dependent calcium-permeable channelsin carrot - NCBI

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Sep 26, 1994 - 'Centre de Biologie du Developpement, UMR 9925, Universite Paul. Sabatier, 118 ..... enzymes (Ranjeva and Boudet, 1987) and for the control.
The EMBO Journal vol.13 no.24 pp.5843-5847, 1994

Recruitment of plasma membrane voltagedependent calcium-permeable channels in carrot cells Patrice Thuleau, Marc Moreau1, Julian I.Schroeder2 and Raoul Ranjeva Centre de Biologie et Physiologie Wgetales, URA-CNRS 1457 and 'Centre de Biologie du Developpement, UMR 9925, Universite Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse cedex, France and 2Department of Biology and Center for Molecular Genetics, University of California San Diego, La Jolla, CA 92093-0116, USA Communicated by C.Paoletti

Numerous biological assays and pharmacological studies have led to the suggestion that depolarizationactivated plasma membrane Ca2+ channels play prominent roles in signal perception and transduction processes during growth and development of higher plants. The recent application of patch-clamp techniques to isolated carrot protoplasts has led to direct voltage-clamp evidence for the existence of Ca2+ channels activated by physiological depolarizations in the plasma membrane of higher plant cells. However, these voltage-dependent Ca2+ channels were not stable and their activities decreased following the establishment of whole-cell recordings. We show here that large pre-depolarizing pulses positive to 0 mV induced not only the recovery of Ca2+ channel activities, but also the activation of initially quiescent voltage-dependent Ca2+ channels in the plasma membrane (recruitment). This recruitment was dependent on the intensity and duration of membrane depolarizations, i.e. the higher and longer the pre-depolarization, the greater the recruitment. Pre-depolarizing pulses to + 118 mV during 30 s increased the initial calcium currents 5- to 10-fold. The recruited channels were permeable to Ba2+ and Sr2+ ions. The data suggested that voltagedependent Ca2+-permeable channels are regulated by biological mechanisms which might be induced by large pre-depolarizations of the plasma membrane. In addition, this study provides evidence for the existence in the plasma membrane of higher plant cells of a large number of voltage-dependent Ca2+ channels of which a major part are inactive and quiescent. It is suggested that quiescent Ca2+ channels can be rapidly recruited for Ca2+-dependent signal transduction. Key words: plant calcium channels/signal transduction

Introduction Cytosolic calcium has been recognized to be crucial in the control of a large number of cellular functions in plant cells, including regulation of enzyme or ion channel activities, cytoskeleton organization and gene expression (Hepler and Wayne, 1985; Leonard and Hepler, 1990). Calcium channels in the plasma membrane of higher plants have been suggested to provide a regulated pathway

for Ca>2 influx. Since their activation could result in massive calcium entry into the cell, thus generating important changes in second messenger concentrations, calcium channels have been considered important targets in signal perception and transduction processes (Johannes et al., 1991; Schroeder and Thuleau, 1991; Bush, 1993; Ranjeva et al., 1993). Consequently, elucidation of molecular mechanisms which may explain how an initial stimulus is coupled to calcium channel activation is of the utmost importance in understanding early events in signal transduction in higher plant cells. Several lines of experimental evidence have shown that signals affecting plant metabolism and development, such as light, touch and gravitropism as well as growth substances, fungal elicitors and phytotoxins, act by interfering directly or indirectly with calcium channels (Bush, 1993; Ranjeva et al., 1993). Interestingly, most of these biotic or abiotic stimuli trigger depolarizations of the plasma membrane (Ishikawa et al., 1983; Spalding and Cosgrove, 1989; Marten et al., 1991; Ullrich and Novacky, 1991; Ehrhardt et al., 1992) and recently the existence of depolarization-activated calcium-permeable channels located in the plasma membrane of higher plants has been demonstrated (Huang et al., 1994; Marshall et al., 1994; Pifieros and Tester, 1994; Thuleau et al., 1994). The application of the whole-cell patch-clamp technique to isolated carrot protoplasts has established that voltagedependent inward calcium currents became activated at a potential positive to -135 mV and maximum currents occurred at -84 mV (Thuleau et al., 1994). Such properties are consistent with the characteristics of plant cells where usually resting membrane potentials are in the range- 140 to -180 mV (Bates and Goldsmith, 1983; Felle, 1988) and where membrane depolarizations positive to -120 mV have been reported as early responses to various stimuli (Kasamo, 1981; Bates and Goldsmith, 1983; Ishikawa et al., 1983; Spalding and Cosgrove, 1989; Marten et al., 1991; Ullrich and Novacky, 1991; Ehrhardt et al., 1992; Wildon et al., 1992). However, these voltage-dependent Ca2+ channels were unstable and became inactive within a few minutes in an irreversible manner (Thuleau et al., 1994). To obtain further insight into the knowledge of voltagedependent calcium channels and their regulation and to directly assess their putative role in signal perception and transduction processes, we have examined the properties of these membrane-bound proteins. In this paper we report our attempts to elucidate mechanisms of activation of plant calcium channels and demonstrate that large transient pre-depolarizations of the plasma membrane potentiate the activity of carrot calcium channels. Recruitment of potentially active but quiescent calcium-permeable channels may be a means for plant cells to gate the flow of calcium in response to a number of depolarizing stimuli.

