ABSTRACT
Hyperpolarization-activated calcium channels (HACCs) are found in the plasma membrane and tonoplast of many plant cell types where they have an important role in Ca2+-dependent signaling. The unusual gating properties of HACCs in plants, i.e., activation by membrane hyperpolarization rather than depolarization, dictates that HACCs are normally open at physiological hyperpolarized resting membrane potentials (the so called pump or P-state), thus, if not regulated, they would be continuously leaking Ca2+ into cells. In guard cells, HACCs are permeable to Ca2+, Ba2+ and Mg2+, activated by H2O2 and the plant hormone abscisic acid (ABA) and their activity is greatly reduced by low amounts of free cytosolic Ca2+ ([Ca2+]Cyt) and hence will close during [Ca2+]Cyt surges. Here we demonstrate that the presence of the commonly used Mg-ATP inside the cell greatly reduces HACC activity especially at voltages ≤ −200 mV and that Mg2+ causes this block. We therefore conclude, firstly, that physiological cytosolic Mg2+ levels affect HACCs gating and that channel opening requires either high negative voltages (≥ −200 mV) and/or displacement of Mg2+ away from the immediate vicinity of the channel. Secondly, based on structural comparisons with Mg2+-sensitive animal inward-rectifying K+ channel, we propose that the likely candidate HACCS described here are cyclic nucleotide gated channels (CNGCs), many of which also contain a conserved di-acidic Mg2+-binding motif within their pores. This conclusion is consistent with the electrophysiological data. Finally, we propose that Mg2+, much like in animal cells, is an important component in Ca2+ signalling and homeostasis in plants.
INTRODUCTION
Ca2+ has long been recognized as an essential component in many plant cellular processes and in order for Ca2+ to function as an intracellular signal, temporal, spatial and stimulus specific changes in [Ca2+]Cyt need to be tightly controlled (1). Ca2+-influx into plant cells is achieved, at least in parts, by three main types of Ca2+-channels (2,3): Firstly, channels that show little or no voltage sensitivity, referred to as non-selective calcium channels (NSCCs) primarily for being active at all voltages and for being less selective for calcium over monovalent cations such as K+ and Na+ (4); secondly, channels that show high voltage-dependence like those activated by depolarization (DACCs) (5) and thirdly others, which are the focus of this report, activated only by membrane hyperpolarization (HACCs) (6).
In guard cells, Ca2+ was shown to be involved in abscisic acid (ABA) signaling and stomatal guard cell movements (7,8) and using the patch clamp technique in both whole cell (WC) and excised configurations (EC), two types of HACCs were identified at the plasma membrane (PM). One type of HACCs is highly selective for Ca2+ (over K+ and Cl−) (9). Its activity is enhanced by ABA (9,10), H2O2 (10), and by external Ca2+ itself, an apparently unique property of this particular plant HACC (11). Meanwhile, increasing [Ca2+]Cyt from 0.2 to 2 μM decreased the open probability (Po) of this HACC by a factor of 10 (9), implying a critical role of [Ca2+]Cyt in the feedback regulation of the channel. The proteins responsible for this type of channel have not being identified to-date. This is not the case for the other type of HACCs where a family of 20 genes is known and their translation products were originally described as being mostly gated open by cyclic nucleotides such as 3’,5’-cyclic adenosine monophosphate (cAMP) or 3’,5’-cyclic guanosine monophate (cGMP) (12), hence their name: cyclic nucleotide gated channels (or CNGCs). Moreover, these plant channels, like their animal functional homologs, poorly discriminate between divalent and monovalent cations. Indeed, when heterologously expressed in oocytes, these channels are not only permeable to Ca2+ (13) but found to be equally permeable to monovalent cations such as K+, Rb+, Na+, Li+ and Cs+ (13,14). Similar inwardly-rectifying currents permeable to either Ca2+, Ba2+ and even Na+, activated by cAMP (15) and cGMP (16) were also characterized in guard cells with the patch clamp technique. The latter work demonstrated that the highly expressed CNGC5 and CNGC6 genes in guard cells are directly responsible for the recorded HACC activity. Likewise, in pollen tubes of Asian pear tree (Pyrus pyrifolia Nakai cv. Hosui), a HACC that conducts indiscriminately either Ca2+ or K+ was shown to be activated by cAMP and down regulated by high [Ca2+]Cyt (17). It is noteworthy that all these type-two HACCs share one common characteristic, they are sensitive to low concentrations of Lanthanides (10,15-17).
