Mechanism of luminal Ca2+ and Mg2+ action on the vacuolar slowly activating channels

Abstract

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca(2+)-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channel's closed conformations. Vacuolar Ca2+ and Mg2+ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a 'permeable blocker'. Vacuolar Ca(2+)-with a much higher affinity than Mg(2+)-slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca(2+)-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channel's closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca2+ at the vacuolar side. The specificity for Ca2+ compared to Mg2+ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca2+ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.

A Novel Calcium Binding Site in the Slow Vacuolar Cation Channel TPC1 Senses Luminal Calcium Levels

“…here with earlier biophysical characterizations of the SV channel (Pottosin et al, 2004). The analysis of patch clamp recordings on Beta vulgaris tap root vacuoles identified two different binding sites for luminal divalent cations.…”

“…In addition, a Ca 2+ -selective binding site outside the membrane spanning part of the channel at the luminal side was characterized. Binding of divalent cations to this luminal site stabilizes the channel's closed conformations, shifting activation potentials to more positive potentials and slowing down channel opening kinetics (Pottosin et al, 2004). It is this luminal binding site that was modified in the TPC1 mutants studied here.…”

“…High cytosolic Ca 2+ concentrations were chosen to saturate the two cytosolic EF-Ca 2+ binding motifs, which in turn promotes high SV channel activity (Hedrich and Neher, 1987). The calcium sensitivity of the vacuolar calcium sensor was investigated at a background of 2 mM Mg 2+ , since it is known that both calcium and magnesium act on the SV channel from the vacuolar side (Pottosin et al, 2004). With submillimolar to low millimolar free Mg 2+ concentrations inside the vacuole (Shaul, 2002), a background of 2 mM vacuolar Mg 2+ resembles physiological conditions.…”

“…Therefore, one best describes its multiphasic activation kinetics on the basis of activation half-times (Pottosin et al, 2004) The D454N/fou2 mutant was characterized as a hyperactive version of the Arabidopsis SV channel (Bonaventure et al, 2007b), appearing entirely resistant toward block by vacuolar calcium, in contrast with wild-type TPC1 (Beyhl et al, 2009). Accordingly, when the D454N/fou2 mutant was expressed in tpc1-2 protoplasts, SV channel activation shifted to less positive membrane potentials and showed accelerated activation kinetics compared with wildtype TPC1.…”

“…The channel protein TPC1 harbors two cytosolic EF-hands for calcium binding, and rising cytoplasmic calcium activates SV currents (Furuichi et al, 2001;Ivashikina and Hedrich, 2005;Ranf et al, 2008;Schulze et al, 2011). On the luminal side, an increase from 0.1 to 1.0 mM calcium inhibits SV channels (Pottosin et al, 2004;Hedrich and Marten, 2011). Screening for mutants impaired in woundinginduced JA biosynthesis identified the Arabidopsis fou2 mutant (Bonaventure et al, 2007a(Bonaventure et al, , 2007b, which harbors a D454N exchange in the SV channel TPC1.…”