Mg²⁺ is a Missing Link in Plant Cell Ca²⁺ Signalling and Homeostasis—A Study on Vicia faba Guard Cells

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 signalling. The unusual gating properties of HACCs in plants, i.e., activation by membrane hyperpolarization rather than depolarization, dictates that HACCs are normally open in the physiological hyperpolarized resting membrane potential state (the so-called pump or P-state); thus, if not regulated, they would continuously leak Ca²⁺ into cells. HACCs are permeable to Ca²⁺, Ba²⁺, and Mg²⁺; activated by H₂O₂ and the plant hormone abscisic acid (ABA); and their activity in guard cells is greatly reduced by increasing amounts of free cytosolic Ca²⁺ ([Ca²⁺]_Cyt), and hence closes during [Ca²⁺]_Cyt surges. Here, we demonstrate that the presence of the commonly used Mg-ATP inside the guard cell greatly reduces HACC activity, especially at voltages ≤ −200 mV, and that Mg²⁺ causes this block. Therefore, we firstly conclude that physiological cytosolic Mg²⁺ levels affect HACC gating and that channel opening requires either high negative voltages (≥−200 mV) or displacement of Mg²⁺ away from the immediate vicinity of the channel. Secondly, based on structural comparisons with a 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 diacidic 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.

1. Introduction

Ca²⁺ has long been recognized as an essential component in many plant cellular processes. 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 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, such as those activated by depolarization (DACCs) [5]; thirdly, others that are activated only by membrane hyperpolarization (HACCs) [6], which are the focus of this report.

In guard cells, Ca²⁺ was shown to be involved in abscisic acid (ABA) signalling and stomatal guard cell movements [7,8]. Using the patch clamp technique in both whole cell (WC) and excised configurations (EC), two types of HACCs were identified in 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 (P_o) 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 activity have not yet been identified. 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, the reason for their name—cyclic nucleotide gated channels (CNGCs). Moreover, these plant channels, similarly to 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 are found to be equally permeable to monovalent cations such as K⁺, Rb⁺, Na⁺, Li⁺, and Cs⁺ [13,14]. Similar inwardly rectifying currents (I) permeable to either Ca²⁺, Ba²⁺, and even Na⁺, which are 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 the 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 downregulated by high [Ca²⁺]_Cyt [17]. It is noteworthy that all these HACCs share one common characteristic—they are sensitive to low concentrations of lanthanides [10,15,16,17].

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⁺ (K_ir) 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 blockage 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 I_K,in was found not to be due to Mg²⁺-dependent blockage [23] but rather due 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 [25,26,27,28]. 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 its involvement in the transport of ammonium (NH₄⁺) or ammonia (NH₃) across the peribacteroid membrane [30]. This is partly achieved through a passive non-selective electrogenic transport system regulated by Ca²⁺. 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 K_ATP, an inward K⁺-rectifier (from the K_ir family of genes) found mostly in cardiac and skeletal muscles, neurons, and pancreatic 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 K_ATP 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 I_K,in [35].

Given the importance of calcium channels in plant cellular signalling, including in guard cell aperture regulation, we address the question of whether internal Mg²⁺ can affect the activity HACCs in guard cell protoplasts (GCPs) and show examples of Gd³⁺-sensitive Ba²⁺ currents (I_Ba) activated by hyperpolarization with and without Mg²⁺ in the patch pipet. We also assess important properties of I_Ba in the absence of Mg²⁺, such as the permeability and sensitivity to some relevant inorganic compounds and physiological effectors, including abscisic acid (ABA) and cAMP.

2. Materials and Methods

2.1. 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 week 3 or 4 in V. faba or from week 5 or 6 in Arabidopsis 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, USA), 0.026% (w/v) Pectolyase Y-23, 0.26% (w/v) BSA, and 1 mM CaCl₂ (pH 5.6), 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, USA).