We know from previous work, particularly in animal systems, that Mg2+ ions are key regulators of many ion channels and receptors (18). For instance, the inwardly rectifying K+ (Kir) channels and TRPV6, a member belonging to a subgroup of transient receptor potential (TRP) cation channels, both show strong Mg2+-dependent gating (19,20). Another example of a voltage-dependent block by Mg2+ ions is the N-methyl-D-aspartate (NMDA) receptor (21,22); in this case extracellular Mg2+ is responsible for this effect. By contrast, in plants, the rectification of IK,in was found not to be due to a Mg2+-dependent block (23) but rather to an intrinsic property of the channel protein itself (24). However, ion channels localized to the tonoplast with a key role in stomatal volume regulation, such as the slow- (SV) and the fast- (FV) activating vacuolar channels, were shown to be affected by cytosolic Mg2+ ions (25–28). Indeed, it was found that, besides Ca2+, Mg2+ also promoted the activation of SV channels affecting their kinetics (time constants of channel activation and de-activation) and voltage-dependent activation characteristics. At the same time, Mg2+ inhibits FV channels, thus reducing K+ leakage from the tonoplast (25). Mg2+ was also shown to inhibit an outward NSCC, termed MgC, which was characterized in the PM of guard and subsidiary cells of Vicia faba and Zea mays (29). Another NSCC example from N2-fixing plants where Mg2+ plays a critical role is related to the transport of ammonium (NH4+)/ammonia (NH3) across the peribacteroid membrane (30). This is achieved in part through a passive non-selective electrogenic transport system, regulated by Ca2+, but more recently, this channel was described as having ‘unusual’ characteristics such as an inward rectification caused by Mg2+ on the cytosolic face and a very low single channel conductance (< 0.2 pS with 150 mM KCl + 10 mM CaCl2 in the pipette and 150 mM NH4+ in the bath) which was found to be inhibited by Mg2+ from the luminal face of the symbiosome (31).
ATP is another major regulator of ion channel gating. One of the best characterized channels is the KATP, an inward K+-rectifier (from the Kir family of genes) found mostly in cardiac and skeletal muscles, neurons and pancreatic β-cells (18). These channels are normally closed in the presence of ATP and only open, to hyperpolarize the cell membrane, when cytosolic ATP levels drop. ADP added in the form of Mg-ADP can restore the activity of KATP that were pre-treated with ATP (32,33). In guard cells, Mg-ATP is required for blue light-activated outward currents (34). Indeed, it was found that 1 to 2 mM Mg-ATP, as well as other intracellular substrates, are required to fully activate a plasma membrane electrogenic ion pump capable of hyperpolarizing the membrane to around −140 mV, a potential well beyond the activation threshold for IK,in (35).
Given the importance and the key role that calcium channels play in plant cellular signaling including guard cell aperture regulation, we address the question of whether internal Mg2+ can affect HACCs activity in guard cell protoplasts (GCPs) and show examples of the Gd3+-sensitive Ba2+-currents (IBa) activated by hyperpolarization with and without Mg2+ in the patch pipet. We also assess important properties of IBa in the absence of Mg2+ such as the permeability and sensitivity to some relevant inorganic compounds and physiological effectors such as abscisic acid (ABA) and cAMP.
