14-3-3 proteins double the number of outward-rectifying K+ channels available for activation in tomato cells

Outward-rectifying K+ channels are modulated in response to environmental s timuli by a range of intracellular factors, such as cytoplasmic Ca2+, pH, k inases and phosphatases. Here we report that voltage-dependent outward-rect ifying K+ channels in tomato cells are also targets for modulation by 14-3- 3 proteins. In whole-cell patch-clamp experiments, recombinant 14-3-3 prote in (tomato isoform TFT7) was introduced into tomato cell protoplasts via th e patch pipette. As a result the steady-state outward K+ current increased twofold and this increase was not dependent upon the presence of cytoplasmi c ATP. A phosphorylated peptide that contained a phosphorylated 14-3-3 targ et-binding motif (RSTS*TP), derived from nitrate reductase, blocked the eff ect of 14-3-3, thus showing the specific nature of 14-3-3 action. Kinetic p arameters of the conductance, like (de)activation kinetics, voltage depende nce of gating and activation potential, were not significantly different be tween control and 14-3-3 infused cells. Analysis of single-channel activity and whole-cell noise indicated that the single-channel conductance was not affected by 14-3-3 infusion. We conclude that 14-3-3 proteins recruit 'sle epy' channels into a voltage-activatable state. The molecular mechanism und erlying the 1 : 1 ratio of constitutively active and 14-3-3 recruited chann els is discussed in the light of known functions of 14-3-3 dimers.