2.2. Solutions

Protoplasts were placed in a 0.5 mL chamber, left to settle down, and then perfused continuously at a flow rate of ≈0.5–1 mL/min. To record I_Ba currents through HACC, we used barium-containing solutions. The bath medium contained (in mM): 100 BaCl₂, 10 Mes (pH 5.5 with Tris base); the pipette contained (in mM): 100 BaCl₂, 4 EGTA, 10 Hepes (pH 7.5 with Tris). For experiments where I_K,in and I_Ba 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 I_K,in, which was replaced by 100 BaCl₂, 10 Mes (pH 5.5 with Tris base) to measure I_Ba. Internal solution (in mM): 1 BaCl₂, 18 KCl, 4 EGTA, 10 Hepes (pH 7.5 with Tris base). Mg-ATP, MgCl₂, or K₂-ATP were added as specified in the figures. Osmolality was adjusted with sorbitol to 310 mOsm.kg⁻¹. For classic solutions used to measure I_K,in 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.

2.3. 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 °C) using standard whole-cell patch clamp techniques, with an Axopatch 200B integrating patch clamp amplifier (Axon Instruments, Inc., Union City, CA, USA). Voltage commands and simultaneous signal recordings and analyses were assessed by a microcomputer connected to the amplifier via a multipurpose input–output device (Digidata 1320A) using pClamp software (versions 8.0 and 10; Axon Instruments, Inc.). After giga ohm seals were formed, the whole-cell configuration was then achieved by gentle suction and the membrane was immediately clamped to a holding voltage (h_v) 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 the 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 in [39]. Ionic activities were calculated using GEOCHEM-EZ [40]. Current–voltage (I-V) relationships for I_Ba and I_K,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 ± SEM.

2.4. Protein Structural Modelling

Models were built using Swiss-Model [41] and structures were visualized with PyMOL (PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC). Homology modelling was supported by PSIPRED [42] and multisequence alignments were produced with MUSCLE [43]. Alignments and additional information of the models are provided in the respective legends.

3. Results

3.1. Removal of Intracellular Mg-ATP Unveils a Larger Instantaneously-Activated, Inwardly-Directed, and Gd³⁺-Sensitive Ba²⁺ Current

We recorded Ba²⁺ currents (I_Ba) in guard cell protoplasts in either the presence or absence of Mg-ATP (Figure 1A). At the end of the trials we also tested for I_Ba sensitivity to Gd³⁺, a potent blocker of I_Ba (Figure 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 (h_V) set to −36 mV. In the presence of Mg-ATP (Figure 1A; left panel), a small instantaneous inward-rectifying current of ≈−20 pA started to activate at around −200 mV, reaching a maximum of −60 pA at −256 mV. As expected, this current was sensitive to the addition of extracellular Gd³⁺ (see Figure 1B; left panel). Meanwhile, in the absence of Mg-ATP (Figure 1A; right panel), much larger (≥8-fold) instantaneous rectifying currents were recorded, which happened to also be Gd³⁺-sensitive (Figure 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 (Figure 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, which 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⁺ (E_Ba ≈ +28 mV), and are far removed from E_Cl (−54 mV). Likewise, using fast depolarization ramps (0.07 V/s) after activating the current I_Ba with a square pulse to −156 mV (see voltage protocol and current trajectories in Figure 1D; left), a reversal potential of +17 mV was measured, again close to E_Ba rather than E_Cl (see Figure 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 (Figure 2).

Once again, the current magnitudes recorded in the absence of Mg-ATP are substantial. For instance, at −196 mV, a −280 pA current is measured in 100 mM [Ba²⁺]_out (Figure 2A), while in 30 mM [Ba²⁺]_out (Figure 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 (Figure 2C) when switching from 100 to 30 mM Ba²⁺, which is accompanied by a negative shift in the apparent reversal potential (E_rev) values. Indeed, when zooming in (Figure 2C, inset), the apparent E_rev shows a negative shift of ≈−14 mV as a result of this Ba²⁺ concentration change. Furthermore, the apparent E_rev in 30 mM [Ba²⁺]_out (≈+8 mV) is still closer to the calculated E_Ba rather than E_Cl, the values for which in this case are +17.7 and −25.7 mV, respectively. This experiment was repeated with 100, 30, and 10 mM BaCl₂ in the bath, with the same qualitative effects observed, i.e., decrease of current amplitude when decreasing the [Ba2+]_o and negative shift in the apparent E_rev.

