Gating of many ion channels is strictly dependent on membrane voltage, and unless the permissible voltage range is applied, no channel activity will be observed. Similarly, ligand-gated channels typically remain inactive without the presence of agonist. However, several types of ion channel have been observed that are active, irrespective of the membrane voltages and without the need for addition of any ligand. Such channels therefore appear to be permanently (constitutively) active at all applied membrane voltages. Nevertheless, the extent of channel activity may still be modulated by many cellular factors and by membrane voltage (Fig. 1). Constitutive NSCCs can therefore be subdivided according to their voltage dependence into voltage-insensitive, depolarization-activated and hyperpolarization-activated NSCCs.
Constitutive NSCCs were among the first cation channels identified in plants. In addition, they were initially considered as constitutive plasma membrane leak conductances (Yurin et al., 1991) or as classes of K+ channel with low selectivity for K+ (Tester, 1990; White & Tester, 1992).
1. Depolarization-activated NSCCs (DA-NSCCs)
The first patch-clamp study on constitutive NSCCs from higher plants was carried out by Stoeckel & Takeda (1989). In the plasma membrane of Haemanthus and Clivia endosperm cells, these authors found NSCCs that were activated by membrane depolarization and stimulated by cytosolic Ca2+. DA-NSCCs were also found in other preparations: Arabidopsis cultured cells (Cerana & Colombo, 1992), Arabidopsis leaf mesophyll (Spalding et al., 1992; Shabala et al., 2006), Arabidopsis root epidermis (Shabala et al., 2006), Arabidopsis guard cells (Pei et al., 1998), Hordeum vulgare root xylem (Wegner & Raschke, 1994; de Boer & Wegner, 1997), Thlaspi spp. mesophyll cells (Piñeros & Kochian, 2003), Arabidopsis pollen tubes (Becker et al., 2004), Phaseolus vulgaris seed coats (Zhang et al., 2002) and Phaseolus vulgaris cotyledon dermal cells (Zhang et al., 2004). Some of the recorded channels showed slow activation kinetics (Cerana & Colombo, 1992; Wegner & Raschke, 1994; de Boer & Wegner, 1997) but in most cases a characteristically fast or ‘instantaneous’ change in current level occurred (Zhang et al., 2000, 2002, 2004; Piñeros & Kochian, 2003; Shabala et al., 2006).
Activation kinetics and voltage dependence of some DA-NSCCs are reminiscent of those observed for outwardly rectifying K+-selective channels (K+ outward rectifiers, KORs). However, in contrast to KORs, DA-NSCCs are insensitive to blocking by extracellular or intracellular Na+ (Shabala et al., 2006). DA-NSCCs can be effectively blocked by extracellular Ca2+ (Shabala et al., 2006), but this has also been observed for many other types of cation channels. Other compounds that may affect DA-NSCCs are TEA+, nifedipine, diltiazem and verapamil (reviewed by Demidchik et al., 2002b; Shabala et al., 2006).
The physiological functions of DA-NSCCs appear related to their ability to catalyse influx and efflux of monovalent cations and influx of divalent cations. The electrochemical gradient for most divalent cations is inwardly directed. This means that DA-NSCCs could form a conduit for the influx of divalent cations. Some indications that Ca2+ influx occurs through DA-NSCCs were found in maize root stelar cells (Roberts & Tester, 1997). In addition, Piñeros & Kochian (2003) demonstrated that passive Zn2+ influx in the Zn-hyperaccumulating Thlaspi caerulescens can occur through DA-NSCC-type conductance.
Involvement of DA-NSCCs in monovalent cation fluxes may be widespread. For example, several studies have shown that DA-NSCCs contribute to K+ loading into the xylem (de Boer & Wegner, 1997) and K+ redistribution in bean seeds (Zhang et al., 2002, 2004). The relatively high permeability for Na+ of many DA-NSCCs suggests they are also important contributors to the uptake and translocation of Na+, and thus their function impacts on salt tolerance. During salt stress, plant cells can rapidly accumulate Na+ to a very high concentration that interferes, in particular, with essential roles of K+ in the cytosol. Thus, maintaining high cytosolic K+/Na+ ratios is an important component of salinity tolerance (Maathuis & Amtmann, 1999; Shabala et al., 2006), and minimizing salt-induced K+ efflux and Na+ influx would contribute to increased tolerance.