EXPERIMENTAL PROCEDURES
Protoplast isolation
Vicia faba L. cv (Bunyan) Bunyan Exhibition seeds were grown on vermiculite under conditions described previously (36). Arabidopsis thaliana (Columbia) seeds were grown on peat pellets (jiffy, Oslo) in a controlled environment growth chamber (Percival, CLF plant climatic, Wertingen) at 22 °C on a 8/16-h light/dark cycle. Guard cell protoplasts (GCPs) were isolated from either 3- to 4- week V. faba or 5- to 6- week A. thaliana plants. GCPs were isolated from abaxial epidermal strips as described previously (37). Briefly, epidermal strips were floated on medium containing 1.8-2.5% (w/v) Cellulase Onozuka RS (Yacult Honsha, Tokyo, Japan), 1.7-2% (w/v) Cellulysin (Calbiochem, Behring Diagnostics, La Jolla, CA), 0.026 % (w/v) Pectolyase Y-23, 0.26 % (w/v) BSA, and 1 mM CaCl2 (pH 5.6) with osmolality adjusted with sorbitol to 360 mOsm.kg−1. After 90-120 min incubation in the dark at 28 °C with gentle shaking, released protoplasts were passed through a 30 μm mesh, and kept on ice for 2 to 3 min before centrifugation (100 g for 4 min at room temperature). The pellet consisting of GCPs was re-suspended and kept on ice in 1 or 2 ml of fresh medium containing 0.42 M mannitol, 10 mM 2-(N-morpholino)ethanesulfonic acid (Mes), 200 μM CaCl2, 2.5 mM KOH (pH 5.55 and osmolality at 466 mOsm.kg−1). Unless stated otherwise, all chemicals were from Sigma (Sigma-Aldrich Co. St Louis, MO).
Solutions
Protoplasts were placed in a 0.5 ml chamber, left to settle down, and then perfused continuously at flow rate of ≈ 0.5-1 ml/min. To record IBa currents through HACC, we used barium containing solutions. The bath medium contained (in mM): 100 BaCl2, 10 Mes (pH 5.5 with Tris base) and the pipette contained (in mM): 100 BaCl2, 4 EGTA, 10 Hepes (pH 7.5 with Tris). Experiments where IK,in and IBa measurements were made on the same GCPs a different bath and internal solutions as follows. Bath (in mM): 30 KCl, 10 Mes (pH 5.5 with Tris base) to measure IK,in and was replaced by 100 BaCl2, 10 Mes (pH 5.5 with Tris base) to measure IBa. Internal solution (in mM): 1 BaCl2, 18 KCl, 4 EGTA, 10 Hepes (pH 7.5 with Tris base). Mg-ATP, MgCl2 and/or K2-ATP were added as specified in the figures. Osmolality was adjusted with Sorbitol to 210mOsm.kg−1. For classic solutions used to measure IK,in (Fig. 4 A, and B), refer to (38). ABA was added externally. All chemicals were from Sigma Chemical, Poole Dorset, UK. The membrane permeable cAMP analog Bt2cAMP was solubilized in deionized water and stored in aliquots of 50–100 μl at a concentration of 0.1 M. Bt2cAMP was diluted to the final desired concentration just a few minutes before its use.
Current-voltage recording and analysis
Patch pipettes (5-10) were pulled from Kimax-51 glass capillaries (Kimble 34500; Kimble, Owens-Illinois) using a two stage puller (Narishige PP-83, Japan). Experiments were performed at room temperature (20 to 22 oC) using standard whole-cell patch clamp techniques, with an Axopatch 200B Integrating Patch Clamp amplifier (Axon Instruments, Inc. Union City, CA, U.S.A.). Voltage commands and simultaneous signal recordings and analyses were assessed by a microcomputer connected to the amplifier via multipurpose Input/output device (Digidata 1320A) using pClamp software (versions 8.0 and 10; Axon Instruments, Inc.). After Giga-Ohm seals are formed, the whole-cell configuration was then achieved by gentle suction, and the membrane was immediately clamped to a holding voltage (hv) of −36 mV. GCPs were continuously perfused throughout the experiment and current recordings began only after at least 5-10 min from going into whole-cell mode to allow for intracellular equilibrium between cytoplasm and patch pipet solution. All current traces shown were low-pass filtered at 2 kHz before analog-to-digital conversion and were uncorrected for leakage current or capacitive transients. Membrane potentials were corrected for liquid junction potential as described (39). Ionic activities were calculated using GEOCHEM-EZ (40). Current-voltage (I-V) relationships for IBa and IK,in were plotted as steady-state currents vs. test potentials when using the square pulse stimulations or utilizing the “Trace vs Trace” feature of Clampfit analysis when using voltage ramps. Unless otherwise stated, every experiment reported here was repeated a minimum of three times and data were graphed as mean±s.e.m.
Homology modeling
Models were built using Swiss Model and structures visualized with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC). Multi-sequence alignments were produced with Muscle (43).
