1,4-Dihydropyridine Binding Sites in Moss Plasma Membranes

Abstract

An increase in cytoplasmic calcium is an early event in hormone (cytokinin)-induced vegetative bud formation in the moss Physcomitrella patens. Whole cell and calcium transport studies have implicated 1,4-dihydropyridine-sensitive calcium channels in this increase in cellular calcium. Controlled changes in cellular calcium concentrations have been identified as important components of signal transduction pathways in plants. Cytoplasmic calcium concentrations are highly regulated; levels are modulated by coordinating passive fluxes and active transport across organellar and plasma membranes (1-4). Cytoplasmic calcium levels have been shown to increase in response to a variety of stimuli including light (5, 6) and hormones (7,8), and small fluctuations in cellular calcium may modulate processes as diverse as secretory activity in the barley aleurone (7), pollen tube growth (9), and phase transition in mitosis (10). Studies examining the effect of calcium channel inhibitors on physiological processes (11-14) have suggested a role for calcium channels in stimulus-induced increases in cytoplasmic calcium levels; however, little is known about the biochemical or molecular properties of these transport systems.Calcium acts as an intracellular messenger in hormone (cytokinin)-induced vegetative bud formation during the development of the filamentous protonemata (the young gametophore) in the moss Physcomitrella patens (15-18). Formation of vegetative buds is an integral part of the moss life cycle leading to the development of the mature gametophore which is essential for subsequent sexual reproduction. Cytokinin applied to moss cells causes profuse premature bud formation (19). Localized increases in calcium take place after addition of cytokinin but precede the cytokinin-induced cell division (15, 18). In moss cells not stimulated by cytokinin, cytoplasmic calcium levels (250 nM) are three orders of magnitude lower than levels in the external medium (0.1-1.0 mM) (18). After addition of cytokinin, cytoplasmic calcium levels increase to 750 nM (18). Whole plant studies indicate that cytokinin-modulated calcium entry takes place via dihydropyridine (DHP) 1 -sensitive channels (20). In moss protonemata, application of DHP calcium channel agonists in the absence of cytokinin stimulates bud initial formation, whereas DHP calcium channel antagonists block cytokinin-induced bud formation (20). We have previously characterized calcium influx into isolated moss protoplasts and have established that the transport activity of the moss calcium channel shares common characteristics with L-type calcium channels in animal cells. Calcium transport in moss is voltagedependent, stimulated by DHP agonists, and inhibited by DHP antagonists, phenylalkylamines, and benzothiazepines (21). A novel feature of the transport activity of this channel is hormonal modulation by cytokinin (21).Cytokinin-induced bud formation in moss is a simple, highly ordered developmental process and is one of few plant responses that allows direct study of stimul...

G Proteins Regulate Dihydropyridine Binding to Moss Plasma Membranes

“…As described previously, nifedipine increases the rate of dissociation of [ 3 H]azidopine in moss membranes (12) (Fig. 3A); similar results were observed for Bay K8644 (Fig.…”

“…In these membranes, DHPs at greater than micromolar concentrations increase the rate of dissociation of radiolabeled DHP, which may be a consequence of a protein conformational change induced by DHP occupancy of low affinity sites (18). In our studies, we have detected only high affinity DHP sites in the plasma membranes of P. patens (12), and in the present study, we present evidence for G protein regulation of this high affinity DHP binding.…”

“…We have previously shown that plasma membranes from the moss P. patens contain a single class of binding sites for the calcium channel blocker [ 3 H]azidopine (12). In the absence of multiple classes of binding sites, other mechanisms must exist to provide a level of channel control during development.…”

“…Whole cell studies of the effect of DHPs on bud formation in moss suggested that hormone-induced bud formation is modulated by calcium entry into cells (8). Subsequent studies characterizing calcium influx into moss protoplasts and DHP binding to moss plasma membranes implicated DHP-sensitive calcium channels in this process (11,12). The data presented in this study support the hypothesis that G proteins regulate DHP binding to high affinity sites that are likely associated with voltage-dependent channels and suggest cytoplasm-inde-pendent regulation.…”

“…Increases in calcium influx into moss protoplasts were seen within 15 s of addition of cytokinin, and calcium influx was inhibited by DHP antagonists and stimulated by DHP agonists (11). DHP-sensitive binding of the DHP arylazide [ 3 H]azidopine to purified moss plasma membranes was found to be cytokinin-dependent (12). While physiological events implicating calcium in hormone-induced bud formation have been described, the biochemical pathways that link signal perception to channel regulation remain largely unknown.…”