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Members of the highly conserved family of 14-3-3 proteins play an important role in various cellular processes. Recent studies, using electrophysiological techniques, show that the 14-3-3 proteins also regulate plasma membrane and vacuolar K+ conducting channels. The molecular mechanism behind the regulatory effect of 14-3-3 proteins on K+ channels remains to be shown. One vacuolar channel down-regulated by 14-3-3 proteins is the slow-activating vacuolar (SV) channel. In Arabidopsis, the protein coded by the KCO1 gene was recently shown to be present in the vacuolar membrane and identified as a component of the SV channel. These two observations raised the question whether the KCO1 protein does interact with 14-3-3 proteins. Therefore, we isolated the barley HvKCO1 gene and the encoded protein indeed contains a canonical 14-3-3 interaction motif, which is conserved in all other KCO1 orthologues from other plant species. Using surface plasmon resonance (SPR) we determined in real-time the affinity between the phospho-peptide derived from the putative KCO1 14-3-3 interaction motif and three barley 14-3-3 proteins. The 14-3-3A protein showed the highest affinity, whereas the binding of all three isoforms was dependent on the presence of either Ca2+ or Mg2+. Interestingly, the barley SV current was strongly reduced by 14-3-3B and C protein, but not by 14-3-3A. This difference between the SPR and patch-clamp data will be discussed, along with the role for Ca2+ in activation of the SV channel by direct interaction and inactivation of the channel by facilitating the binding of 14-3-3 to the channel. |
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