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Keywords:

  • anionic channels;
  • calcium regulation;
  • Lilium longiflorum;
  • patch clamp;
  • pollen

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Currents through anion channels in the plasma membrane of Lilium longiflorum pollen grain protoplasts were studied under conditions of symmetrical anionic concentrations by means of patch-clamp whole-cell configuration.
  • With Cl-based intra- and extracellular solutions, three outward-rectifying anion conductances, ICl1, ICl2 and ICl3, were identified. These three activities were discriminated by differential rundown behaviour and sensitivity to 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), which could not be attributed to one or more channel types. All shared strong outward rectification, activated instantaneously and displayed a slow time-dependent activation for positive potentials. All showed modulation by intracellular calcium ([Ca2+]in), increasing intensity from 6.04 nM up to 0.5 mM (ICl1), or reaching a maximum value with 8.50 μM (ICl2 and ICl3).
  • After rundown, the anionic currents measured using NO3-based solutions were indistinguishable, indicating that the permeabilities of the channels for Cl and NO3 are similar. Additionally, unitary anionic currents were measured from outside-out excised patches, confirming the presence of individual anionic channels.
  • This study shows for the first time the presence of a large anionic conductance across the membrane of pollen protoplasts, resulting from the presence of Ca2+-regulated channels. A similar conductance was also found in germinated pollen. We hypothesize that these putative channels may be responsible for the large anionic fluxes previously detected by means of self-referencing vibrating probes.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Pollen grains are the carriers of male gametes in flowering plants. After landing on the stigma, they germinate and develop a cytoplasmic extension – the pollen tube – that grows up to the ovule, where the sperm cells are released. This event is a key point in plant sexual reproduction. The pollen tubes are an extreme example of cellular polarity and exclusive apical growth, and have been extensively used for basic cell biology studies involving cellular growth and morphogenesis (Boavida et al., 2005; Michard et al., 2009).

The use of noninvasive methods has demonstrated a close link between intracellular ion fluctuations and ionic fluxes across the plasma membrane, and the cellular phenomena that occur during the formation and elongation of the pollen tube (Mascarenhas, 1993; Feijóet al., 1995, 2001, 2004; Cheung, 1996; Hepler et al., 2001). For example, oscillatory H+ gradients have been shown to exist and to be intimately associated with polarized pollen tube growth. A constitutive alkaline band in the clear zone and a growth-dependent acidic tip were later shown to be based molecularly on the differential distribution of a P-H+-ATPase (Feijóet al., 1999; Certal et al., 2008).

Several groups have detected the presence of [Ca2+] tip focused gradients (e.g. Holdaway-Clarke et al., 1997; Feijóet al., 2001; Holdaway-Clarke & Hepler, 2003; Michard et al., 2009). Additionally, ion-specific self-referencing electrodes have been used to study ionic fluxes during pollen tube growth, demonstrating an uneven distribution of the different fluxes. Non-oscillatory ionic fluxes of typical permeable ions in cells, such as H+, Ca2+, K+ and Cl, have been observed from the grain and along the tube shank, while the tip zone displays distinctive oscillatory fluxes for the same ions, and usually in the reverse direction to that on the shank (Feijóet al., 1999; Moreno et al., 2007). These oscillatory fluxes have been shown to be essential to tube growth and show a similar period of oscillation to that of growth, although with distinct phase delays (Holdaway-Clarke et al., 1997; Messerli et al., 1999; Feijóet al., 2001; Zonia et al., 2002; Holdaway-Clarke & Hepler, 2003). Early studies by Weisenseel & Jaffe (1976) demonstrated that pollen tube germination and growth have an absolute requirement for extracellular K+, Ca2+, a trace of boron, and a somewhat acidic pH. Conversely, substitution experiments with nonpermeable anions (cellobinate or methylsulfonate) showed that inorganic anions were not required for the growth of the pollen tubes; but it should be noted that these authors never estimated the chloride concentration in the germination medium, and therefore were not able to determine the contributions of reagent contamination or pollen release. Nevertheless, there are several indications of the presence of anionic fluxes from the pollen tube. Trofimova & Molotkovskii (1993) reported that the activation of Nicotiana tabacum pollen grains was accompanied by Cl efflux. Zonia et al. (2001, 2002) have confirmed the occurrence of large Cl fluxes from the tip of the growing pollen tube of Lilium longiflorum and of Nicotiana tabacum, leading to the accumulation of anions in the extracellular medium. In particular, Cl fluxes exceeded by a factor of 10 other previously reported H+ and Ca2+ fluxes in the same species. Furthermore, substitution of nitrate by chloride in the germination medium was the only treatment that induced a preference of growth reorientation under an electric field towards the cathode (Malhóet al., 1992). Recent studies by Breygina et al. (2009a,b), measuring chloride concentration and plasma membrane voltage polarization, showed that cytoplasmic Cl is involved in the polarization of the lily pollen tube during germination. This group showed that the anion channel inhibitor 5-nitro-2-(phenylpropylamino)-benzoate (NPPB) inhibited Cl release from the grain (during hydration) and from the tube tip, and stopped cell growth. These effects were also achieved by the extracellular addition of high [Cl], possibly by equilibrating the intracellular [Cl]. This study also demonstrated an NPPB-induced depolarization of the membrane, with additional disruption of the functional compartmentalization of the cytoplasm necessary for polar growth.

Doubts about the nature of the Cl currents measured by Zonia et al. (2002) were raised by Messerli et al. (2004), who suggested that the ion-selective Cl probes are sensitive to the concentration of buffer and indirectly detect changes in H+ gradients in the bathing solution, and therefore the Cl fluxes measured were contaminated by H+ fluxes. It should be noted, however, that Messerli et al. (2004)did not reproduce the exact measurement conditions used by Zonia et al. (2002), and did not take fully into account the pharmacological evidence. In addition, transcriptomic studies have demonstrated a number of anion/chloride membrane transporters and channels to be specifically and highly expressed in the pollen of Arabidopsis (Pina et al., 2005). The promoter of a cation-chloride co-transporter (CCC) was also found to be highly expressed in pollen (Colmenero-Flores et al., 2007). The contradictory interpretations of chloride fluxes in pollen call for a direct analysis of plasma membrane chloride channel activity. Arguments have also been made elsewhere that the issues of the existence and relevance of chloride currents in pollen tubes have not yet been resolved (Hepler et al., 2006; Kunkel et al., 2006; Moreno et al., 2007).

There have been great efforts to identify the putative channels underlying the detected macroscopic fluxes in pollen. Most of the information gathered relates to the identification of passive K+ currents with different characteristics by means of patch-clamp techniques. Three types of K+ channel in lily have been reported by Obermeyer & Kolb (1993) and suggested to be involved in inward influx during pollen tube growth. Two outward channels have also been reported (Obermeyer & Kolb, 1993; Obermeyer & Blatt, 1995). Griessner & Obermeyer (2003) detected, for the first time, both inward and outward K+ currents in the plasma membrane of protoplasts from L. longiflorum pollen tubes. The inward currents showed different activation kinetics from, and higher current density than, those detected in the grain. K+ channels have also been found in pollen protoplasts from Arabidopsis (Fan et al., 2001) and Brassica chinensis, which are distinctly regulated by pH and/or Ca2+ (Fan et al., 1999, 2003; Fan & Wu, 2000). Mouline et al. (2002) cloned, for the first time, a pollen-specific K+ channel of the Shaker family (SPIK) from Arabidopsis and showed that disruption of the SPIK coding sequence strongly affected inwardly rectifying K+ channel activity in the pollen plasma membrane, resulting in impaired pollen tube growth. Dutta & Robinson (2004) detected K+ and Ca2+ single-channel currents from outside-out patches from L. longiflorum pollen grain and tube tip protoplasts, which were attributed to putative stretch-activated channels. A hyperpolarization-activated inward Ca2+ channel regulated by actin filaments was identified in Arabidopsis (Wang et al., 2004).