During salt stress, both intra- and extracellular [Na+] are likely to be high. In such conditions, KORs are substantially blocked by Na+, preventing loss of cellular K+ (Shabala et al., 2006). Elevated external Ca2+ will further block KORs (Sokolik & Yurin, 1986; Yurin et al., 1991; Marten et al., 1999; Shabala et al., 2006). In such conditions, DA-NSCCs become a more prominent component of the membrane permeability to monovalent cations, potentially allowing K+ loss and entry of Na+ (Shabala et al., 2006) (Fig. 2).
Figure 2. Inhibition of cation channels leads to the amelioration of Na+ toxicity in higher plants. External Ca2+, and intracellular and extracellular Na+ can all have inhibitory effects on cation channels. Na+ blocks the K+-selective outwardly rectifying channel (KOR), thus preventing cellular K+ loss. Ca2+ blocks KOR, voltage-independent nonselective cation channels (VI-NSCCs) and depolarization-activated nonselective cation channels (DA-NSCCs), thus preventing excessive Na+ influx and some K+ efflux.
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Na+ entry constitutes an efficient and relatively cheap form to restore the osmotic balance and, as such, plays an important role in plant salinity tolerance. However, excessive Na+ influx causes Na+ toxicity, and relatively high DA-NSCC K+/Na+ selectivities may therefore provide adaptive advantages in this respect. A comparative study on Arabidopsis thaliana and its halophytic relative Thellungiella halophila showed an approximately fourfold higher PK/PNa of DA-NSCC-like channels in roots of Thellungiella compared with Arabidopsis (Volkov et al., 2004; Volkov & Amtmann, 2006; Wang et al., 2006). The PK/PNa of other NSCCs that are involved in Na+ influx was also found to be lower in Thellungiella halophila.
2. Voltage-independent NSCCs (VI-NSCCs)
The open probability of VI-NSCCs is not modulated by membrane voltage, suggesting these proteins lack any domains for sensing changes in membrane voltage. Thus, most VI-NSCCs equally conduct outward and inward current. VI-NSCCs are not inhibited by organic antagonists of Ca2+ channels such as nifedipine and verapamil, or by conventional blockers of K+ channels (TEA+, TTX, Cs+, Li+ and Na+). However, they are sensitive to Gd3+ and La3+. VI-NSCCs can be subdivided into two groups on the basis of blockage by cations such as Ca2+, Ba2+, Mg2+ and Zn2+. One class is partially blocked, whereas another class is both insensitive and permeable to these divalent cations. Inhibition by quinine is another aspect that pertains to some VI-NSCCs (Demidchik & Tester, 2002), but not others (White & Lemtiri-Chlieh, 1995).
Most studies on VI-NSCCs have been carried out using root cells (White & Lemtiri-Chlieh, 1995; Roberts & Tester, 1997; Tyerman et al., 1997; White, 1997; Buschmann et al., 2000; Maathuis & Sanders, 2001; Demidchik & Tester, 2002; Demidchik et al., 2002a; Volkov et al., 2004; Murthy & Tester, 2006; Shabala et al., 2006; Volkov & Amtmann, 2006), which reflects the potential physiological significance of this transport system for the nonspecific uptake of cations. VI-NSCCs and VI-NSCC-like conductances have also been found in guard cells (Véry et al., 1998), Arabidopsis mesophyll cells (Shabala et al., 2006), cultured Arabidopsis cells (Amtmann et al., 1997), Arabidopsis pollen tubes (Becker et al., 2004), expanding Pisum sativum leaf epidermis (Elzenga & van Volkenburgh, 1994) and motor cells of Samanea saman (Yu et al., Moran, 2001), whereas some of the earlier recordings were obtained using green algae Nitella flexilis (Yurin et al., 1991; Sokolik, 1999).
Although conclusive evidence is still lacking, it is widely considered that the main physiological function of VI-NSCCs is to catalyse uptake of cations. This may include monovalent cations like Na+ and NH4+ that do not permeate selective K+ channels, auxiliary uptake capacity for K+ when K+-selective channels are inhibited or not expressed, and the uptake of divalent cations such as Ca2+ and Mg2+ for which no selective pathways have been found in plant membranes at resting membrane potentials. This function can be highly beneficial where the acquisition of essential nutrients such as Ca2+, Mg2+, K+, NH4+, Mn2+ and Zn2+ is concerned, but also creates the potential for influx of harmful ions such as Na+, Cs+, Pb2+, Hg+ and Cd2+. Examples of both processes are discussed later.
