The Ins and Outs of Cellular Ca²⁺ Transport

Introduction

Some chemical facts match calcium well with the signaling functions it performs throughout plant biology. It is the third most abundant metal in the Earth’s crust and it readily forms a precipitate with phosphate. To avoid precipitation of the sparingly soluble Ca₃(PO₄)₂ in the cytoplasm, a low cytosolic free Ca²⁺ concentration (e.g., < 0.1 μM) must be maintained despite a thousand-fold higher external concentration and a very negative membrane potential (e.g., < −180 mV). The resulting inward-directed electrochemical potential difference for Ca²⁺ across the plasma membrane (Δμ_Ca) is −52 kJ mol⁻¹ given these typical values, meaning that Ca²⁺ may flow into the cytoplasm through passive transporters such as ion channels without breaking any rules. It also means Ca²⁺ must be pumped up a 52 kJ mol⁻¹ ‘hill’ in order to move out. To put this hill in perspective, the free energy (ΔG) of ATP hydrolysis is −49 kJ mol⁻¹ in a typical cellular condition. The same thermodynamic analysis applied to membranes bounding other cellular compartments shows that with respect to the cytoplasm, IN is always ‘downhill’, and OUT is always ‘uphill’.

A transient rise in cytosolic free Ca²⁺ concentration that can function as an intracellular signal is generated when influx temporarily exceeds efflux. Two models have been proposed to explain how a transient change in concentration of a single ion can regulate so many aspects of plant development, including abiotic and biotic stress responses, tip growth, and gravitropism [1]. In a “Ca²⁺ signature” model, variations in a Ca²⁺ transient’s magnitude, duration, and/or repetition frequency are proposed to encode specific information that activate or inhibit different signal transduction networks. In a simple switch model [1], Ca²⁺ transients with different shapes all function in an equivalent fashion as simple switch. The key distinction is that specific information in a Ca²⁺ switch is not encoded through the complexity of the message, but rather lies in the unique status of the receiver. Both models are likely relevant to different signaling pathways in plants. At the core of both models is the subject of this review - proteins that transport Ca²⁺ uphill and downhill across membranes to create and shape Ca²⁺ transients [2,3].

Influx: Taking stock of the paths

Eflux: Changing a “signature”, or loading a compartment?

Pumps and antiporters provide two types of energized transport systems that move Ca²⁺ out of the cytoplasm after a Ca²⁺ release. The known Ca²⁺ antiporters all belong to a family of CAXs (Ca²⁺ exchangers), which in some cases have been shown to exchange Ca²⁺ for a counter ion such as H⁺ [36]. With antiporters, the downhill movement of the counter ion provides the driving force for Ca²⁺ transport. CAXs are considered high capacity, low affinity efflux systems. There are two types of Ca²⁺ pumps, ACAs (auto-inhibited Ca²⁺-ATPases) and ECAs (ER-type Ca²⁺-ATPases) belonging to a family of P-type ATPases that move ions against their concentration gradients using the energy from ATP hydrolysis [37]. These are considered low capacity, high affinity efflux systems. In addition, a Zn transporting heavy metal pump associated with the chloroplast (AtHMA1) [38] has been reported to transport Ca²⁺ [39]. This latter example highlights that Ca²⁺ efflux might also occur through unexpected transport systems that have yet to be characterized.

Because pumps and antiporters have the potential to change the magnitude and duration of a Ca²⁺ signal, knockouts or over-activation were initially expected to reveal dramatic effects on plant growth and development. Such predictions have yet to be validated. While several interesting phenotypes have been documented, such as partial male sterility and increased sensitivities to abiotic stresses [40] there have been no reports of a “dramatic” phenotype, such as a lethal knockout. In contrast, lethal mutations have been reported in animals [41]. It is not clear if plants have more gene redundancies or alternative efflux systems, or if plants are less reliant on using efflux systems to shape specific Ca²⁺ signatures for essential developmental events (i.e., plants might use Ca²⁺ switches more than Ca²⁺ signatures).