RESULTS
Removal of intracellular Mg-ATP unveils a larger instantaneously-activated, inwardly-directed and Gd3+- sensitive Ba2+-current
We recorded Ba2+-currents (IBa) in guard cell protoplasts either in the presence or absence of Mg-ATP (Fig. 1A) and at the end of the trials we also tested for IBa sensitivity to Gd3+, a potent blocker of IBa (Fig. 1B). The recorded currents were generated in response to a square pulse protocol from +64 mV to −256 mV in increments of −20 mV with the holding voltage (hV) set to −36 mV. In the presence of Mg-ATP (Fig.1 A; left panel), a small instantaneous inward-rectifying current of ≈ −20 pA started to activate around −200 mV to only reach a maximum of −60 pA at −256 mV. This current was, as expected, sensitive to the addition of extracellular Gd3+ (see Fig. 1B; left panel). Meanwhile in the absence of Mg- ATP (Fig. 1A; right panel), much larger (≥ 8-fold) instantaneous rectifying-currents, which happened to be also Gd3+-sensitive were recorded (Fig. 1B; left traces). The current-voltage (I-V) relationships in the presence (●; n=3) or absence (○; n =7) of Mg-ATP are represented in Figure 1C (left panel). We also plotted the effect of Gd3+ on the I-V relationships in the presence (■) or absence (□) of Mg-ATP (Fig. 1C; right panel). These results highlight that when omitting Mg-ATP from the intracellular medium, a larger Gd3+- sensitive inwardly-rectifying Ba2+-current is unveiled that activates at significantly less negative voltages (see the shift to the right of ≥ −100 mV in the I-V plot). Furthermore, the currents recorded in 0 Mg-ATP seem to reverse near the calculated Nernst equilibrium potential for Ba2+ (EBa ≈ +28 mV), and are far removed from ECl (−54 mV). Likewise, using fast depolarization ramps (0.07 V/sec) after activating the current IBa with a square pulse to −156 mV (see voltage protocol and current trajectories in Fig. 1D; left), a reversal potential of +17 mV was measured, again close to EBa rather than ECl (see Fig. 1D, zoomed I-V plot). This is also indicative of the higher permeability of this conductance to Ba2+ as compared to Cl−.
To test whether changing external Ba2+ concentration will affect current magnitude as well as the I-V relationship, GCPs were patched in whole cell mode using the Mg-ATP free internal solution (Fig. 2). Once again, the current magnitudes recorded in the absence of Mg-ATP are quite substantial. For instance, at −196 mV, a −280 pA current is measured in 100 mM [Ba2+]out (Fig. 2A) while in 30 mM [Ba2+]out (Fig. 2B) the same voltage gives rise to a current value of −220 pA. All current magnitudes at any given voltage are decreased when switching to lower [Ba2+] in the bath. The corresponding I-V plots appear as shifting to negative values (Fig. 2C) when switching from 100 to 30 mM Ba2+ and this is accompanied by a negative shift in the apparent reversal potential (Erev) values. Indeed, when zooming in (Fig. 2C, inset), the apparent Erev shows a negative shift of ≈ −14 mV as a result of this Ba2+ concentration change. Furthermore, the apparent Erev in 30 mM [Ba2+]out (≈ +8 mV) is still closer to the calculated EBa rather than ECl which are in this case +17.7 mV and −25.7 mV respectively. This experiment was repeated with 100, 30 and 10 mM BaCl2 in the bath and the same qualitative effects were seen i.e., decrease of current amplitude when decreasing the [Ba2+]o and negative shift in the apparent Erev (data not shown).
HACC permeability sequence to divalent cations in the absence of Mg-ATP: Ba > Ca ≈ Sr ≈ Mn ≫ Mg
Guard cell permeability to other divalent cations such as Ca2+, Sr2+, Mn2+ and Mg2+ in the absence of Mg-ATP was also tested (Fig. 3). As expected, HACC was permeable to Ca2+ (Fig. 3A) and found to have similar permeability to both Sr2+ (Fig. 3A) and Mn2+ (Fig. 3B). Meanwhile Mg2+ did not permeate HACC (Fig. 3A). The I-V plots (Fig. 3A and B) summarizes the permeability data: i.e., the lack of HACC permeability to Mg2+ as well as the much larger permeability to Ba2+ when compared to either Ca2+, Sr2+ or Mn2+. The effect of Mn2+ ions over time is reported in Figure 3B and highlights the unique behavior of this ion. Unlike Ca2+ or Sr2+, Mn2+ (Fig. 3B) triggered a transient block of HACC followed by some current recovery while still washing out the Ba2+ and replacing it with 100 mM Mn2+. This transient block effect was repeated on two other GCPs but was never seen with either Ca2+ or Sr2+; nor was it seen with Mg2+ even after 20 minutes of washing out Ba2+.