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0.5 soc Fig. 1. Recruitment of voltage-dependent calcium channels in carrot cells by pre-depolarization. (A) Voltage-dependent inward calcium currents were recorded 2 min after the establishment of the whole-cell configuration by depolarization of the membrane potential from the holding potential at - 160 mV to the peak current potential at -80 mV. The top insert shows the voltage pulse protocol. (B) Washout of calcium channel activities. Inward calcium currents were recorded on the same protoplast as in (A) 10 min after the establishment of whole-cell recordings. (C) After calcium channel activities were fully washed-out, inward calcium currents were recorded as in (A) immediately after a 30 s prepulse to + 18 mV. (D) Inward calcium currents were recorded as in (A) after a 30 s prepulse to + 118 mV.

10 min, as illustrated by the reduction in inward currents (Figure IB). Wash-out could not be reversed by hyperpolarization in the range from - 120 to - 160 mV lasting for up to 10 min (Thuleau et al., 1994). In contrast, by pre-depolarizing the cell for 30 s to + 18 mV, we observed a reactivation of voltage-dependent channel activities (n = 59; Figure IC). A pre-depolarization of the plasma membrane to + 118 mV lasting 30 s resulted in an increase of initial calcium currents by 6.6-fold ( +4, n = 29; compare Figure IA and D). Potentiation by a large pre-depolarization did not change the biophysical characteristics of inward currents. Thus, when depolarizing voltage-ramps, from a holding potential of - 160 to +50 mV, were applied to carrot protoplasts before and immediately after a 30 s pre-depolarizing voltage step to + 138 mV, no change was observed either in the activation potential and the peak current potential of the channels or in the reversal potential of voltagedependent currents (Figure 2). These data suggested that the pre-depolarizing treatment triggered activation of calcium-permeable channels sharing similar properties. Therefore the pre-depolarization of the plasma membrane led not only to the recovery of initial voltage-dependent calcium channel activities, but also to the recruitment of quiescent Ca2+ channels.

Results

Recruitment of voltage-dependent calciumpermeable channels depends on the intensity and duration of the pre-depolarization

Large pre-depolarizations of the plasma membrane recruit voltage-dependent calciumpermeable channels Patch-clamp studies on carrot cells were performed under conditions which allow the distinction of Ca2+ channels from other ion channel types (Thuleau et al., 1994). Figure 1 A shows voltage-dependent calcium inward currents measured by patch-clamp techniques on carrot protoplasts, 2 min after the establishment of whole-cell recordings. When the membrane potential was stepped from the holding potential of -160 mV to the peak current potential -80 mV, inward calcium currents were activated. However, the activities decreased slowly ('wash-out') and in a large number of cells the wash-out was complete within

To examine in more detail the recruitment of calciumpermeable channels, the plasma membrane of carrot cells was successively pre-depolarized for durations of 30 s to different potentials ranging from -22 to +158 mV and the potential was stepped back to the holding potential of - 160 mV. Voltage-dependent calcium channel activities were immediately measured by a depolarizing step from the holding potential to -80 mV. The recruitment of calcium-permeable channels occurred for pre-depolarizations positive to 0 mV (n = 6) (Figure 3A and B). Inward calcium currents increased with the intensity of predepolarizing pulses and reached a maximum at a membrane potential of -+ 140 mV (Figure 3A and B). In addition, time-dependent properties of recruitment

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Fig. 3. Voltage-dependence of recruitment of voltage-dependent calcium channels. (A) Inward calcium currents were recorded during a voltage step from 160 to -80 mV after successive 30 s prepulses to different membrane potentials ranging from -22 to + 158 mV. After each recording the membrane potential was held at 160 mV for 3 min. (B) Currents measured at the end of voltage pulses shown in (A) are plotted as a function of applied pre-depolarizing pulses. -

