JXB Advance Access published online on October 1, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern225
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Homeostatic control of slow vacuolar channels by luminal cations and evaluation of the channel-mediated tonoplast Ca2+ fluxes in situ
1Centro Universitario de Investigaciones Biomédicas. Universidad de Colima, 28045 Colima, Col., México
2School of Agricultural Science, University of Tasmania, Tas7001, Australia
* To whom correspondence should be addressed. E-mail: pottosin{at}ucol.mx
Ca2+, Mg2+, and K+ activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca2+, by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca2+ activity was 
0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca2+ in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca2+) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca2+ within a physiological range of concentrations (0.1–1.0 mM). Aggregation of the physiological vacuolar Na+ (60 mM) and Mg2+ (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca2+ variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca2+ activates the SV channel in a voltage-independent manner with Kd=0.7–1.5 µM. Comparison of the vacuolar Ca2+ fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 µM) cytosolic Ca2+, only 0.5% of SV channels are open. This mediates a Ca2+ release of only a few pA per vacuole (0.1 pA per single SV channel). Overall, our data suggest that the release of Ca2+ through SV channels makes little contribution to a global cytosolic Ca2+ signal.
Key words: Calcium channel, calcium signalling, patch-clamp, SV channel, tonoplast, vacuole
Received 12 June 2008; Revised 5 August 2008 Accepted 6 August 2008