Here we used the patch-clamp technique to investigate Cl currents across the plasma membrane of pollen protoplasts from L. longiflorum. In addition, we studied the possible regulation of the currents by [Ca2+]in, a cation known to form a constitutive elevated domain at the tip which is essential for directed apical growth (Pierson et al., 1994, 1996; Holdaway-Clarke et al., 1997; Messerli & Robinson, 1997). The whole-cell currents can be split into three anion conductances which cross the membrane of the pollen protoplasts through putative anionic channels and which show distinct regulation by [Ca2+]in. Outside-out isolated patches displayed isolated single-channel anionic currents with at least three conductive states, which could underlie the macroscopic whole-cell currents. The currents measured may account for the extracellular fluxes measured by Zonia et al. (2002) and thus their possible physiological role is discussed.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Protoplast isolation

Lilium longiflorum (Thunb.) pollen grains were collected from the anthers, aliquoted and stored at −20°C. On the day of the experiment, protoplast isolation was performed according to Tanaka et al. (1987), Fan et al. (2001) and Mouline et al. (2002), with modifications. The pollen grains were hydrated for 10 min at 20°C in 2 ml of standard solution: 1 mM KNO3, 0.2 mM KH2PO4, 1 mM MgSO4, 1 μM KI, 0.1 μM CuSO4, 5 mM CaCl2, 0.5 M glucose, 1 M sorbitol and 5 mM MES (2-(N-morpholino)ethanesulfonic acid), pH 5.8, adjusted with TRIS (tris(hydroxymethyl)aminomethane) (osmolarity = 1.5 Osmol kg−1). Hydrated pollen grains were centrifuged at 63 g for 5 min and the pellet was resuspended in 2 ml of enzyme solution: 1% (w/v) cellulase RS ‘Onozuka’ (Duchefa, Haarlem, the Netherlands), 0.5% (w/v) macerozyme R-10 (Duchefa, Haarlem, the Netherlands), and 0.2% (w/v) BSA in standard solution. Enzymatic digestion of the cell wall was performed for 80 min, at 30°C with mild agitation. The enzymatic solution was removed by centrifugation at 63 g for 5 min (supernatant), and the pellet was further washed twice with bathing solution B1 (Table 1) which osmolarity was adjusted to 1.5 Osmol kg−1 with sorbitol. The protoplasts were further resuspended in 300 μl of solution B1 and the suspension was kept on ice for patch-clamp measurements for up to 8 h after isolation. Alternatively, pollen grains were germinated for 40 min in 1.6 mM H3BO3, 1 mM KCl, 50 μM CaCl2, 5% sucrose (w/v), and 50 μM MES, pH 6.3, after the hydration step and before the enzymatic digestion (which was reduced to c. 30 min). This procedure resulted in pollen grains with protruding pollen tubes (c. 200 μm in length) suitable for patch-clamp measurements.

Table 1.   Recording solutions
 NMG-ClNMG-NO3CaCl2Ca(NO3)2MgCl2MgATPTEA-ClTEA-NO3GdCl3EGTAMESHEPES[Ca2+]free
  1. P1–4, internal solutions (pipette); B1–5, external solutions (bathing). The concentrations are in mM, unless otherwise stated. The pH was adjusted to 7.2 (pipette) and 5.8 (bathing) with N-methyl-d-glucamine (NMG) and the osmolarity was adjusted with sorbitol to 700 mOsmol kg−1. The free [Ca2+]in in the pipette solutions was estimated using the software webmaxclite v1.15, available online from http://www.stanford.edu/~cpatton/webmaxc/webmaxclite115.htm. NMG+ was used to replace the permeant cations Na+ and K+; tetraethylamonium (TEA+) and gadolinium (Gd3+) were used to block K+ and Ca2+ currents, respectively (Fan et al., 2001; Dutta & Robinson, 2004; Wang et al., 2004).

P1134.450.35556.04 nM
P2125555558.50 μM
P312256.55550.54 mM
P44.41350.35556.04 nM
B11095312015 
B211212015 
B3110312015 
B4114312015 
B51055312015 

Electrophysiology

Currents from pollen protoplasts (Øc. 20 μm) were measured under voltage-clamp conditions with standard whole-cell recording techniques (Hamill et al., 1981) using a patch-clamp amplifier, Axopatch-1D or Axopatch 200A (Axon Instruments, Foster City, CA, USA). Seal resistance was > 0.5 GΩ. The electrical signal was sampled at 50 kHz and filtered at 5 kHz. Whole-cell series resistance, and pipette and cell membrane capacitive transients were partially subtracted from the records by the amplifier circuitry before sampling. Series resistance correction was 70–80%. Clampex 8.0 software (Axon Instruments) was used to generate the command potentials and to collect the current data. The voltage protocols used to study current activation and channel tail currents are shown as insets in Figs 1(a) and 2(a), respectively. Unitary currents were recorded from outside-out patches at fixed membrane voltage values ranging from −150 to +150 mV, for 20 s. Data were acquired at 50 kHz and low-pass filtered at 4 kHz. The composition of intracellular and extracellular solutions used is shown in Table 1. The inhibitory effect of NPPB on the currents was tested. A stock solution in dimethyl sulfoxide (DMSO) was kept at 4°C (25 mM) and freshly dissolved in the bathing solution before use. Micropipettes, showing resistances of 7–9 MΩ (whole-cell) or 25 MΩ (outside-out patches) when filled with appropriate solution, were pulled from a glass capillary (GB150T-8P; Science Products, Hofheim, Germany) with a PB-7 vertical puller (Narishige, Tokyo, Japan). The reference electrode was an Ag/AgCl wire embedded in a 0.5 M KCl/agar bridge. Bath solutions were changed by injection at a continuous flow rate of c. 20 cm s−1 through a 500-μm i.d. tube, placed at one extreme of a 1-ml circular measuring chamber, and removal at the same rate from the opposite side. All experiments were performed at room temperature. Currents from germinated pollen were measured as described for pollen grain protoplasts, with P1/B1 solutions (nM Ca2+; Table 1). The giga-ohm seals were formed 20–100 μm away from the tip.

image

Figure 1. Typical chloride whole-cell currents from a pollen grain protoplast of Lilium longiflorum, measured with P1/B1 solutions (control group; nM [Ca2+]in). The currents were elicited from a holding potential of −100 mV with the voltage protocol depicted in (a(i)). (a) (i) The initial current recorded immediately after entering the whole-cell configuration (Iinitial). (ii) The current recorded after rundown (Ifinal). (iii) The current recorded after inhibition by 500 μM NPPB (ICl2). (iv) The current lost during rundown (ICl1) obtained by subtraction of Iinitial and Ifinal. (v) Inhibited current (ICl3) obtained by subtraction of Ifinal and ICl2. (b) Current–potential (I/V) relationships of Iinitial (squares), Ifinal (circles) and ICl2 (triangles).