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of calcium-permeable channels were studied by holding the membrane potential to a pre-depolarizing potential of + 138 mV for different durations before the activation of voltage-dependent Ca2+ channels. Recruitment of calciumpermeable channels was apparent after a 5 s pre-depolarizing voltage pulse to + 138 mV (n = 6; Figure 4A and B). Furthermore, calcium inward currents increased proportionally to the duration of the pre-depolarizing treatment to reach a maximum after a 25 s prepulse (Figure 4A and B). Therefore, recruitment of voltage-dependent calcium channels was strictly dependent on the intensity and duration of pre-depolarizing pulses, with a 30 s prepulse to + 138 mV leading to the maximum recruitment of calcium-permeable channels present in the plasma membrane. Recruited voltage-dependent calcium channels inactivate with time After a 30 s pre-depolarizing pulse to +118 mV, the membrane potential was stepped back to the holding potential of -160 mV and was immediately held to -80 mV for 30 s. In these conditions a decay of calcium currents was observed, with a half-time of 12 s (±5 s, n = 5) (Figure 5). Following this decay in calcium

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Fig. 4. Time-dependence of recruitment of voltage-dependent calcium channels. (A) Inward calcium currents were recorded during a voltage step from -160 to -80 mV after successive prepulses to + 138 mV applied for different durations ranging from 5 to 30 s. After each recording the membrane potential was held at - 160 mV for 3 min. (B) Currents measured at the end of voltage pulses shown in (A) are plotted as a function of the duration of applied pre-depolarizing pulses.

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currents, recruited currents could not be restored by subsequently holding the membrane at the hyperpolarizing

potential of -160 mV. Conversely, if the membrane potential was depolarized to +118 mV for 30 s, 100% of initial recruited currents could be recovered (data not shown). Occasionally, it has been possible by this device to keep voltage-dependent calcium channels active for at least 1 h without any wash-out (data not shown). Recruited voltage-dependent calcium channels are permeable to other divalent cations In previous studies we have shown that voltage-dependent calcium channels were permeable to other divalent cations 5845

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prepulse ** Fig. 6. Divalent cation selectivity of recruited voltage-dependent calcium-permeable channels. Whole-cell currents measured before and immediately after a prepulse of 30 s to + 118 mV, during a 3.7 s voltage-ramp between - 160 and + 50 mV. Carrot protoplasts were bathed in a solution containing 10 mM HEPES, 1.6 mM Ca(OH)2, pH 6.7; 2 mM MgCl2; sorbitol osmolality 600 mosmol/kg and either 30 mM BaCI2 (A) or 30 mM SrCl2 (B). The internal solution was as described in Materials and methods.

(Thuleau et al., 1994). To examine whether recruitment of calcium-permeable channels occurred in the presence of other divalent cations, Ca2+ was replaced in the external medium by either Ba2+ or Sr2+. When depolarizing voltage-ramps were applied to carrot protoplasts bathed in a solution containing either 30 mM BaCl2 or 30 mM SrCl2, voltage-dependent inward currents similar to those recorded in the presence of 30 mM CaCl2 were observed (Figure 6). The peak current for Ba2+ and Sr2+ occurred at -84 + 13 (n = 5) and -82 + 7 mV (n = 4) respectively. In addition, voltage-dependent barium and strontium currents reversed at +31 ± 15 (n = 5) and +24 ± 8 mV (n = 4) respectively (Figure 6A and B). These results are similar to those obtained when calcium was the charge carrier (Thuleau et al., 1994; compare Figures 2 and 6). In addition, if prior to depolarizing voltage-ramps the cells were pre-depolarized at a potential of + 118 mV during 30 s, recruitment of voltage-dependent channels permeable to Ba2+ and Sr2+ was observed (Figure 6A and B). The depolarizing prepulse induced a 4-fold (+2, n = 3) activation of voltage-dependent Ba2+ inward currents while increasing voltage-dependent Sr2+ inward currents to levels up to 6.5 times (+2.5, n = 3) the initial level (Figure 6A and B). Therefore, the recruitment of voltage-dependent calcium-permeable channels appeared independent of the permeating ion and occurred for Ba2+ or Sr2+ ions.