3.2. HACC Permeability Sequence for 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 (Figure 3).

As expected, HACC was permeable to Ca²⁺ (Figure 3A) and found to have similar permeability to both Sr²⁺ (Figure 3A) and Mn²⁺ (Figure 3B). Meanwhile, Mg²⁺ did not permeate HACC (Figure 3A). The I-V plots (Figure 3A,B) summarize 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⁺ (Figure 3B) triggered a transient blockage of HACC followed by some current recovery, while still washing out the Ba²⁺ and replacing it with 100 mM Mn²⁺. This transient blockage effect was repeated on two other guard cell protoplasts but was never seen with either Ca²⁺ or Sr²⁺, nor was it seen with Mg²⁺, even after 20 min of washing out the Ba²⁺.

3.3. HACC Permeability Sequence for 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 as K⁺ (Figure S1A), Na⁺ (Figure S1B), and Cs⁺ (Figure S1C), but not tetraethylammonium (TEA⁺) (Figure S1D). 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 also does not seem to permeate through this HACC. Indeed, when 100 mM Cl^- was added at the same time as TEA⁺ (see Figure S1D), 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⁺).

3.4. Effect of Blockers of I_Ba in the Absence of Mg-ATP and Comparison with the Effect on I_K,in

In order to further characterize this HACC, which was 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 I_Ba, as well as on I_K,in, (the other major conductance that activates upon hyperpolarization in guard cells) (Figure 4).

One of the most conspicuous effects lies in the potent effect of Gd³⁺ in blocking I_Ba (Figure 4A), even when used at relatively low concentrations (20 to 100 µM), while the same concentrations of Gd³⁺ had no effect on I_K,in (Figure 4A). An even higher concentration of Gd³⁺ (500 µM) did not affect I_K,in (data not shown). La³⁺ also blocked I_Ba 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 I_K,in, hardly any effect on I_Ba was registered (Figure 4B). Even though Cs⁺ was used at concentrations up to 1 mM, it had no or only a small effect on I_Ba, while one-tenth of this amount (0.1 mM) is sufficient to block a large proportion (≈ 80% or more) of I_K,in (Figure 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 (Figure 4C). Increasing Mn²⁺ concentration to 10 mM shows that Mn²⁺ is not an efficient blocker of I_Ba as compared to Gd³⁺ or La³⁺, and 10 mM Mn²⁺ only causes an extra 20% I_Ba inhibition (see IV plot in Figure 4C). Finally, 10 mM tetraethylammonium chloride (TEA), a concentration that was shown to block 70% to 80% of I_K,in in intact guard cells [44], had no effect whatsoever on I_Ba (Figure 4D) measured in 0 Mg-ATP.

3.5. Rapid Enhancement of I_Ba by ABA

Given that in V. faba guard cells a HACC was implicated downstream of ABA in stomatal movements, we tested whether ABA affects I_Ba activated in the absence of internal Mg-ATP. We patch-clamped guard cells to measure I_Ba currents under baseline conditions, i.e., zero Mg-ATP inside and no added ABA outside (Figure 5).

After about 10 min, 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 I_Ba currents is seen at all voltages between −100 and −200 mV (≥1.3- to 1.5-fold) after only 5 min of ABA treatment (Figure 5B), and a near doubling of the size of the I_Ba currents occurs at 10 min (Figure 5A,B). The enhancement of I_Ba in response to ABA, especially at 10 min, spans from −60 to −200 mV, and also appears to shift the activation threshold of I_Ba (Figure 5B) to the right. This suggests that ABA not only enhances calcium entry through HACCs but can also mobilize calcium entry at less negative voltages.

3.6. Characterization of the Effect of Mg²⁺ on I_Ba and I_K,in

To answer whether internal Mg²⁺ alone causes the block of I_Ba when we add Mg-ATP, GCPs were patched either without Mg-ATP or without ATP, but with added Mg²⁺ (added as MgCl₂) (Figure 6).

