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

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.