Discussion Calcium channels in the plasma membrane have been suggested to provide a major pathway for calcium influx into higher plant cells (Schroeder and Hagiwara, 1990;

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Thuleau et al., 1990, 1993; Cosgrove and Hedrich, 1991). Recently direct application of patch-clamp techniques to isolated carrot protoplasts has led to the first identification of voltage-dependent calcium-permeable channels which can be activated by physiologically occurring depolarizations of the plasma membrane during signal transduction (Thuleau et al., 1994). However, prior to this study, insights into mechanisms of enhancement of calcium channel activity were lacking. This paper describes experimental evidence showing that a major part of voltagedependent calcium channels present in the plasma membrane is quiescent but can be activated by repetitive large pre-depolarizations. Assuming cellular buffering capacities of 95% (Becker et al., 1989), the increase of calcium inward currents due to channel recruitment would dramatically increase the rate of increase in the cytosolic free Ca2+ concentration to >1 tM/s. This rate of increase in the cytosolic free Ca2+ is large enough to trigger a number of cellular calcium-dependent processes but would become lethal if the stimulation is sustained for a while. Voltage-dependent calcium channels inactivate with time but may be reactivated by large depolarizing conditioning pulses as described for various animal models (Artalejo et al., 1992; Sculptoreanu et al., 1993a,b). At present, the only clear-cut particularity of recruitment of calcium channels in carrot cells is that substituting barium for calcium has no effect on either inward current intensities or on the inactivation of calcium channels. Concerning the biochemical basis of recruitment and inactivation, at the present stage a couple of possibilities may be put forward. The first one should involve a post-translational modification of calcium channels or associated proteins (e.g. protein phosphorylation/ dephosphorylation) as established for a number of plant enzymes (Ranjeva and Boudet, 1987) and for the control of potassium channel activities in plants (Luan et al., 1993; Thiel and Blatt, 1994). Furthermore, such a hypothesis should be consistent with data reported for animal systems where it has been clearly shown that recruitment and inactivation of voltage-dependent calcium channels are controlled by reversible protein phosphorylation (Artalejo et al., 1992; Sculptoreanu et al., 1993a,b). The second possibility may be a direct voltage-dependent conformational change of the protein eliciting the same type of results induced by phosphorylation. Thus it has been shown that smooth muscle calcium channels expressed in Chinese hamster ovary cells were subject to voltage-dependent recruitment but not to protein phosphorylation (Kleppisch et al., 1994). The pre-depolarizing pulses necessary to fully recruit voltage-dependent calcium channels in carrot cells are unlikely to occur in normal physiological conditions. Nevertheless, depolarizations to potentials of up to +50 mV have been reported in plant cells (Williamson and Ashley, 1982). Furthermore, initial activation of higher plant voltage-dependent Ca2+ channels (Thuleau et al., 1994) can also transiently depolarize the plasma membrane to positive potentials due to the ¢ 1000-fold gradient of Ca>2 ions across the plasma membrane. The present data show that brief depolarizations positive to 0 mV suffice to enhance Ca2+ channel activity. They show further that a large number of quiescent but recruitable Ca2+ channels exist in the plasma membrane of carrot cells. It is possible

Recruitment of voltage-dependent Ca2+ channels

that further modulatory mechanisms, in addition to the rapid voltage-dependent recruitment revealed here, can contribute to Ca2+-dependent signal transduction in higher plant cells. In conclusion, the data reported here establish that potentially active voltage-dependent calcium channels may be rapidly recruited in the plasma membrane of plant cells. Regardless of the actual physiological stimuli and the biochemical events underlying this process, the use of molecular electrophysiology has unveiled a novel aspect of the activation properties of plant calcium channels and will allow further insight into the understanding of signal perception and transduction in higher plants.

Materials and methods Protoplasts were isolated from carrot cells (Daucus carota L.) as described previously (Graziana et al., 1988; Thuleau et al., 1988). During patch-clamp recordings, protoplasts were bathed in an external solution containing 30 mM CaC12 (30 mM BaCI2 or 30 mM SrCl2 when indicated), 2 mM MgCI2, 10 mM HEPES, 1.6 mM Ca(OH)2, pH 6.7. The osmolality was adjusted to 600 mosmol/kg by the addition of D-sorbitol. The pipette solution which equilibrated with the cytoplasm contained 2 mM MgCI2, 5 mM Tris2-EGTA, 10 mM MgATP, 0.1 mM CaCI2, 10 mM HEPES-Tris, pH 7.2, and D-sorbitol 620 mosmol/kg. Whole-cell patch-clamp experiments were performed as described by Hamill et al. (1981). Recordings were performed and low pass-filtered with an RK 300 amplifier (Biologic Science Instruments, Claix, France). The application of voltage protocols and subsequent data analysis were performed using a Lab Master DMA interface (Axon Instruments, Foster City, CA) and the patch-clamp software pCLAMP 5.5.1 (Axon Instruments). Membrane potentials were corrected for voltage drops induced by the access resistance and for liquid junction potentials as described previously (Neher, 1992; Ward and Schroeder, 1994). Equilibrium potentials were calculated after correction for ionic activities in solution (Robinson and Stokes, 1955). The temperature was 21'C.