HACC permeability sequence to monovalent cations in the absence of Mg-ATP: K≈Na>Ba>Cs≫TEA
We observed that this Gd3+-sensitive HACC is also permeable to some physiologically relevant monovalent cations such K+ (Suppl. Fig. 1A), Na+ (Suppl. Fig. 1B) and Cs+ (Suppl. Fig. 1C) but not tetraethylammonium (TEA+)(Suppl. Fig. 1D). These data indicate that the Gd3+-sensitive current characterized in this work does not select for small mono or divalent cations (except for the case of Mg2+ and the bigger cation TEA+). Chloride too does not seem to permeate through this HACC. Indeed, when 100 mM Cl− was added at the same time as TEA+ (see Suppl. Fig. 1D), no current could be detected, indicating that Cl− is as impermeable as TEA+. Qualitatively, the same effect was seen in all patched GCPs (n = 5 for K+ and Na+; n = 2 for Cs+ and TEA+).
Effect of blockers of IBa in the absence of Mg-ATP and comparison with the effect on IK,in
In order to further characterize this HACC, readily unmasked when Mg-ATP was omitted from the patch pipet, the effect of some classical blockers such as the lanthanides (La3+ and Gd3+), Mn2+, Cs+ and TEA+ were tested on IBa as well as on IK,in, (the other major conductance that activates upon hyperpolarization in guard cells). One of the most conspicuous effects lies in the potent effect of Gd3+ in blocking IBa (Fig. 4A) even when used at relatively low concentrations (20 to 100 μM) while the same concentrations of Gd3+ had no effect on IK,in (Fig. 4A). An even higher concentration of Gd3+ (500 μM) did not affect IK,in (data not shown). La3+ also blocked IBa measured in 0 Mg-ATP, but we found that much higher concentrations of La3+ (0.2 to 0.5 mM) are needed to achieve the same block as compared to Gd3+ (data not shown). Experimenting with cesium, a blocker of IK,in, hardly any effect on IBa was registered (Fig. 4B). Even though Cs+ was used at concentrations up to 1 mM, it had only a small effect, if any, on IBa while the tenth of this amount (0.1 mM) is sufficient to block a large proportion (≈ 80% or more) of IK,in (Fig. 4B). Furthermore, Mn2+ used at 2 mM inhibited HACC by ≈ 37% (at V = −196 mV) when the charge carrier (in this case Ba2+) was still present in the bath (Fig. 4C). Increasing Mn2+ concentration to 10 mM shows that Mn2+ is not an efficient blocker of IBa as compared to Gd3+ or La3+ and 10 mM Mn2+ only causes an extra 20% IBa inhibition (see IV plot in Fig. 4C). Finally, 10 mM tetraethylammonium chloride (TEA), a concentration that was shown to block 70 to 80 % of IK,in in intact guard cells (44), had no effect whatsoever on IBa (Fig. 4D) measured in 0 Mg-ATP.
Rapid enhancement of IBa by ABA
Given that in guard cells, a HACC was implicated downstream of ABA in stomatal movements, we tested whether ABA affects IBa activated in the absence of internal Mg-ATP. We patch clamped guard cells to measure IBa currents under baseline conditions, i.e., zero Mg-ATP inside and no added ABA outside (Fig. 5). After about 10 minutes, the time usually necessary to reach steady-state conditions, we switched the perfusion solution to the one containing 20 μM ABA. A rapid and pronounced increase of IBa currents is seen at all voltages between −100 and −200 mV (≥ 1.3 to 1.5 fold)after only 5 minutes of ABA treatment (Fig. 5B) and a near doubling of the size of the IBa currents occurs at 10 minutes (Fig. 5, A and B). The enhancement of IBa in response to ABA, especially at 10 minutes, spans from −60 to −200 mV and also appears to shift the activation threshold of IBa (Fig. 5B) to the right. This suggests that ABA not only enhances calcium entry through HACC but can also mobilize calcium entry at less negative voltages.
