Homeostatic control of slow vacuolar channels by luminal cations and evaluation of the channel-mediated tonoplast Ca2+ fluxes in situ.
Ca(2+), Mg(2+), and K(+) activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca(2+), by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca(2+) activity was approximately 0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca(2+) 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 Ca(2+)) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca(2+) within a physiological range of concentrations (0.1-1.0 mM). Aggregation of the physiological vacuolar Na(+) (60 mM) and Mg(2+) (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca(2+) variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca(2+) activates the SV channel in a voltage-independent manner with K(d)=0.7-1.5 microM. Comparison of the vacuolar Ca(2+) 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 microM) cytosolic Ca(2+), only 0.5% of SV channels are open. This mediates a Ca(2+) release of only a few pA per vacuole (approximately 0.1 pA per single SV channel). Overall, our data suggest that the release of Ca(2+) through SV channels makes little contribution to a global cytosolic Ca(2+) signal.
Pérez
V
V
Centro Universitario de Investigaciones Biomédicas. Universidad de Colima, 28045 Colima, Col., México.
Wherrett
T
T
Shabala
S
S
Muñiz
J
J
Dobrovinskaya
O
O
Pottosin
I
I
D027461:Beta vulgaris / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D003600:Cytosol / Q000502:physiology; D006706:Homeostasis; D015640:Ion Channel Gating; D007473:Ion Channels / Q000502:physiology*; D017136:Ion Transport; D008564:Membrane Potentials; D014617:Vacuoles / Q000502:physiology*
D002412:Cations; D007473:Ion Channels; D002118:Calcium