All VI-NSCCs studied so far have been shown to be permeable to K+ (Demidchik et al., 2002b) and with the exception of VI-NSCCs in pea leaf epidermis (Elzenga & van Volkenburgh, 1994) and maize root cortex (Roberts & Tester, 1997), K+ is typically the most permeant ion. In most conditions, K+-selective inward rectifiers (KIRs) dominate passive K+ influx into root cells (Maathuis & Sanders, 1993), a contention that was given extra credence after Hirsch et al. (1998) showed that a loss of function in the main root KIR, AKT1, significantly reduced K+ uptake. However, a large component of the low-affinity K+ uptake remains in akt1-1 mutants, and although it is not clear what the exact mechanism is for this influx, VI-NSCCs in conjunction with KORs (Maathuis & Sanders, 1997) could mediate a significant part of this. Thus, VI-NSCCs may provide extra K+ uptake capacity, especially in conditions where the main low-affinity uptake pathway is inhibited.
As was discussed for DA-NSCCs, VI-NSCCs are prime candidates for Na+ entry. Indeed, Na+ permeability of VI-NSCCs has been demonstrated in a range of tissues and species (Stoeckel & Takeda, 1989; Elzenga & van Volkenburgh, 1994; White & Lemtiri-Chlieh, 1995; Amtmann et al., 1997; Roberts & Tester, 1997; Tyerman et al., 1997; Véry et al., 1998; Maathuis & Sanders, 2001; Demidchik & Tester, 2002) and there is now substantial evidence that root Na+ influx is to a large extent catalysed by VI-NSCCs (Maathuis & Sanders, 2001; Demidchik & Tester, 2002; Demidchik et al., 2002b; White & Davenport, 2002; Tester & Davenport, 2003; Maathuis, 2006a; Shabala et al., 2006). This notion is further supported by the observation that Ca2+ has a direct blocking effect on Na+ currents through VI-NSCCs (see Fig. 2) and on Na+ influx into intact tissue (Tyerman et al., 1997; Essah et al., 2003). The latter phenomenon is thought to be a main part of the ameliorative action of Ca2+ on salinity. Na+ influx in intact tissue is also sensitive to VI-NSCC blockers such as quinine, lanthanides and histidine modifiers (Essah et al., 2003; Wang et al., 2006).
Plants need large amounts of Ca2+, and understanding the mechanism of Ca2+ uptake from the soil is therefore of great value (Marschner, 1995; Welch, 1995). No Ca2+-selective channel has been found in plant membranes (Bothwell & Ng, 2005) and plant VI-NSCCs may be important in plant Ca2+ nutrition as a pathway for Ca2+ uptake, a notion that appears to contradict the blocking effect of Ca2+ on NSCCs (Fig. 2). The Ca2+ block derives from its high-affinity binding to the channel pore. However, the affinity of the pore for Ca2+ is low enough still to allow considerable Ca2+ permeation. A key role of VI-NSCCs in nutritional Ca2+ influx was demonstrated in Arabidopsis root epidermal cells (Demidchik et al., 2002a): 45Ca2+ flux measurements showed that Ca2+ uptake was blocked by Gd3+, a nonspecific blocker of VI-NSCCs. In addition, [Ca2+]cyt measured in Arabidopsis roots by Ca2+/aequorin chemiluminometry revealed a linear voltage dependence, consistent with the voltage independence of VI-NSCCs. A further important feature of VI-NSCCs is their enhanced activity in cells of the elongation zone where Ca2+ influx is particularly high. A reason for this requirement may be the Ca2+-dependent exocytosis that sustains cell expansion.