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image

Figure 2. Whole-cell deactivation currents. (a) Typical raw data for the whole-cell tail current (Iinitial). (b) Current–potential (I/V) curve of Iinitial (squares), Ifinal (circles) and ICl2 (triangles). Currents were measured with the protocol shown as an inset in (a), in P1/B1 solutions, and crossed the axis close to the expected value for ECl−.

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Data analysis

Analysis was performed with clampfit 8.0 (Molecular Devices, Sunnydale, CA, USA) on raw data for cells that had stable giga-ohm seals. Liquid junction potentials were calculated with the clampex 8.0 software (Molecular Devices, Sunnydale, CA, USA) and corrected for all whole-cell recordings. All linear and nonlinear data fitting was performed with the origin 6.1 software (OriginLab Corporation, Northampton, MA, USA). Statistical significances were determined using the Wilcoxon test with spss Statistics software (IBM, Armonk, NY, USA) and differences were considered significant if < 0.05. For each experimental condition, data from different cells were averaged. The data shown are mean ± SE (n), where n is the number of cells obtained for a particular experiment. Asymptotic forward (outward) and backward (inward) conductances (determined for the most positive and negative portions of the Current–Voltage (I/V) relationship, respectively, where the current values are directly proportional to the electromotive force) were estimated using a least-mean-square linear fit. Equilibrium potentials for the ions in solution were calculated with the Nernst equation. The relative permeability was determined by measuring the shift in reversal potential (Erev) upon changing the solution on one side of the membrane from a solution containing mainly Cl ions to a solution with a majority of NO3 anions. The permeability ratio was estimated by rearranging the Goldman–Hodgkin–Katz equation: PNO3/PCl exp(ΔVrevF/RT), where ΔVrev is the difference between the reversal potential with the NO3 and that with Cl; F is Faraday’s constant; R is the gas constant; and T is temperature in degrees Kelvin. Conductance–Voltage (G/V ) curves were derived from I/V relationships according to G = Iss/(Vm  Erev), where Iss is the steady-state current at the end of the test potential Vm, and Erev is the reversal potential of the current. Conductance values were normalized for the maximum response and fitted with a Boltzmann type equation:

  • image( Eqn 1)

(Vh, the potential for the half-maximal chord conductance; VS, the slope factor of the curve; A1 and A2, its minimum and maximum attainable values, respectively.) The time-dependent current activated by a +160-mV pulse was best fitted with a sum of two exponential functions and an instantaneous current component (Iinst):

  • image( Eqn 2)

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Anionic currents across channels of the plasma membrane of pollen protoplasts of lily show evidence for the co-existence of three different activities of outward rectifiers

In this study we applied the whole-cell configuration of the patch-clamp technique to protoplasts of pollen grains from L. longiflorum to investigate anionic currents using symmetrical Cl concentrations ([Cl]) on both sides of the plasma membrane (see Table 1 for detailed medium composition). Under these experimental conditions (control), the pipette solution (P1; intracellular) and bathing solutions (B1; extracellular) have similar [Cl] and it is expected that the membrane conductance will be dominated by Cl, the main permeable ion in solution. To study these currents, the cell membrane was clamped at a holding potential of −100 mV, followed by test potentials (Vm) ranging from −180 mV to +180 mV, with 20-mV increments (Fig. 1a(i), inset). This voltage protocol elicited steady-state currents, showing outward rectification, with the positive currents (anions entering the cell) being greater than the negative currents (anions exiting the cell). The reversal potential of all I/V curves was close to the expected equilibrium potential for Cl under these experimental conditions (ECl = −0.92 mV).

In all protoplasts, a gradual decrease in the maximum amplitude of the currents after breaking into the whole-cell configuration was observed (rundown of the current, from Fig. 1a(i) to (ii)). This behaviour is a common feature of currents from channels showing intracellular regulation by effectors, probably because of their dilution during the period required to equilibrate the cytoplasmic content of the cell with the solution from the pipette (Marty & Neher, 1995), already described for anionic channels (Becq, 1996; Binder et al., 2003). The current lost by rundown (ICl1; Fig. 1a(iv)) may be attributed to a population of channels present in the plasma membrane. Alternatively, the gradual removal of a modulating factor could affect the conductance of all channel populations in the membrane by altering their open probability, thereby impacting steady-state currents at any given voltage. After complete rundown, a stable level of current was achieved. As it is known that NPPB, a known inhibitor of anion channels, blocks tobacco and lily pollen tube growth (Zonia et al., 2002; Breygina et al., 2009a) and Cl release from tobacco pollen grain (Breygina et al., 2009a), this inhibitor was then tested on the remaining anionic current. The application of NPPB allowed us to discriminate between two additional populations of anionic currents, one that was insensitive to the inhibitor (ICl2) and one that was completely abolished in the presence of the inhibitor (ICl3). All currents measured had an instantaneous component for all tested membrane potentials, followed by two slower time-dependent activation components at the most positive potentials tested, one occurring in < 150 ms and a slower one with time constants of 896 ± 188, 440 ± 38, and 608 ± 78 ms for ICl1, ICl2 and ICl3, respectively. Fig. 1(a(i–iii)) shows a sequence of typical currents recorded from one protoplast, where the panels represent the current detected immediately after entering whole-cell configuration (Iinitial), after rundown (Ifinal) and after the application of 500 μM NPPB to the extracellular side (ICl2), respectively. The representative I/V curves are shown in Fig. 1(b). The current values for each membrane potential were averaged from the last 50 ms for each potential pulse. The current lost by rundown (ICl1) and the current inhibited by NPPB (ICl3) were isolated by point-by-point subtraction of the raw data of Iinitial − Ifinal and Ifinal − ICl2, respectively (Fig. 1a(iv-v)). The percentage of rundown of the activation currents was 37 ± 7% (−160 mV) and 27 ± 5% (+160 mV) (Table 3). The inhibition by 500 μM NPPB was 23 ± 6% (−160 mV) and 27 ± 7% (+160 mV).

For a better understanding of the behaviour of the channels responsible for the observed currents under different values of membrane potential, we converted the Cl currents to their corresponding chord conductance values (see the Materials and Methods section) and plotted the normalized values against the membrane potential. Fig. 3 shows the results obtained for ICl1, ICl2 and ICl3. The data could be described by a Boltzmann-type equation, which describes how the conductance responds to different membrane potentials. The slope factors (Vs) and the potential for the half-maximal chord conductance (Vh) from the fits are presented in Table 2, showing that ICl1 has a distinct sensitivity to variations of membrane potential values from those of ICl2 and ICl3 (which are indistinguishable from this point of view).

image

Figure 3. Voltage dependence of the average chord conductances (G/Gmax) for the control group (P1/B1; = 7). ICl1, triangles; ICl2, squares; ICl3, diamonds. Data were normalized for the maximum values and fitted with Eqn 1 in the Materials and Methods section. Fitting parameters are shown in Table 2.

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Table 2.   Fitting parameters of the voltage dependence of anionic chord conductances for the different [Ca2+]in
 nM [Ca2+]in (mV)μM [Ca2+]in (mV)mM [Ca2+]in (mV)Germinated pollen (mV)
VhVsnVhVsnVhVsnVhVsn
  1. Experimental conditions: nM [Ca2+]in (P1 or P4), μM [Ca2+]in (P2) and mM [Ca2+]in (P3). Chloride group: currents measured in B1, and substituted to B3 (ICl-NO3). Nitrate group: currents measured in B4, and substituted to B5 (INO3-Cl). Fitting parameters were obtained from Equation 1 as described in the Materials and Methods section. Data are given as mean ± SE. *, significant differences inside the same experimental group; **, significant differences between different experimental groups; < 0.05.

