Effects of cytoplasmic Mg2+ on slowly activating channels in isolated vacuoles of Beta vulgaris

The slow vacuolar (SV) channel can mediate a large part of the ionic current in plant tonoplasts, but its actual physiological role is still unclear. We demonstrate that in vacuoles from the taproots of sugar beet (Beta vulgaris L.), besides Ca2+, cytoplasmic Mg2+ also plays an important role in promoting the activation of the SV channel. An increase in Mg2+ concentration decreases the time constants of channel activation and deactivation, and determines a consistent shift, towards negative voltages, of the conductance characteristic; as an example, when the free concentration of Mg2+ was increased from the micromolar range up to 10 mM the activation shifted by about -60 mV. The experimental results obtained, which are based on a fast perfusion procedure allowing us to change the solution bathing the vacuole in a few milliseconds, suggest that magnesium-binding is a faster process than the voltage-activation gating of the channel, which constitutes the rate-limiting step controlling channel opening. Interestingly, the activation of the channel mediated by Mg2+ depends on the cooperative binding of at least three magnesium ions. We verified that cytoplasmic magnesium favours the activation of SV channels in the presence of nanomolar cytoplasmic calcium concentrations. A critical discussion on the Calcium Induced Calcium Release (CICR) mechanism proposed for the SV channel is presented.