Characterization of the effect of Mg2+on IBa and IKin
To answer whether internal Mg2+ alone causes the block of IBa when we add Mg-ATP, GCPs were patched either without Mg-ATP or without ATP but with added Mg2+ (as MgCl2). Furthermore, and as a control for ‘ion transport functionality’ of the patched GCPs, we used conditions that allow measurements of not just IBa, but also to record IK,in. The experiments were started in conditions allowing to probe for IK,in with KCl (30 mM) in the bath and this was then followed by replacing the KCl with a solution containing BaCl2 (100 mM). This was done first in the absence of both intracellular Mg2+ and ATP (Fig. 6A) and then repeated on another batch of guard cells with internal medium containing 1 mM MgCl2 but no ATP (Fig. 6B). Firstly, in zero Mg-ATP the only current that activated in response to hyperpolarization was IBa (Fig. 6A). IK,in could not be activated. Secondly, when only Mg2+ was included in the pipet solution (no ATP added), IK,in could now be activated in 30 mM K+, however, when switching the bath from K+- to Ba2+-containing media, IBa currents vanished (Fig. 6C) indicating that including only Mg2+ in the patch pipet can cause the block of IBa at voltages where they are normally activated in 0 Mg-ATP.
Could cAMP activate HACC in guard cells despite the presence of intracellular Mg2+?
We know from our own previous work (15) that cAMP activated a Gd3+-sensitive HACC in guard cells while recording in Mg2+- and ATP-free media (see also, Suppl. Fig. 2A) but since we now show that this conductance discriminates poorly between divalent and monovalent cations, a hallmark characteristic of all animal as well as plant cyclic nucleotides gated channels (CNGCs) (13,45), we sought to check if the Gd3+-sensitive IBa is also gated by cAMP in conditions where intracellular Mg2+ is present and IBa is already blocked (Suppl. Fig. 2B). In the absence of db2cAMP (the lipophilic permeable analog of cAMP), and as expected only a small background IBa current is seen (≤10 pA around −190 mV). After perfusing with db2cAMP (1 mM), a substantial increase in IBa amplitude between voltages from around −30 to −190 mV was observed (>60 pA around −190 mV). Keeping db2cAMP in the bath and adding Gd3+ (50 μM) resulted in a total block of the current (≤4 pA around −190 mV). This may indicate that GCPs harbor CNGCs that can be activated by cAMP despite the blocking effect by Mg2+.
Identification of candidate Mg2+-dependent cation channels
Crystallographic and functional analyses of a strong inward-rectifier K+ channel (Kir2.2) from chicken (46) showed that the rectification characteristic can be explained by Mg2+-binding to negatively charged regions in the pore (formed by D173) with possible additional contributions from acidic residues within the cytoplasmic regulatory domains (D256 and E300/E225) (Suppl. Fig. 3). To explore the possibility that a similar mechanism allows Mg2+ inward rectification for a subset of candidate plant channels that display the electrophysiological properties described here (i.e. activation by hyperpolarization and cAMP, absence of cation selectivity and inhibition by lanthanides), we built homology models of the pore-forming residues for AtCNGCs (47). The models were built using structures of the human hyperpolarization-activated channels HCN1, based on a ~22 % sequence identity. The obtained models clearly showed that AtCNGCs do not have a Mg2+-binding site corresponding to the location of Kir2.2 D173 inside the pore region (Fig. 7A, left arrow, and Suppl. Fig. 3). However, a tandem glutamic acid motif that could form a possible di-acidic Mg2+-binding site is found in most AtCNGCs located a little downstream of the pore in the so-called C-linker disc (Fig. 7). Akin to Kir2.2 D173 and E300/E225, this di-acidic AtGNCG motif forms a positively charged opening with distances of 7.3–8.3Å between neighbouring charges and a diameter of ~11Å [for Kir2.2 D173 these values are 7.3–7.4Å and 10.4–11.8Å, respectively (PDB 3jyc)]. As in Kir2.2, these distances between carboxyl groups are too large for a direct ion coordination, suggesting that Mg2+ is bound through bridging water molecules (46). Interestingly, this di-acidic motif is not present in AtGNCG2, which has been shown to be an atypical family member with respect to ion selectivity (49). The di-acidic motif is also absent in HCN1, for which Mg2+ inward rectification has not been documented (Fig. 7C).