3. Hyperpolarization-activated NSCCs (HA-NSCCs)
Plants do not appear to have voltage-gated Ca2+-selective channels (Bothwell & Ng, 2005), but recordings on many plant membranes do show the presence of channels that conduct Ca2+ and these are believed to be important in the generation of Ca2+ signals. Although some of these activate via depolarization (Thion et al., 1998), most activate at very negative membrane voltages and are often described as hyperpolarization-activated Ca2+ channels (HACaCs) (Gelli & Blumwald, 1997; Gelli et al., 1997; Hamilton et al., 2000; Kiegle et al., 2000; Véry & Davies, 2000; Demidchik et al., 2002a, 2007). Unfortunately, a proper examination of monovalent ion selectivity in HACaCs is often lacking, although earlier reports (Schroeder & Hagiwara, 1990) and selectivity analyses of plant Ca2+-permeable cation channels in lipid bilayers (Aleksandrov et al., 1976; White & Tester, 1992; White & Davenport, 2002) and algal cells (Lunevsky et al., 1980, 1983) showed considerable permeability to monovalent ions. It is therefore likely that most HACaCs are in fact Ca2+-permeable HA-NSCCs and we will regard both terms as interchangeable.
Hyperpolarization-activated Ca2+ channels can catalyse the large Ca2+ influx that is required for plant elongation growth of root hairs and cells in the elongation zone (Kiegle et al., 2000; Véry & Davies, 2000; Demidchik et al., 2002a, 2007), and, similar to Ca2+-permeable VI-NSCCs, HACaCs are more active in growing tissues (Véry & Davies, 2000; Demidchik et al., 2007). In many cells, the resting membrane potential may be too positive for significant HACaC activity (Maathuis & Sanders, 1993; Demidchik et al., 2002a), but in such conditions Ca2+-permeable VI-NSCCs may elevate [Ca2+]cyt, which shifts the activation potential of HA-NSCCs to more positive voltages (Fig. 3). Alternatively, HACaC activity could be increased through the hyperpolarizing action of plasma membrane H+-ATPases (Miedema et al., 2001). The stimulation of HACaC activity by elicitors suggests that certain classes of HA-NSCCs are involved in Ca2+ signalling, which is part of the early response to pathogen attack (Gelli & Blumwald, 1997; Gelli et al., 1997).
Figure 3. Constitutive Ca2+-permeable cation channels in the plasma membrane of higher plants. Left: the mechanism of possible involvement of voltage-independent nonselective cation channels (VI-NSCCs) in activation of hyperpolarization-activated Ca2+ channels (HACaCs) that results in a large elevation of [Ca2+]cyt and stimulation of exocytosis and cell elongation growth. Right: current–voltage relationships (I–V curves) of constitutive Ca2+-permeable cation channels. Notably, elevation of [Ca2+]cyt shifts the HACaC I-V curve to a more positive voltage range. DACaC, depolarization-activated Ca2+ channel.
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Interestingly, HACaC activity in root epidermal protoplasts appears approx. 40–60 min after formation of the whole-cell configuration (Demidchik et al., 2002a). This behaviour suggests that HACaCs either can be inserted into the plasma membrane or are activated by some unknown factor such as reactive oxygen species (ROS). Plant annexins have been shown to catalyse transmembrane Ca2+ fluxes (Clark & Roux, 1995) and, in the presence of Ca2+ and ROS, animal annexins move to the plasma membrane and form Ca2+-permeable channels (Gerke & Moss, 2002). Animal annexin-type Ca2+ conductances are similar to HACaCs: they are activated by intracellular Ca2+ and hyperpolarization. Thus, annexins could function as HACaC-like Ca2+ channels in plants (Clark & Roux, 1995; White et al., 2002).
Apart from Ca2+ nutrition and signalling, HA-NSCCs may also have specialized tasks in specific membranes. A very special type of HA-NSCCs was found in the peribacteroid membrane that surrounds bacterial symbionts in nitrogen-fixing root nodules (Tyerman et al., 1995). These hyperpolarization-activated channels showed permeability to monovalent and divalent cations, required cytosolic Mg2+ for their inward rectification and were inhibited by polyamines and Ca2+ in the symbiosome lumen (Whitehead et al., 1998, 2001; Roberts & Tyerman, 2002). The peribacteroid membrane controls fluxes of fixed nitrogen (NH3 or NH4+) between bacteria and the host cells (Obermeyer & Tyerman, 2005) and the HA-NSCCs found in this membrane mediate the NH4+ flux, thus playing an important role in the nitrogen metabolism of legumes (Tyerman et al., 1995). Interestingly, the HA-NSCCs present in the peribacteroid membrane have an extremely low unitary conductance of approx. 0.11 pS, which could only be determined using noise spectrum analysis. Such low unitary conductance is atypical for most ion channels but more often associated with carriers, or ion channels with complex gating (Tyerman et al., 1995; Obermeyer & Tyerman, 2005).