Immunosuppressants implicate protein phosphatase regulation of K+ channels in guard cells.

Abstract

The elevation of Ca2+ levels in the cytoplasm inactivates inward-rectifying K+ channels that play a central role in regulating the apertures of stomatal pores in higher plants. However, the mechanism for the Ca2+-mediated inhibition of K+-channel function is unknown. Using patch-clamp techniques, we show that cyclophilin-cyclosporin A and FK506-binding protein-FK506 complexes, which are highly specific inhibitors of protein phosphatase 2B (calcineurin), block Ca2+-induced inactivation of K+ channels in Vici4kfaba guard cells. A constitutively active calcineurin fragment that is Ca2+-independent inhibits K+-channel activity in the absence of Ca2 . We have also identified an endogenous Ca2 -dependent phosphatase activity from V. faba that is inhibited by the cyclophilin-cyclosporin A and FK506-binding protein-FK506 complexes. Our rmdings implicate a Ca2+-dependent, calcineurin-like protein phosphatase in a Ca2+ signaltransduction pathway of higher plants. Ca2+, an important second messenger in plant as well as in animal cells (1, 2), regulates the aperture of stomatal pores in higher plants (3, 4), thereby controlling CO2 uptake during photosynthesis and transpirational water loss (5, 6). Opening of stomatal pores is largely mediated by a rapid increase in the intracellular concentration of K+ in guard cells (7). Inward K+ channels and, hence, K+ influx in these cells are blocked by elevated concentrations of cytoplasmic Ca2+ (3). A possibly analogous system in animals is the dihydropyridinesensitive Ca2+ channel, which is inactivated by a Ca2+-dependent mechanism in excitable cells (8). It has been suggested that the Ca2+, calmodulin-dependent protein phosphatase calcineurin may limit Ca2+ influx through the neuronal cell membrane by dephosphorylating the Ca2+ channel, thereby inactivating it (9). Although Ca2 , calmodulindependent protein kinases and phosphatases have been well studied in animal systems (10, 11), our understanding of their possible role in higher plants is poorly developed.Recent investigations of signal-transduction pathways sensitive to the immunosuppressants cyclosporin A (CsA) and FK506 have provided a family of reagents that can be used to identify cellular functions of calcineurin (12). These immunosuppressants bind to their cellular receptors (immunophilins), thereby forming immunophilin-ligand complexes. Although FK506-binding protein (FKBP12)-FK506 and cyclophilin (CyP)-CsA complexes (but not their individual components) are potent and specific inhibitors of calcineurin, variants of these complexes, having only minor structural differences in the ligand, exhibit strikingly different behavior toward calcineurin and, therefore, toward calcineurinmediated pathways (13). Collectively, these reagents reveal a fingerprint of calcineurin that has been used to identify a role for this phosphatase in mediating Ca2+-dependent signal transmission from the T-cell receptor (12)(13)(14)(15)(16)(17)(18)(19). We have used patch-clamp techniques to monitor resultant alterations in ion-channel ac...

Signaling Events Leading to Crassulacean Acid Metabolism Induction in the Common Ice Plant

“…6C). CsA and FK506 block Ca 2ϩ -induced inactivation of guard cell K ϩ channels, which in turn prevents K ϩ efflux and stomatal closure (Luan et al, 1993). Thus, enhanced Ppc1 transcript accumulation in response to CsA may merely be an indirect effect of increased water loss due to a disruption in stomatal closure.…”

“…Pharmacological and biochemical studies support the apparent involvement of Ca 2ϩ -dependent calcineurin-like PP activity in regulating guard cell K ϩ channel activity (Luan et al, 1993;Allen and Sanders, 1995). Enhanced salt tolerance in transgenic tobacco plants expressing a reconstituted, constitutively active yeast calcineurin indicates that Ca 2ϩ /CaM-dependent calcineurin, as in yeast, are involved in osmotic stress adaptation in plants (Pardo et al, 1998).…”

“…Recent studies have implicated Ca 2ϩ /CaM-dependent PP2B (calcineurin) as playing a pivotal role in signal cascades involving guard cell behavior (Luan et al, 1993;Allen and Sanders, 1995) and salt stress adaptation (Pardo et al, 1998). Disruption of the Arabidopsis SOS3 gene, which encodes a protein resembling yeast calcineurin subunit B and animal calcium sensor proteins that stimulate PPs or inhibit protein kinases, results in hypersensitivity to Na ϩ and Li ϩ , suggesting that a signaling pathway involving calcineurin or related phosphatase activities regulates K ϩ and Na ϩ transport processes (Liu and Zhu, 1998).…”

Roles of ion channels and transporters in guard cell signal transduction

“…Experiments using phosphatase and kinase inhibitors during whole cell recordings have long suggested the importance of phosphorylation/dephosphorylation in the regulation of plant K + channels, e.g. the guard cell inward K + current was reduced in response to protein phosphatase 2B inhibitors [54]. Moreover, protein phosphatase 1 and 2A inhibitors, calyculin A and okadaic acid, inhibited inward and outward K + currents in V. faba guard cells [55].…”

Roles of Higher Plant K+ Channels

“…These cellular regulation mechanisms of guard cell K' channels and their physiological implications for stomatal movements are reviewed in more detail elsewhere (Assmann, 1993;Ward et al, 1995), but examples are: (a) ABAinduced Ca2+ elevation and the inhibition of stomatal opening by Ca2+, which correlates with a down-regulation of K+i, channels by elevated Ca2+ concentrations (Schroeder and Hagiwara, 1989); (b) enhancement of K+,,, channel activity by cytosolic alkalinization can be triggered by ABA-induced alkalinization (Irving et al, 1992;Blatt and Armstrong, 1993); and (c) the activity of Kfi, channels is increased by extracellular acidification, which can provide an additional mechanism, enhancing proton pumpinduced stomatal opening (Blatt, 1992).…”