesophyll protoplasts has the effect of shifting KDC1 localization from endomembranes to the plasma membrane. Patch-clamp experiments performed on tobacco mesophyll protoplasts expressing AKT1 alone or in combination with KDC1∷GFP showed voltage-activated inward potassium currents with different properties. In particular, the addition of Zn2+ to the bath solution induced a clear decrease of the potassium currents in protoplasts transformed with AKT1 alone, whereas a current potentiation (indicative of KDC1 presence) was observed in protoplasts co-transformed with AKT1 + KDC1∷GFP. Split-Ubiquitin assay experiments performed in yeast cells confirmed the interaction between AKT1 and KDC1.
KeywordsCarrot Shaker Root GFP Plasma membrane Split-Ubiquitin
We thank G. Duby, H. Sentenac and J-B. Thibaud for providing us pFunct-AKT1 and pFunct-GFP vectors. We thank Dr. I. Szabo’, Dr. M. Zoratti and Dr. U. De Marchi for providing their instrumentation for patch-clamp experiments in Padova. This research was supported by the “Ministero dell’Istruzione e della Ricerca”, “Fondi per gli Investimenti della Ricerca di Base” to F.G. and F.L.S, Project N-RBAUO183A9 and by the US Department of Energy (DOE-DE-FG02-03ER15449) to J.I.S.
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