DISCUSSION
In order to record ion-currents (for instance IK,in or IK,out) from plant cells in the “whole-cell” patch configuration, it is standard, for reasons highlighted in the introduction (see also: “Methods” section in (50)), to include ATP and Mg2+ either in the form of [Mg-ATP] or [MgCl2 + K2-ATP]. In contrast, the composition of the internal solution used to characterize the hyperpolarization-activated Ca2+-current (HACC or IBa) is more variable especially with regards to ATP and Mg2+. One notices for instance that ATP and Mg2+ are either both included (9,11,17,51,52) or completely omitted from the internal solution (10,16,53–55). Here, we carefully addressed the role and consequences of Mg2+ inclusion, either in the form of Mg-ATP or MgCl2. We report experiments designed to assess the effect of Mg2+ on IBa current activated by hyperpolarization in GCPs, being aware that other cell types,, e.g., root cells, might indeed show different responses. We show that omitting Mg-ATP unmasked the presence of an instantaneously-activating inwardly-rectifying conductance. This ‘newly unveiled’ conductance still retains most of the biophysical and pharmacological characteristics that are hallmarks of the classical ICa-type (9,10) and CNGCs which we referred to here as either IBa or HACCs (13,14). Like IK,in, this conductance is activated by hyperpolarizing-going voltages and shows some voltage-dependent rectification (but not as strongly as compared to IK,in for instance; see Fig. 4A). Also, typical of HACCs, we found that besides Ba2+, other divalent cations such as Ca2+, Sr2+ and Mn2+ also permeate this channel but at a much slower rate than Ba2+ does, hence the smaller currents resolved even though using the same amounts of divalent cations in the bath. More importantly, this HACC is activated by cAMP as well as permeable to monovalent cations (see Suppl. Fig. 1 and 2), which are definite attributes of CNGCs. HACCs including CNGCs are specifically blocked by low concentrations of extracellular Gd3+ that are far less effective in blocking IK,in (see Fig. 4A). Equally important, we found that the unmasked IBa is also enhanced by ≈ 1.3- to 1.5-fold in response to 5 minutes treatment with ABA and up to 2-fold after 10 minutes. Similarly, it was shown that ABA increases a PM ICa-type whole-cell current in Arabidopsis guard cells by ≈ 2- to 3-fold within 5 minutes of treatment (9). Note that the ABA effect reported here was obtained not only in Mg2+-free but even more importantly in ATP-free internal solution, thus indicating that ATP is not as crucial for this channel as was suggested by an earlier report (56). This is, in fact, in agreement with many other reports showing that indeed ABA can increase cytosolic Ca2+ levels through activation of PM calcium channels activated by hyperpolarization in Arabidopsis thaliana guard cells (10,57,58). Furthermore, ATP was neither required for cGMP-nor cAMP- activated (49,50) Ca2+-permeable cation channels in many different plant cell types (mesophyll, guard cells or pollen tubes) that also show many of the HACC characteristics. This observation is in contrast to the above-mentioned report where ATP and subsequent protein (de)-phosphorylation was described as a prerequisite for an ABA effect on calcium channels (56). This indicates that there may be more than one subtype of calcium channels co-existing in the PM and/or that additional modes of regulation of these Ca2+-permeable channels are operating which might require ATP- and protein kinases-dependent signaling (56,59).
One discrepancy that stands out in our report is that Mg2+ was shown to permeate HACCs in guard cells (10) and in root hairs (51), whereas external Mg2+, in our experiments, did not appear to permeate this channel (Fig. 3A). This is even more intriguing, considering the fact that cGMP was recently shown to activate an inward rectifying current (also Lanthanide-sensitive) with Mg2+ as a charge carrier (16). Hence this is another hint that we may be dealing with more than one sub-type of calcium channels. In animal cells, Mn2+ was described as both a blocker of Ca2+-channels, if Ca2+ is present in the bath, while in the absence of Ca2+, Mn2+ permeates the channel (60,61). These data might infer that HACCs, even though sharing many similarities in terms of their biophysical and pharmacological characteristics, might slightly differ from one cell type to another depending on tissue type and/or plant species.