Furthermore, and as a control for the “ion transport functionality” of the patched GCPs, we used conditions that allow measurements of not just I_Ba, but also to record I_K,in. The experiments were started in conditions allowing to probe for I_K,in with KCl (30 mM) in the bath, which 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 (Figure 6A), and then repeated on another batch of guard cells with internal medium containing 1 mM MgCl₂ but no ATP (Figure 6B). Firstly, in zero Mg-ATP, the only current that activated in response to hyperpolarization was I_Ba (Figure 6A). I_K,in could not be activated. Secondly, when only Mg²⁺ was included in the pipet solution (no ATP added), I_K,in could then be activated in 30 mM K⁺. However, when switching the bath from K⁺- to Ba²⁺-containing media, I_Ba currents vanished, indicating that including only Mg²⁺ in the patch pipet can cause the blockage of I_Ba at voltages where it is normally activated in 0 Mg-ATP.

3.7. Can cAMP Activate HACCs in V. faba 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 (Figure 7A). However, since we had shown that this conductance discriminates poorly between divalent and monovalent cations, a hallmark characteristic of all animal and plant cyclic nucleotide gated channels (CNGCs) [13,45], we sought to check if the Gd³⁺-sensitive I_Ba is also gated by cAMP in conditions where intracellular Mg²⁺ is present and I_Ba is already blocked (Figure 7B).

In the absence of db₂cAMP (the lipophilic permeable analog of cAMP), and as expected only a small background I_Ba current is seen (≤10 pA around −190 mV). After perfusing with db₂cAMP (1 mM), a substantial increase in I_Ba 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⁺.

3.8. Identification of Candidate Mg²⁺-Dependent Cation Channels in Arabidopsis thaliana

Given the data from the physiological experiments, we undertook a search for candidate Mg²⁺-dependent cation channels in plants (Figure 8).

Crystallographic and functional analyses of a strong inward-rectifying K⁺ channel (K_ir2.2) 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) (Figure S2). 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 [47,48]. 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 K_ir2.2 D173 inside the pore region (Figure 8A, left arrow, and Figure S2). However, a tandem glutamic acid motif that could form a possible diacidic Mg²⁺ binding site is found in most AtCNGCs located a little downstream of the pore, in the so-called C-linker disc (Figure 8). Akin to K_ir2.2 D173 and E300/E225, this diacidic AtGNCG motif forms a positively charged opening, with distances of 7.3–8.3Å between neighbouring charges and a diameter of ~11Å (for K_ir2.2 D173, these values are 7.3–7.4Å and 10.4–11.8Å, respectively (PDB 3jyc)). As in K_ir2.2, these distances between carboxyl groups are too large for direct ion coordination, suggesting that Mg²⁺ is bound through bridging water molecules [46]. Interestingly, this diacidic motif is not present in AtGNCG2, which has been shown to be an atypical family member with respect to ion selectivity [49]. The diacidic motif is also absent in HCN1, for which Mg²⁺ inward rectification has not been documented (Figure 8C).

4. Discussion

In order to record ion currents (for instance I_K,in or I_K,out) from plant cells in the “whole-cell” patch configuration, for reasons highlighted in the introduction (also ass Methods section in [50]), it is standard to include ATP and Mg²⁺ in either 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 I_Ba) is more variable, especially with regards to ATP and Mg²⁺. For example, one can notice that ATP and Mg²⁺ are either both included [9,11,17,51,52] or completely omitted from the internal solution [10,16,53,54,55]. Here, we summarize the role and consequences of Mg²⁺ inclusion, either in the form of Mg-ATP or MgCl₂. Incidentally, there are no data on cytosolic magnesium concentrations or the distribution of magnesium within the different cellular compartments of guard cells. However, we think it is reasonable to assume that intracellular free [Mg²⁺] is between 400 and 600 µM, which the total magnesium, including the complexed form, may be as high as 10 mM.

An overview of some of the pharmacological properties related to the HACC (I_Ba) and I_K,in are presented in Figure 9.