Chloride
 ICl113.9 ± 13.4*49.8 ± 3.99*714.4 ± 8.80*,**38.04 ± 5.42*930.3 ± 6.59*,**49.7 ± 7.63*37.6 ± 34.0*48.8 ± 10.34
 ICl2102.8 ± 6.8561.6 ± 1.03776.8 ± 4.46**69.8 ± 2.25**8102.8 ± 9.1661.3 ± 2.593103.8 ± 9.563.8 ± 5.24
 ICl393.7 ± 10.061.1 ± 2.60776.7 ± 14.152.9 ± 6.408120.7 ± 4.4458.8 ± 2.72387.7 ± 15.859.0 ± 7.93
 Ifinal115.8 ± 3.2464.9 ± 2.209   
 ICl-NO3119.7 ± 4.9073.3 ± 3.089   
Nitrate
 INO3142.4 ± 14.58*48.2 ± 1.53*4   
 INO3-final107.2 ± 11.7669.7 ± 1.624   
 INO3-Cl123.5 ± 8.5073.7 ± 0.824   

Current tail analysis confirmed the nature of the Cl currents. The results from a typical experiment performed with the same intracellular and extracellular solutions as those described in Fig. 1 are shown in Fig. 2. In this experiment, the cell membrane was clamped from −100 to +180 mV to activate the outward current for 1 s, followed by the test potentials (Vm) ranging from −140 to +140 mV, with 20-mV increments. With this kind of protocol (Fig. 2a, inset), the channels present in the membrane are at their maximum conductance state before jumping to the different test pulses. The I/V curves of the peak tail currents were linear with the applied potential and showed a reversal potential close to 0 mV, near to ECl−, as expected under anionic symmetric conditions (Fig. 2b). Furthermore, the tail currents of ICl1, ICl2 and ICl3 also underwent rundown and inhibition by NPPB, and were consistent with the values we obtained from the activation results. These results are evidence for the presence of three different populations of anion conductances, with the overall behaviour of an outward rectifier, that is, promoting chloride influx.

We attempted to observe single-channel events from outside-out patches. We were able to detect pulses of current corresponding to a channel with a major conductivity state and two substates, which could account for the populations of whole-cell macroscopic currents. It was not possible to determine whether the observed currents crossed the membrane through three different channels, or if they were produced by different conductive states of a particular channel (or channels). These issues may be resolved in time, when the molecular identity of the membrane protein(s) responsible for the currents is determined. The molecular identification of the channels is a project that was beyond the scope of the present work. For convenience, we shall continue using the ICl1, ICl2 and ICl3 designations, which result from empirical data.

The magnitude of outward currents depends on the external chloride concentration

In the experiments described, we used Cl as the major anion in the bathing solution. To confirm the nature of the Cl currents, we tested a lower [Cl]out, which resulted in an expected reduction in the outward currents (reduced entrance of chloride to the cell), while the inward currents remained relatively unchanged, as usual for channels permeable to chloride. Fig. 4 shows the average curves, obtained after rundown, for the current measured under control conditions and after exchanging to a bathing solution containing only 27 mM Cl (P1/B1 [RIGHTWARDS ARROW] P1/B2; see Table 1). The reversal potential of the whole-cell currents became more positive in comparison to the symmetrical conditions (P1/B1). Nevertheless, it did not reach the expected value for the ECl− (+40.5 mV). On average, the reversal potential was 19.7 ± 2.3 (= 6). The deviation was probably attributable to the contribution of the NO3 (ENO3 = 0 mV), which under these conditions (low [Cl]out) becomes more relevant. These results showed that the measured currents were not specific for chloride, but in conditions where chloride was the major anion present, its flux seemed to account for the overall behaviour of the currents.

image

Figure 4. Effect of [Cl]out on whole-cell currents. Average current–potential (I/V) curves of currents measured with P1/B1 (Ifinal; squares; [Cl]out = 140 mM) and after bathing solution exchange to B2 (Ilow-Cl; circles; [Cl]out = 27 mM) are shown. The arrow indicates the expected ECl− with P1/B2.

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Relative permeabilities of putative plasma membrane channels to Cl and NO3 are similar

To assess the relative permeabilities of the channels to Cl and NO3 (PNO3/PCl), Cl was partially replaced by NO3 in the bathing solution (P1/B1 [RIGHTWARDS ARROW] P1/B3; see Table 1 and Fig. 5). The reverse experiment was also performed, where the starting point was NO3-based solutions followed by the partial replacement of NO3 in the bathing solution by Cl (P4/B4 [RIGHTWARDS ARROW] P4/B5; see Table 1). The exchange of solution was performed after complete rundown had occurred. The average amplitude of the currents measured under symmetrical Cl (P1/B1) and NO3 (P4/B4) (see Table 3 for values of steady-state currents) and the corresponding forward and backward conductances (Table 4) were not significantly different, in accordance with the channels not discriminating between the two anions. No substantial changes were observed in the reversal potential of the I/V curves, a further indication of the currents being dominated by the passage of anions. The value found for PNO3/PCl determined from the deviation of the averaged Vrev (as described in the ‘Data analysis’ section of the Materials and Methods) was 1.13 (= 9) and 1.46 (= 4) for the first and second substitution experiments, respectively. The Boltzmann parameters obtained from the chord conductances were similar before and after Cl substitution by NO3 (Table 2), showing that the dependence of the anionic conductance on the membrane potential was not altered by the presence of different anions.

image

Figure 5. Relative permeability of the channels to chloride and nitrate. (a, b) Typical whole-cell currents measured with (a) P1/B1 (high [Cl]out) and (b) after bath solution exchange to B3 (high [NO3]out). (c) Average current–potential (I/V) curves of Ifinal (squares) and ICl-NO3 (circles; = 9).

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Table 3.   Steady-state chloride and nitrate currents, measured with different [Ca2+]in
 nM [Ca2+]inμM [Ca2+]inmM [Ca2+]inGerminated pollen (mV)
−160 mV+160 mVn−160 mV+160 mVn−160 mV+160 mVn−160 mV+160 mVn
  1. Experimental conditions: nM [Ca2+]in (P1 or P4), μM [Ca2+]in (P2) and mM [Ca2+]in (P3). Chloride group: currents measured in B1, and substituted to B3 (ICl-NO3). Nitrate group: currents measured in B4, and substituted to B5 (INO3-Cl). ‘% rundown’ refers to the current lost during rundown, and ‘%Δ substitution’ refers to the current variation after bathing solution substitution. Data are given as mean ± SE. *, significant differences; < 0.05.