In plants, genetic evidence that the information content of a Ca²⁺ signal can be controlled by a Ca²⁺ efflux system has recently emerged from three studies. Knockout of PCA1, which encodes a vacuolar Ca²⁺ pump in the moss Physcomitrella patens, increases the magnitude and duration of a NaCl-triggered Ca²⁺ signal and decreases NaCl tolerance of the organism [42]. Secondly, antisense suppression of the NbCA1-encoded endomembrane Ca²⁺ pump in tobacco increased the magnitude and duration of a Ca²⁺ signal triggered by fungal or viral pathogens, or the cryptogein elicitor, and accelerated pathogen-triggered programmed cell death [43]. The third recent example comes from studying a double knockout of aca4 and 11, the two vacuolar Ca²⁺ pumps in Arabidopsis [44]. Knockout plants showed a high frequency of hypersensitive-like lesions (i.e., lesion mimic mutant). Lesions were dependent on salicylic acid (SA), whose production was previously shown to be regulated by Ca²⁺ signals [45,46]. The vacuoles of aca4/11 mesophyll cells in which lesions originate were shown to have normal Ca²⁺ levels, indicating that other transporters maintain Ca²⁺ loading levels [47] and that lesions result from an altered Ca²⁺ signal rather than vacuolar Ca²⁺ deficiency. Ca²⁺ imaging experiments in the aca4/11 double mutant are needed to assess the signal-shaping roles of these pumps.

It is important to note that phenotypes associated with Ca²⁺ efflux systems can have two different mechanistic origins; one by changing the dynamics of a Ca²⁺ transient, the other by changing the Ca²⁺ levels in a specific compartment (i.e. “Ca²⁺ nutrition”). In considering Ca²⁺ nutrition, increased or decreased loading of Ca²⁺ into a specific compartment might alter biochemical reactions or macromolecular interactions, and thereby disrupt processes such as vesicle trafficking or cell wall biogenesis [48,49]. Thus, changes in Ca²⁺ nutrition could indirectly cause many phenotypes, even in situations where Ca²⁺ imaging provides exciting evidence for an altered Ca²⁺ signature (e.g., as imaged in cells deficient in PCA1 and NbCA1). Thus, delineating the actual cause of a phenotype in an efflux mutant will always be very difficult.

Together, the death promoting phenotypes associated with reduced activities of PCA1, NbCA1, and ACA4/11 provide strong support for a model in which endomembrane Ca²⁺ pumps can function to dampen the magnitude and duration a Ca²⁺ signal (Figure 1). In the absence of these specific pumps, it appears that a Ca²⁺ transient can morph into a “cell-death Ca²⁺ signature”. An interesting question is why don’t other Ca²⁺ efflux pathways compensate for a missing pump? We offer two answers for consideration. First, location might be critical. Many Ca²⁺ signals are thought to be highly localized. Thus, a signal emanating from the vacuole might be too far away to be quickly sequestered by efflux systems associated with the ER (or vice versa). Second, in specific cell types under specific conditions, other efflux systems might be down-regulated, thereby blocking their ability to dampen the rise of a cell death Ca²⁺ signature. It is noteworthy that ACA and CAXs have known autoinhibitors [37,50], and are therefore subject to regulation that can decrease their transport activities under certain situations.

The Future – The Genesis of Switches and Signatures

While deficiencies in identified efflux pathways appear to potentiate the emergence of a Ca²⁺ cell death signature, the influx pathways that initiate those signals are yet to be defined. They could be among the above discussed transporters or among the additional Ca²⁺ pathways still to be discovered either through finding new candidates, new properties of proteins better known for other functions, or functions at other cellular membranes. For example, the AtSKOR and OsHKT2;4 transporters are known more as K⁺ transporters but they also have measureable Ca²⁺ permeability [51,52] as do the multifunctional, membrane associated annexins [53]. GLR channels may have important functions not only at the plasma membrane [54], and TPC1 may not be restricted to the tonoplast [55,56]. Identifying the various contributors to Ca²⁺ circuits and their localizations is necessary but the desired level of understanding will require learning how they are regulated to generate information-rich Ca²⁺ signatures, or simple Ca²⁺ switches.