We report experiments designed to assess the effect of Mg²⁺ on I_Ba 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 I_Ca-type [9,10] and CNGCs, which we referred to here as either I_Ba or HACCs [13,14]. Similarly to I_K,in, this conductance is activated by hyperpolarizing-going voltages, and shows some voltage-dependent rectification (but not as strongly as compared to I_K,in, for example; see Figure 4A). Additionally, 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 the same amounts of divalent cations in the bath were used. More importantly, this HACC is activated by cAMP, as well as permeable to monovalent cations (see Figure S1), 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 I_K,in (see Figure 4A). Equally important, we found that the unmasked I_Ba is also enhanced by ≈1.3- to 1.5-fold in response to 5 min treatment with ABA and by up to 2-fold after 10 min. Similarly, it was shown that ABA increases a PM I_Ca-type whole-cell current in Arabidopsis guard cells by ≈2- to 3-fold within 5 min of treatment [9]. Note that the ABA effect reported here was obtained not only in Mg²⁺-free internal solution, 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 agreement with many other reports showing that ABA can indeed increase cytosolic Ca²⁺ levels through activation of PM calcium channels via 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 channel co-existing in the PM or that additional modes of regulation of these Ca²⁺-permeable channels are in operation, which might require ATP- and protein-kinase-dependent signalling [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 in our experiments external Mg²⁺ did not appear to permeate this channel (Figure 3A). This is even more intriguing when considering that cGMP was recently shown to activate an inward rectifying current (which was also lanthanide-sensitive), with Mg²⁺ as a charge carrier [16]. Hence, this is another hint that we may be dealing with more than one subtype of calcium channel. In animal cells, Mn²⁺ was described as a blocker of calcium channels if Ca²⁺ is present in the bath, while in the absence of Ca²⁺ the Mn²⁺ permeates the channel [60,61]. These data might infer that HACCs, despite sharing many similarities in terms of their biophysical and pharmacological characteristics, might slightly differ from one cell type to another depending on the tissue type or plant species.

Therefore, 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 (Figure 6).

The current activated by voltages lower than −200 mV is small but significant (Figure 1A). At the present time, we have no evidence to support that this instantaneous, rectifying, Mg²⁺-resistant Ba²⁺ current would be carried by a different population type of HACCs (the channel type that was unmasked when Mg²⁺ was omitted). If anything, this current could still be carried by the same type of channel, since addition of 20 µM Gd³⁺ to the bath was still able to swiftly and efficiently block this current.

Our data describe that in guard cells, Mg²⁺ blocks I_Ba by shifting the I-V relationship and its activation threshold to more negative voltages (Figure 1C). This effect is reminiscent of the inhibitory effects by Mg²⁺ on many ion channels, which have been interpreted as “charge screening effects” [18]. Indeed, the rectification of the inwardly rectifying K⁺ (K_ir) channels is due to a voltage-dependent blockage 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]; 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 I_K,in gating. Indeed, unlike earlier reports, we found that activation of I_K,in was dependent on Mg²⁺ being present inside the patch pipet (Figure 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. This is consistent with our structural modelling, which revealed the presence of diacidic 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] (Figure S2B). Given the position of the diacidic 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 crosstalk would provide a mechanistic explanation for our observation that cAMP overrides the channel blockage produced by 1mM MgCl₂ (no added ATP) (Figure S2C), thereby preventing uncontrolled Ca²⁺ leakage.

5. Conclusions

Here, we firstly propose that in Vicia faba guard cells, Mg²⁺ can limit or prevent continuous Ca²⁺-leakage, possibly through all HACCs (including CNGCs) at resting membrane potentials, thereby being part of the (intra)cellular calcium signaling processes [66]. Furthermore, CNGCs have also been recognized as having a critical role in Ca²⁺-dependent plant defense signalling and responses [67,68]. Secondly, we propose that the activation of these channels requires mechanism(s) by which Mg²⁺ binding is altered. In CNGCs, such a mechanism is conceivably enabled by cAMP binding, thereby assigning Mg²⁺ an important role in calcium homeostasis and calcium-dependent downstream processes. Finally, the effect reported here suggests that Mg²⁺ has a role in cellular Ca²⁺ homeostasis, similarly to in animal cells.