Chloride
 ICl1 (pA)432 ± 1902086 ± 86611716 ± 2074220 ± 53210985 ± 2936721 ± 150531334 ± 5706178 ± 11984
 ICl2 (pA)451 ± 1893314 ± 13186717 ± 1844342 ± 10586143 ± 181919 ± 2723239 ± 273683 ± 6194
 ICl3 (pA)77 ± 28*688 ± 2046136 ± 40*1626 ± 493*643 ± 9*1148 ± 2193184 ± 671958 ± 7413
 % rundown37 ± 727 ± 51156 ± 746 ± 7681 ± 767 ± 7372 ± 955 ± 44
 %Δ substitution5 ± 924 ± 49   
 ICl1 (pA/pF)31 ± 16148 ± 721135 ± 12197 ± 831064 ± 16447 ± 81353 ± 23247 ± 484
 ICl2 (pA/pF)19 ± 8143 ± 55621 ± 6125 ± 32610 ± 3139 ± 37310 ± 1147 ± 254
 ICl3 (pA/pF)4 ± 233 ± 1164 ± 0.869 ± 3063 ± 0.982 ± 2237 ± 378 ± 303
Nitrate
 INO31 (pA)300 ± 2582812 ± 12754   
 INO3-final (pA)536 ± 2694185 ± 18604   
 INO3-Cl (pA)569 ± 2853806 ± 17884   
 % rundown27 ± 945 ± 74   
 %Δ substitution4 ± 317 ± 74   
Table 4.   Values of backward and forward conductances determined from currents measured with different [Ca2+]in
 nM [Ca2+]inμM [Ca2+]inmM [Ca2+]inGerminated pollen
gB (nS)gF (nS)gF/gBngB (nS)gF (nS)gF/gBngB (nS)gF (nS)gF/gBngB (nS)gF (nS)gF/gBn
  1. Experimental conditions: nM [Ca2+]in (P1 or P4), μM [Ca2+]in (P2) and mM [Ca2+]in (P3). Chloride group: currents measured in B1, and substituted to B3 (ICl-NO3). Nitrate group: currents measured in B4, and substituted to B5 (INO3-Cl). Data are given as mean ± SE.

  2. *, significant differences; < 0.05.

  3. gB, backward conductance (negative conductance); gF, forward conductance (positive conductance); nS, nanoSiemens.

Chloride
 ICl10.72 ± 0.2313.79 ± 5.2826 ± 1071.79 ± 0.4627.45 ± 4.3824 ± 792.38 ± 0.9253.95 ± 13.4530 ± 932.48 ± 0.7841.38 ± 8.8962 ± 504
 ICl21.62 ± 0.6733.14 ± 12.82*32 ± 972.18 ± 0.6540.42 ± 10.0323 ± 390.23 ± 0.0320.69 ± 3.6687 ± 530.58 ± 0.1236.14 ± 5.9266 ± 114
 ICl30.40 ± 0.14*6.56 ± 1.9135 ± 1670.65 ± 0.21*16.29 ± 5.2735 ± 890.15 ± 0.0613.50 ± 2.96132 ± 6130.94 ± 0.2417.64 ± 4.8621 ± 73
 Ifinal0.51 ± 0.2320.66 ± 7.6949 ± 109      
 ICl-NO30.61 ± 0.29*17.10 ± 6.82*31 ± 69      
Nitrate
 INO310.36 ± 0.1523.37 ± 9.2775 ± 244      
 INO3-final1.60 ± 0.8441.62 ± 18.1838 ± 104      
 INO3-Cl1.83 ± 0.9440.71 ± 18.6530 ± 94      

The three anion activities (ICl1,ICl2 and ICl3) are differentially modulated by different intracellular calcium concentrations ([Ca2+]in)

The possible effect of [Ca2+]in was investigated in three independent groups of cells by changing the [Ca2+]in from a nM concentration (6 nM; control; P1/B1) to a concentration of 8.5 μM (P2/B1) or 0.5 mM (P3/B1). For comparative purposes, the averaged values of the current measured at ± 160 mV from the three experimental groups are depicted in Table 3. Globally, all the currents were affected by Ca2+ concentration, but the behaviour of the individual components varied considerably: the amplitude of ICl1 increased with increasing [Ca2+]in from the nM concentration to 0.5 mM, while ICl2 and ICl3 showed their maximum values at the μM intracellular concentration and inhibition at the mM concentration, with a more pronounced effect on ICl2. The corresponding averaged values for the backward and forward conductances, which followed the same trend, are shown in Table 4.

To extend our understanding of the actual effect of the different [Ca2+]in, we plotted the normalized values of the chord conductances against the membrane potential. The slope factor (Vs) and the potential for the half-maximal chord conductance (Vh) from the fits of the data obtained with different [Ca2+]in are presented in Table 2. For all [Ca2+]in tested, G/Gmax(ICl1) displayed a different potential for both the half-maximal chord conductance (Vh) and the slope factor (Vs) from the other two currents, with a shift for less depolarizing potentials (with a maximum shift for μM [Ca2+]in). This result confirmed that ICl1 had a different dependence on the membrane potential from the other two currents, for all [Ca2+]in tested. The current ICl1 also responded differently to [Ca2+]in, showing a significantly different Vh in the μM and mM [Ca2+]in experiments. The values of Vs and Vh for ICl2 varied significantly in the μM [Ca2+]in experimental conditions, indicating that the sensitivity of this current population to variations in the membrane potential may be regulated by [Ca2+]in. Unlike the other two current populations, ICl3 presented a similar sensitivity to variations in the membrane potential at all [Ca2+]in. Experimentally it was not possible to go to membrane potential values higher than +200 mV and we had to assume that the maximum G/Gmax was near to the maximum attainable value (ICl2 and ICl3). For these two currents, Vs and Vh are an approximation.

We then tested the relative contribution of ICl3 to the Ca2+ sensitivity response. NPPB was added at increasing concentrations. The maximum inhibition with NPPB was attained at 500 μM for the control group (P1/B1). Fig. 6 shows the average percentage of inhibition obtained with 500 μM NPPB for the three experimental groups. We observed that, in the presence of μM and mM [Ca2+]in, the maximum attainable inhibition required higher inhibitor concentrations (1750 and ≥ 2000 μM, respectively). Corresponding control experiments with DMSO were performed with nM (= 7) and μM [Ca2+]in (= 6). We observed that, for concentrations of NPPB > 500 μM, the effect of the solvent had to be taken into consideration. We cannot discount the possibility of intracellular calcium directly affecting the apparent affinity of the channels to the inhibitor. Nevertheless, all data obtained support the regulation of the activity of the three populations of channels by [Ca2+]in (current amplitude, conductances and Boltzmann parameters). The maximum values of inhibited current obtained with the three calcium concentrations were similar, confirming that NPPB inhibited the same population of channels (ICl3) under the three different experimental conditions. This current corresponds to c. 25% of the current remaining after the occurrence of rundown.

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Figure 6. Average 5-nitro-2-(phenylpropylamino)-benzoate (NPPB) inhibition (500 μM) of chloride currents measured in the presence of different [Ca2+]in (P1, P2 and P3). nM [Ca2+]in, white bars; μM [Ca2+]in, wide hatched bars; mM [Ca2+]in, narrow hatched bars; germinated pollen, narrow horizontal bars. *, significant differences; < 0.05.

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Anionic channel currents measured from germinated pollen also present three different activities of outward rectifiers, similar to those from hydrated pollen

Anionic currents were also investigated in germinated pollen. To access the pollen tube plasma membrane, the germinated grains were submitted to enzymatic digestion of the cell wall for a short incubation period of c. 40 min, and protoplasts corresponding to the pollen tube zone were selected on the basis of size and cellular content, being clearly distinguishable from the sporoplast. This protocol does not, however, allow discrimination between the membrane from the sides of the tube and from the tip, and we therefore had to assume that, on the balance of probability, the currents were from the tube membrane. Nevertheless, the protocol for the isolation of the anionic currents was the same as previously described, and the currents observed were overall similar (= 4). Fig. 7 shows the anionic currents measured with P1/B1 solutions (nM [Ca2+]in) under exactly the same experimental conditions as those described for the currents shown in Fig. 1. The three detected anionic activities showed strong outward rectification, and the current–voltage relationships reversed near 0 mV, as expected for anionic currents measured with symmetric Cl-based solutions. Tables 2 and 4 contain the values for the Boltzmann parameters and for the conductances, respectively, calculated for these currents. These values are not significantly different from those obtained for the currents measured from the pollen grain, suggesting that the same putative channels are responsible for the currents, both in the grain and in the tube.

