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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.

Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K(+)) is essential. Therefore, we have addressed the question of how the activity of K(+) permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K(+) uptake and reduces the activity of K(+) (in) channels as measured with the patch-clamp technique. In contrast, fusicoccin (FC), a well-known stimulator of germination, stimulates radicle growth, net K(+) uptake and reduces the activity of K(+) (out) channels. Both types of channels are under the control of 14-3-3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14-3-3 affected both channels in an opposite fashion: whereas K(+) (in) channel activity was fully dependent upon 14-3-3 proteins, K(+) (out) channel activity was reduced by 14-3-3 proteins by 60%. Together with previous data showing that 14-3-3 proteins control the activity of the plasma membrane H(+)-ATPase, this makes 14-3-3 a prime candidate for molecular master regulator of the cellular osmo-pump. Regulation of the osmo-pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.

In addition to their regulation of cytoplasmic enzymes, the 14-3-3 proteins are important regulators of membrane localised proteins. In particular, many of the cells' ion pumps and channels are either directly or indirectly modulated by 14-3-3 proteins. Binding of 14-3-3 can lead to the activation of pump activity as in the case of the plasma membrane H+-ATPase or inhibition as in the case of the F-type ATP synthase complexes. 14-3-3 binding can also lead to surprising results such as the recruitment of 'sleepy' outward rectifiying K+ channels in tomato cells. Our present knowledge extends to an initial understanding of isoform-specific binding of 14-3-3 to certain membrane proteins and a perception of the protein kinases and phosphatases that maintain the regulatory process in a state of flux.

The bradykinin-induced rise in intracellular Ca 2+ concentration ([Ca 2+ ] i ) and the bradykinin receptor involved in this response were characterized in bovine pulmonary artery endothelial cells. It was found that bradykinin induces an intracellular biphasic Ca 2+ response, consisting of a transient peak followed by an elevated plateau phase. Both bradykinin and the bradykinin B 1 receptor agonist, des-Arg 9 -bradykinin, induced a concentration-dependent increase in [Ca 2+ ] i , but the bradykinin-induced rise was much greater. Moreover, the bradykinin-induced [Ca 2+ ] i rise could be inhibited by the bradykinin B 2 receptor antagonists, d -Arg 0 [Hyp 3 , Thi 5,8 , d -Phe 7 ]bradykinin and Hoe 140 ( d -Arg[Hyp 3 , Thi 5 , d -Tic 7 , Oic 8 ]bradykinin), but not by the bradykinin B 1 receptor antagonist, des-Arg 9 -[Leu 8 ]bradykinin. From these results it can be concluded that a bradykinin B 2 receptor is involved in this response. Furthermore, we found that the tachykinin NK 1 receptor antagonist, RP67580 ([imino 1 (methoxy-2-phenyl)-2 ethyl]-2 diphenyl 7,7 perhydroisoindolone-4 (3aR, 7aR)), and its negative enantiomer, RP68651 (2-[1-imino 2-(2 methoxy phenyl) ethyl] 7,7 diphenyl 4-perhydroisoindolone (3aS–7aS)), could inhibit the bradykinin-induced [Ca 2+ ] i response, although no functional tachykinin NK 1 receptors were found. Binding studies evidenced no binding of RP67580 or RP68651 to the bradykinin receptor. We conclude that RP67580 inhibits the bradykinin-induced rise in [Ca 2+ ] i via a bradykinin B 2 receptor-independent mechanism.

The conductance of the vacuolar membrane at elevated cytosolic Ca2+ levels is dominated by the slow activating cation selective (SV) channel. At physiological, submicromolar Ca2+ concentrations the SV currents are very small. Only recently has the role of 14-3-3 proteins in the regulation of voltage-gated and Ca2+-activated plasma membrane ion channels been investigated in Drosophila, Xenopus and plants. Here we report the first evidence that plant 14-3-3 proteins are involved in the down-regulation of ion channels in the vacuolar membrane as well. Using the patch-clamp technique we have demonstrated that 14-3-3 protein drastically reduces the current carried by SV channels. The current decline amounted to 80% and half-maximal reduction was reached within 5 s after 14-3-3-addition to the bath. The voltage sensitivity of the channel was not affected by 14-3-3. A coordinating role for 14-3-3 proteins in the regulation of plasma membrane and tonoplast ion transporters is discussed.