Development. Current Topics in Plant Physiology. Vol. 4, American Society of Plant Physiologists, Rockville, MD. Luan,S., Li,W., Rusnak,F., Assmann,S.M. and Schreiber,S.L. (1993) Proc. Natl Acad. Sci. USA, 90, 2202-2206. Marshall,J., Corzo,A., Leigh,R.A. and Sanders,D. (1994) Plant J., 5, 683-694. Marten,I., Lohse,G. and Hedrich,R. (1991) Nature, 353, 759-762. Neher,E. (1992) Methods Enzymol., 207, 123-131. Pifieros,M. and Tester,M. (1994) Planta, in press. Ranjeva,R. and Boudet,A.M. (1987) Annu. Rev. Plant Physiol., 38,73-93. Ranjeva,R., Thuleau,P. and Schroeder,J.I. (1993) Curr. Opin. Biotechnol., 4, 172-176. Robinson,R.A. and Stokes,R.H. (1955) In Electrolyte Solutions. New York Academy of Science, NY, pp. 480-499. Schroeder,J.I. and Hagiwara,S. (1990) Proc. NatI Acad. Sci. USA, 87, 9305-9309. Schroeder,J.I. and Thuleau,P. (1991) Plant Cell, 3, 555-559. Sculptoreanu,A., Rotman,E., Takahashi,M., Scheuer,T. and Catterall,W.A. (1993a) Proc. Natl Acad. Sci. USA, 90, 10135-10139. Sculptoreanu,A., Scheuer,T. and Catterall,W.A. (1993b) Nature, 364,

240-243. Spalding,E.P. and Cosgrove,D.J. (1989) Planta, 178, 407-410. Thiel,G. and Blatt,M.R. (1994) Plant J., 5, 727-733. Thuleau,P., Graziana,A., Canut,H. and Ranjeva,R. (1990) Proc. Natl Acad. Sci. USA, 87, 10000-10004. Thuleau,P., Graziana,A., Ranjeva,R. and Schroeder,J.I. (1993) Proc. Natl Acad. Sci. USA, 90, 765-769. Thuleau,P., Graziana,A., Rossignol,M., Kauss,H., Auriol,P. and Ranjeva,R. (1988) Proc. Natl Acad. Sci. USA, 85, 5932-5935. Thuleau,P., Ward,J.M., Ranjeva,R. and Schroeder,J.I. (1994) EMBO J., 13, 2970-2975. Ullrich,C.I. and Novacky,A.J. (1991) Plant Physiol., 95, 675-681. Ward,J.M. and Schroeder,J.I. (1994) Plant Cell, 6, 669-683. Wildon,D.C., Thain,J.F., Minchin,P.E.H., Gubb,I.R., Reilly,A.J., Skipper,Y.D., Doherty,H.M., O'Donnell,P.J. and Bowles,D.J. (1992) Nature, 360, 62-65. Williamson,R.E. and Ashley,C.C. (1982) Nature, 296, 647-651. Received on August 9, 1994; revised on September 26, 1994

Acknowledgements In memoriam Claude Paoletti. This work was supported in part by NSF grant 904.977 (J.I.S.) and by the Centre National de la Recherche Scientifique, the Universite Paul Sabatier Toulouse and by grants from the European Union (AAIR III Program) to R.R.

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Felle,H. (1988) Planta, 174, 143-153. Graziana,A., Fosset,M., Ranjeva,R., Hetherington,A.M. and Lazdunski,M. (1988) Biochemistry, 27, 764-768. Hamill,O.P., Marty,A., Neher,E., Sakmann,B. and Sigworth,F.J. (1981) Pflugers Arch. Ges. Physiol., 391, 85-100. Hepler,P.K. and Wayne,R.O. (1985) Annu. Rev. Plant Physiol., 36, 397439. Huang,J.W., Grunes,D.L. and Kochian,L.V. (1994) Proc. Natl Acad. Sci. USA, 91, 3473-3477. Ishikawa,H., Aizawa,H., Kishira,H., Ogawa,T. and Sakata,M. (1983) Plant Cell Physiol., 24, 769-772. Johannes,E., Brosnan,J.M. and Sanders,D. (1991) BioEssays, 13, 331336.

Kasamo,K. (1981) Plant Cell Physiol., 22, 1257-1267. Kleppisch,T., Pedersen,K., Strubing,C., Bosse-Doenecke,E., Flockerzi,V., Hofmann,F. and Hescheler,J. (1994) EMBO J., 13, 2502-2507. Leonard,R.T. and Hepler,P.K. (1990) Calcium in Plant Growth and

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