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Figure 7. Typical chloride whole-cell currents from a germinated pollen grain of Lilium longiflorum, measured with P1/B1 solutions (nM [Ca2+]in). The currents were elicited from a holding potential of −100 mV with the voltage protocol depicted in (a(i)). (a) (i) The initial current recorded immediately after entering the whole-cell configuration (Iinitial). (ii) The current recorded after rundown (Ifinal). (iii) the current recorded after inhibition by 500 μM NPPB (ICl2). (iv) The current lost during rundown (ICl1) obtained by subtraction of Iinitial and Ifinal. (v) Inhibited current (ICl3) obtained by subtraction of Ifinal and ICl2. (b) Current–potential (I/V) relationships of Iinitial (squares), Ifinal (circles) and ICl2 (triangles).

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The values obtained for the percentage of rundown were 72.01 ± 9.3% (−160 mV) and 54.89 ± 4.4% (+160 mV) (Table 3). The values found are greater than those obtained with nM [Ca2+]in, and approximate those obtained from hydrated pollen protoplasts with higher [Ca2+]in. In contrast, the currents ICl2 and ICl3 obtained from germinated pollen gave values that approximate those obtained from a hydrated grain with the lower [Ca2+]in tested. This result suggests that the relative contributions of ICl1, ICl2 and ICl3 to the total current crossing the cell membrane at a given time may differ between the hydrated grain and tube growth (probably as a consequence of regulatory mechanisms other than Ca2+). Consistent with this finding, the percentages of inhibition with 500 μM NPPB were 43.10 ± 11.9% (−160 mV) and 36.53 ± 13.9% (+160 mV), significantly different from the values obtained with hydrated pollen protoplasts (Fig. 6).

Anionic single-channel currents across the plasma membrane of lily pollen protoplasts

Single-channel anionic currents were measured from isolated outside-out membrane patches of pollen grain protoplasts with the same intra- and extracellular solutions as those used for whole-cell currents (P1/B1). Before the measurement of single-channel currents, we confirmed the outside-out configuration by measuring the currents in response to the application of the activation voltage protocol. Fig. 8(a) shows a typical set of currents measured at different membrane potentials, jumping from a holding potential of −100 mV. These currents, although much smaller in amplitude than those obtained with the whole-cell configuration, showed the typical outward rectification, and the tail transient currents resulting from the deactivation of the channels. To study single-channel events we used longer recordings and higher sampling rates. Fig. 8(b) shows the currents measured from an outside-out patch measured at different membrane potential values (+150, 0 and −150 mV). For potentials ≥ +50 mV we systematically observed bursts of activity with flickering, which displayed greater amplitude with increasing positive potential values (increasing electrochemical gradient for Cl). This behaviour was consistent during the 20 s of the recordings and was observed in all the tested cells (= 38). For negative potentials, we observed a noisier baseline for membrane potentials ≤ −100 mV, which was probably an indication of channel activity. In two cells, we detected single-current events for membrane potentials ≤ −100 mV. We observed only two or three longer opening events per 20-s recording, while positive currents were active during the entire measurement period. The measured currents displayed opposite directions for positive and negative potentials, according to the applied electrochemical gradients. Concurrently, we never observed unitary currents at 0 mV, which is the reversal potential expected for the currents (and which corresponds to the equilibrium potentials for Cl and NO3, the two anions present in solution).

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Figure 8. Typical anionic currents from an excised outside-out patch of a pollen grain protoplast of Lilium longiflorum (control group; nM), measured with P1/B1 solutions. (a) The currents were measured as described in Fig. 1. (b) Single-channel events acquired at −100, 0 and +100 mV membrane potentials. C, closed state of the channel; O, open state(s) of the channel.

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The anionic nature of the unitary currents was confirmed by the use of NPPB (= 2), a known inhibitor of chloride channels which inhibited a population of the whole-cell currents (ICl3). Fig. 9 shows currents measured from one cell in the absence (control) and the presence of 500 μM NPPB. The inhibitor caused a shift of the baseline towards values closer to zero for both positive and negative membrane potentials, consistent with an inhibitory effect. This result suggests the presence of small conductance constitutive channels which maintain constant macroscopic levels of anionic current across the plasma membrane of pollen grain protoplasts. We could, on occasion, observe single-channel events resulting from the inhibition.

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Figure 9. Outside-out anionic currents recorded at 100, 0 and +100 mV membrane potentials before (a) and after the application of 500 μM 5-nitro-2-(phenylpropylamino)-benzoate (NPPB) to the extracellular side (b). The inhibitor reduces the overall amplitude of the current baseline, and exposes single-channel events. C, closed state of the channel; O, open state of the channel.

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Although the electrical signals were not suitable for an open probability analysis of the currents, we were still able to detect unitary currents showing different levels of conductance. Fig. 10 shows one such recording. As the simultaneous opening of three channels is an improbable occurrence, it is likely that these levels of current represent different conductance substates from one channel (C[RIGHTWARDS ARROW]O3). This also holds for the simultaneous double closing (O2[RIGHTWARDS ARROW]C). Although noisier, the currents measured at positive membrane potentials also presented, at times, different levels (not shown).

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Figure 10. An example of a burst of channel activity showing three conductance states. The currents were measured from an outside-out excised patch held at −100 mV. C, closed state of the channel; O, open state(s) of the channel.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

By employing the patch-clamp technique in the whole-cell configuration, we identified three distinct anionic current populations regulated by [Ca2+]in (ICl1, ICl2 and ICl3) sharing strong outward rectification. Throughout this work we have treated ICl1, ICl2 and ICl3 as individual currents. Each of these currents comprises instantaneous and slow activation components (Iinst, I1 and I2) undergoing similar magnitudes of rundown and inhibition. Similarly, Marten et al. (1999) detected whole-cell K+ currents resulting from the heterologous expression of AKT3 (Arabidopsis Potassium Transporter 3, a potassium channel from vascular tissue of Arabidopsis) in Xenopus oocytes which presented instantaneous and time-dependent components. These authors also described similar inhibition profiles and rundown behaviour for the two current components through the potassium channel. In addition, the heterologous expression of the Ca2+-activated channel mTMEM16A in HEK293 cells produced whole-cell currents with a similar profile to those detected here, with faster and slower activation components (Schroeder et al., 2008). Under control conditions (P1/B1), ICl1 was identified as the current lost by rundown, possibly being modulated by an unknown intracellular regulator that was diluted in the pipette solution replacing the intracellular ionic contents. ICl3 was identified as the current that was inhibited by NPPB, and ICl2 was the current that remained after the loss of ICl1 and that was insensitive to NPPB. We do not exclude the possibility that ICl1 could also be inhibited by NPPB. Nevertheless, the attempts to seal in the presence of NPPB proved unsuccessful. All I/V curves showed Vrev close to the expected ECl− and the positive currents diminished with low [Cl]out, with a corresponding shift of Vrev towards positive potentials, strong indications that this anion is the major contributor to these currents. Tail analysis confirmed the anionic nature of the currents, with the I/V curves being linear, reversing at the equilibrium potential expected for Cl and NO3, undergoing rundown and being partially inhibited by NPPB.

