title: 14-3-3 proteins double the number of outward-rectifying K+ channels available for activation in tomato cells
abstract: Outward-rectifying K+ channels are modulated in response to environmental stimuli by a range of intracellular factors, such as cytoplasmic Ca2+, pH, kinases and phosphatases. Here we report that voltage-dependent outward-rectifying 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 protein (tomato isoform TFT7) was introduced into tomato cell protoplasts via the patch pipette. As a result the steady-state outward K+ current increased twofold and this increase was not dependent upon the presence of cytoplasmic ATP. A phosphorylated peptide that contained a phosphorylated 14-3-3 target-binding motif (RSTS*TP), derived from nitrate reductase, blocked the effect of 14-3-3, thus showing the specific nature of 14-3-3 action. Kinetic parameters of the conductance, like (de)activation kinetics, voltage dependence of gating and activation potential, were not significantly different between 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 'sleepy' channels into a voltage-activatable state. The molecular mechanism underlying the 1 : 1 ratio of constitutively active and 14-3-3 recruited channels is discussed in the light of known functions of 14-3-3 dimers.
authors.affiliation: 1Department of Developmental Genetics, Vrije Universiteit, BioCentrum Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands, 2Department of Structural Biology, Vrije Universiteit, BioCentrum Amsterdam, De Boelelaan 1087, 1081.
authors.initials: PP
authors.lastname: Booij
authors.initials: MR
authors.lastname: Roberts
authors.initials: SA
authors.lastname: Vogelzang
authors.initials: R
authors.lastname: Kraayenhof
authors.lastname: De Boer AH