So, the first key finding is that omitting Mg-ATP from the intracellular medium unmasks a larger Gd3+-sensitive non-selective cation conductance that is also regulated by cAMP and ABA. The mechanism consists of shifting the I-V characteristic to the right where less negative voltages can mobilize cations (including Ca2+) through the channel. The second key finding is the demonstration that Mg2+ alone can block this conductance (Fig. 6).
The current activated by voltages more negative than −200 mV is small, but significant (Fig. 1A). At the present time, we have no evidence to support that this instantaneous, rectifying and Mg2+-resistant Ba2+-current would be carried by a different population type of HACCs (the channel type unmasked when Mg2+ was omitted). If anything, this current could still be carried by the same type of channels since addition of 20 μM Gd3+ to the bath is still able to swiftly and efficiently block this current.
In summary, our data describe for the first time that in guard cells, Mg2+ blocks IBa by shifting the I-V relationship and its activation threshold to more negative voltages (Fig. 1C). This effect is reminiscent of the inhibitory effects by Mg2+ on many ion-channels that have been interpreted as ‘charge screening effects’ (18). Indeed, the rectification of the inwardly rectifying K+ (Kir) channels is due to a voltage-dependent block by cytosolic Mg2+ (and polyamines) thereby blocking outward K+-efflux. Upon hyperpolarization, Mg2+ is ejected from the pore, which appears to result in a time-dependent opening of the channel (62,63). Likewise, TRPV6 shows Mg2+-dependent gating that contributes to its strong inward rectification (20) and it was suggested that Mg2+ can block the channel by binding to a site within the transmembrane electrical field where it interacts with other permeant cations (20). It is also conceivable that other mechanisms could be operating such as electrostatic interaction between Mg2+ and some PM lipids, such as Phosphatidylinositol 4,5-bisphosphate (or PIP2). It was demonstrated that increasing the amount of membrane PIP2 results in decreasing the sensitivity of KCNQ channel to inhibition by Mg2+ (64). In addition, some Ca2+-channels were found to require PIP2 for their normal function (65). This begs the question of whether guard cell PM Ca2+-channels are also PIP2-sensitive. Our results also raise the question of whether Mg2+ could be equally important for IK,in gating. Indeed, and unlike earlier reports, we found that activation of IK,in was dependent on Mg2+ being present inside the patch pipet (Fig. 6).
Furthermore, the activation of the above HACCs by hyperpolarization and cAMP, the absence of cation selectivity and inhibition by lanthanides is consistent with the hypothesis that channels responsible for the observed effects are CNGCs and this is consistent with our structural modelling that has revealed the presence of di-acidic motifs in the pore forming helix of a subset of AtCNGCs. Acidic residues pointing towards the inner side of the pore, have previously been shown to confer Mg2+-dependence to inward rectifying K+ channels in animals (47) (Suppl. Fig. 3A). Given the position of the di-acidic motif in the cytoplasmic side of the pore, it is conceivable that Mg2+-binding can be affected by changes in pore opening, for example introduced by cAMP binding to the cytoplasmic region of AtCNGCs. Such a crosstalk would provide a mechanistic explanation for our observation that db2cAMP overrides the channel blockage produced by 1mM MgCl2 (no added ATP) (Suppl. Fig. 2B).
We therefore propose, firstly, that Mg2+ can limit, if not prevent, continuous Ca2+-leakage possibly through all HACCs (including CNGCs) at resting membrane potentials and secondly, that the activation of these channels requires mechanism(s) by which Mg2+-binding is altered, such in the case of adding cAMP (Suppl. Fig. 2B), in turn giving Mg2+ an important role in calcium homeostasis and calcium-dependent signaling.
Author Contributions
FL-C and CG conceived the study, FL-C performed the experiments and analyzed the data. CG and SA performed the structural analyses. All authors contributed to the writing of the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Acknowledgements
This research has been supported by the King Abdullah University of Science and Technology (KAUST). We are indebted to Professor Enid MacRobbie (Department of Plant Science, University of Cambridge, UK) for allowing us to use some of the data gathered by FL-C while in her laboratory (research was supported by BBSRC Grant P05730 to E.M.). We also thank Prof. Mark Tester for his invaluable comments.