Nevertheless, the three current populations displayed distinctive features, with ICl1 showing features that were clearly distinct from those of the other two. ICl1 increased with higher [Ca2+]in, displaying maximum currents in the presence of 0.5 mM [Ca2+]in, while ICl2 and ICl3 had higher values for μM [Ca2+]in. As the time constants that characterized the rise of the currents did not vary with the tested [Ca2+]in, it is likely that Ca2+ regulates the number of open channels contributing to the current at a given moment. The G/V curve for ICl1 was different from those of ICl2 and ICl3, with different potential values for the half-maximum chord conductance (Vh) and the slope factor (Vs). The currents measured with symmetrical NO3 showed a shift of the midpoint potential (Vh) by c. +30 mV, also indicating a different sensitivity of the channels to the potential in the presence of this anion. The average inhibition caused by 500 μM NPPB for ICl3 (measured with nM [Ca2+]in; +160 mV) was significantly different from the inhibition values obtained for the test groups (measured with μM and mM [Ca2+]in).

To control the [Ca2+]in tested, we used a very low [Ca2+]out (3 mM) and an additional general blocker for Ca2+ currents (Gd3+; Wang et al., 2004) to prevent the entrance of Ca2+ into the cell. The presence of Gd3+ also facilitated giga-seal formation (Dunina-Barkovskaya et al., 2004). Under these conditions, the success of sealing and the maintenance of stable giga-seals was low (10%), but once a high resistance seal was established it remained stable for several hours. While not physiological, in this study we used a broad range of potential values for studying the currents, a common procedure in this kind of patch-clamp approaches. The extreme positive values were necessary to attain linearity of the currents with the applied potentials. These values could then be used for the determination of the conductance ratios and the respective Boltzmann parameters that were used for comparison purposes between experimental groups. Although we had no indications of seal destabilization in the transition to whole cell, because of the strong outward rectification observed, we had to consider the possibility of the negative currents being attributable to the passage of ions directly between the pipette and the bathing solutions. Attempts to subtract the possible leak current led to an overestimation of the current values, which became positive for negative potentials. Additional experimental evidence showed that the negative currents measured were certainly passing through channels in the membrane: they underwent rundown and were partially inhibited by NPPB, just as observed for the positive currents. Therefore, the anionic channels described here displayed the ability to conduct ions both inwardly and outwardly, according to the anionic electrochemical gradient found under natural conditions.

We consistently observed that the currents suffered rundown over time (c. 90 min). Binder et al. (2003) observed a similar behaviour over time while studying the Cl currents in protoplasts from the marine alga Valonia utricularis and attributed it to a retarded equilibration of the cytosol with the pipette solution, as indicated by alterations of the Vrev of the detected currents until it stabilized near the predicted ECl−. In this study, we observed no drifts of the reversal potential of the measured currents from ECl−, which may indicate that the equilibration is faster in these protoplasts. This also applied to the currents measured with symmetrical NO3. Alternatively, the natural intracellular anion concentration may be similar to those in the pipette solution. We observed that the currents from occasional outside-out patches had the same characteristics as, although smaller amplitudes than, those measured from full protoplasts. A previous study by Dutta & Robinson (2004) showed no Cl currents in outside-out patches from lily pollen protoplasts, although the same group could measure unitary current events attributable to K+ and Ca2+. As we were able to measure whole-cell currents of very small amplitude in outside-out patches, it is possible that these authors may have misinterpreted the macroscopic even current levels with the absence of channel activity (Fig. 8a). Although uncommon, macroscopic currents have been observed from outside-out patches (e.g. Whitehead et al., 1998; Artigas & Gadsby, 2002). Here, we obtained values for whole-cell currents measured from protoplasts of between 1.3 and 15 nA, in response to a voltage command of +160 mV. The currents measured under similar conditions from outside-out patches were smaller, as expected, although in some cases they reached c. 1 nA. Another experimental difference that could explain the absence of Cl channels was the use of extremely high [Ca2+]out (40 mM) by Dutta & Robinson (2004). There is evidence of inward K+ currents in Arabidopsis pollen grain protoplasts regulated by [Ca2+]out (Fan et al., 2001).

Whole-cell data do not allow us to determine if these currents cross the membranes of these cells through one or more than one type of channel. Additionally, each of these three isolated populations may represent different channels sharing similar conductance and/or pharmacological characteristics. The identification of single-channel events from isolated outside-out patches provided some hints about the nature of the outward rectifying whole-cell currents. This experimental configuration has been successfully used to identify unitary currents through plant channels (Hedrich & Jeromin, 1992; Piñeros & Kochian, 2003; Wang et al., 2004; Wu et al., 2010). As we detected macroscopic currents from spontaneous outside-out vesicles that formed during whole-cell measurements with 8–10 MΩ pipettes, our strategy was to use pipettes with a smaller aperture than those used to study the whole-cell currents (25 MΩ), in order to obtain smaller patches of membrane that could, eventually, show single-channel events. Our results demonstrated the presence of single-channel anion currents across the plasma membrane of lily pollen protoplasts displaying at least three conductive substates. The detected single event currents ranged from a few pA up to 30 pA, for currents measured at ± 100 mV. These events superimpose a baseline which probably represents the summation of currents from a number of channels. The direction and amplitude of the currents followed what was expected according to the electrochemical gradient for the anions in solution (with Cl as the major permeant anion). The effect of the inhibitor NPPB confirmed the anionic nature of the currents. The results suggest the presence of open channels at all times for the different potentials, in agreement with the measured whole-cell currents. Moreover, the measurements were made with the same intra- and extracellular solutions used for studying the macroscopic anion currents, and therefore it is likely that they contribute to these currents.

The whole-cell current ICl1 showed Boltzmann parameters that were clearly distinct from those of the other two current populations for all the experimental conditions tested. Moreover, ICl2 and ICl3 had similar dependences of the conductance on the applied Vm values (Fig. 3, Table 2). The most straightforward interpretation of these results is that there are two channel types in the membrane of lily pollen protoplasts with different sensitivities to the membrane potential. The single-channel currents depicted in Fig. 10 seem to result from the activity of one type of channel, the electrical behaviour of which is characterized by at least three conductive states. Although it is unlikely, these states could also derive from the activity of two or more channels. Only the positive molecular identification of the channel (or channels) responsible for the observed currents will provide, hopefully in the near future, a definitive answer to these questions.

Several anion channels have been identified in the plasma membrane of plant cells. These have been associated with various processes in plants, such as stomatal closure, hormone signalling, membrane excitability, cellular osmoregulation, growth regulation and anionic nutrition (reviewed in Barbier-Brygoo et al., 2000; Roberts, 2006; Angeli et al., 2007). The regulation of stomatal opening and closure in Vicia faba has been extensively studied and involves the coordination of a variety of ionic fluxes. Namely, the S-type anionic channel is responsible for the entrance of anions during stomatal closure. This channel is highly regulated by [ATP]in (phosphorylation), [Ca2+]in, and ABA. Other anion channels have been identified in the plasma membrane of these cells. The R-type, involved in membrane excitability and auxin sensing, is also activated by [ATP]in (nucleotide binding) and [Ca2+]in, and regulated by intracellular pH and extracellular anions. Both show a higher permeability to NO3 than to Cl. Most of the described anion channels are inward rectifiers (channels through which the exit of anions from the cell is greater than their entrance). Some outward rectifiers have been detected in root cells and have been suggested to be involved in repolarization of the membrane potential after sodium uptake by roots in saline soils (Skerret & Tyerman, 1994). The anionic selectivity of the channels to NO3 and Cl, as well as their pharmacology and regulation, depends on species and cell type.

The majority of the currents reported here were measured from protoplasts of hydrated pollen grains, before the onset of germination. These results, together with the single-channel currents measured from the same cellular preparation, demonstrate beyond doubt the existence of anionic fluxes across channels in the plasma membrane of ungerminated grains. Moreover, we report anionic currents from germinated pollen which probably result from the same kind of putative channels responsible for the currents across the plasma membrane of the hydrated grain. It has been established that pollen tube germination does not require extracellular anions (Weisenseel & Jaffe, 1976). Nevertheless, our results, combined with those of Zonia et al. (2002) and Breygina et al. (2009a), suggest that pollen grains may have considerable anionic reserves, which are readily available for transport across the membrane. If this is the case, the channels responsible for the currents we observed could be involved in the exit of anions during grain hydration and tube growth processes. Although displaying the same general characteristics (e.g. strong outward rectification, deactivation transient currents), our results suggest that the relative contributions of the three current populations to the total amount of anionic current are different before and after tube germination. The existence of Ca2+ domains has been demonstrated by means of imaging techniques, with intracellular nM values for [Ca2+]in in most of the tube, and an increasing concentration gradient towards the tip up to μM concentrations (Pierson et al., 1994; Holdaway-Clarke et al., 1997; Messerli & Robinson, 1997; Messerli et al., 2000). There are suggestions that [Ca2+]in may reach mM domains close to the plasma membrane at the tip. On the basis of our results, one can speculate that the channels responsible for ICl1 may be involved in the anionic apical effluxes detected at the tip of the growing tube (Zonia et al., 2002) where [Ca2+]in reaches its maximum. We do not exclude, however, the possibility that the anionic currents from lily protoplasts may additionally be distinctly regulated by other intracellular or extracellular effectors, reflecting the different stages of pollen physiology. For instance, it has been shown that inositol 3,4,5,6-tetrakisphosphate modulates the oscillatory Cl efflux at the pollen tube apex, thereby playing a possible role in tube growth and volume regulation (Zonia et al., 2002). A recent study (Chen et al., 2010), in which concurrent measurements of anionic currents and [Ca2+]inin vivo were performed, suggests the existence of three modes by which anionic S-type currents may be regulated, one of which is Ca2+-dependent. Additionally, both Ca2+-dependent and Ca2+-independent currents showed an extra regulation involving protein dephosphorylation, investigated with okadaic acid, a protein phosphatase antagonist.

We speculate that many of the channels already present in the plasma membrane of the grain protoplasts may suffer a spatial redistribution to specific regions of the membrane during pollen tube germination and growth or that they undergo recycling. Previous studies in the growing pollen tube showed proton fluxes (per area unit) similar to those detected in the hydrated grain, probably attributable to relocation/recycling of the transporters, or de novo synthesis, balancing transporter concentrations (Feijóet al., 1999; Certal et al., 2008). The results presented here seem to confirm that the same putative channels could be responsible for the currents measured from hydrated pollen and during pollen tube growth, but responding to the natural spatial and temporal variation of [Ca2+]in (and other factors). At this point we cannot discriminate between a redistribution of the channels initially present in the membrane and the de novo synthesis of these membrane proteins. We observed a great variation in current density between cells, probably reflecting differences in the number of active channels in the membrane. The error associated with the mean I/V curves obtained with the measured currents normalized to their respective capacity values was greater than the error associated with the nonnormalized currents, particularly in ICl1. Therefore, we chose to use the original current values. For comparison purposes, the normalized values are shown in Table 3. Under physiological conditions it is expected that the membrane potential of the hydrated grain and that of the pollen tube are intrinsically negative (Feijóet al., 1995; Breygina et al., 2009a,b). We have estimated the equivalent ionic fluxes to the Cl currents measured with nM Ca2+, by assuming a spherical shape for the membrane surface and calculating the corresponding current density (pA cm−2). The values were transformed to fluxes by dividing them by the product of the unitary charge for Cl and the Faraday constant. For the negative membrane potential values (−160 to −60 mV), we obtained values for the fluxes ranging on average from 300 to 4000 pmol cm−2 s−1, according to a given radius (10 to 3 μm, respectively). These values were estimated from Iinitial and are comparable to those reported by Zonia et al. (2002) which were measured with vibrating probes and which showed an oscillatory efflux that ranged from 50 to 8000 pmol cm−2 s−1, under a low extracellular Cl concentration (c. 1.1 mM). We followed the same methodology to estimate the fluxes for the germinated pollen, assuming a tube diameter of 15 μm with a length of 200 μm. The values ranged from 90 to 167 pmol cm−2 s−1, the upper limit being much lower than the peak value reported by Zonia et al. (2002). This could reflect the different anionic electrochemical gradients. Alternatively, one must take into account that the net fluxes obtained with the vibrating probes were not measured under voltage control and reflected the sum of both electrogenic and nonelectrogenic transport systems. The whole-cell currents measured from the germinated pollen tube were greater than those measured from the grain (c. 3 times) but the estimated fluxes for the germinated pollen tube were smaller (3.3 times for −60 mV and 23 times for −160 mV). These observations further support a possible relocation of the channels throughout a greater membrane surface, or a preferential migration towards the tube resulting in the clustering of channels. This would also explain the macroscopic currents measured from outside-out patches. Here we were unable to address this point further without establishing the actual localization of the channel(s).

The currents described here share similarities with the few outwardly rectifying depolarization-activated anion channels (OR-DAACs) already described in plants. Nevertheless, the kinetic properties of the currents studied here are distinctive from the former. An anion channel that only allows outward current flow has been identified in protoplasts derived from wheat (Triticum aestivum) roots. It activates in < 100 ms and is positively regulated by nM [Ca2+]in (Skerret & Tyerman, 1994). Outward rectifier channels in roots of L. albus display a slower kinetics (seconds) which is dependent on the holding potential (Zhang et al., 2004). The currents measured here from lily protoplasts were not dependent on the holding potential (−100, 0 and +100 mV; data not shown). The channels described are involved in stabilization of the plasma membrane voltage in different ionic environments. The T. aestivum OR-DAAC is probably involved in Cl uptake in high-salinity conditions, to maintain a negative membrane potential after the influx of Na+; the L. albus OR-DAAC is involved in the adaptation to phosphorus deficiency. We suggest that the putative anionic channels from the plasma membrane of lily pollen protoplasts may be specific to these cells and have a unique function during hydration and tube growth, possibly keeping the membrane potential within negative physiological values, while counterbalancing the cationic movements across the membrane, and maintaining the electroneutrality of the medium. By following these equilibration movements, it is expected that chloride also plays a role in the transport and equilibration of water, as previously demonstrated (Zonia et al., 2002).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by funding from the Fundação para a Ciência e a Tecnologia, Portugal, through the Project PTDC/QUI/64359/2006 awarded to A.B. and the PhD fellowship SFRH/BD/27399/2006 awarded to B.T. The laboratory of J.A.F. is supported by the Centro de Biologia do Desenvolvimento (FCT U664) and FCT grants BIA-BCM/108044/2008.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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