Ionic Channels of the Sugar Beet Tonoplast are Regulated by a Multi-ion Single-file Permeation Mechanism |
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Authors: | F Gambale M Bregante F Stragapede AM Cantu′ |
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Institution: | (1) Istituto di Cibernetica e Biofisica, Via DeMarini 6, I-16149, Genova, Italy, IT |
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Abstract: | Ionic channels of the sugar beet tonoplast were studied using the patch-clamp technique. At micromolar concentrations of
cytosolic calcium, several (at least four) distinct single-channel current levels were routinely identified. On the basis
of channel voltage dependence, kinetic properties and conductance of single openings, the largest channel (103 ± 2 pS in symmetric
150 mm KCl) corresponds to the slow vacuolar (SV) channel already identified by Hedrich and Neher (1987). The majority of the whole-vacuole
current was ascribed to this time-dependent slow-activating channel elicited by positive vacuolar potentials. The channel
of intermediate amplitude (41 ± 1 pS in 150 mm KCl) did not show any voltage dependence and delay in the activation upon the application of voltage steps to both positive
and negative transmembrane potentials. Owing to its voltage independence this channel was denominated FV1. The opening probability
of the SV-type channel increased by increasing the cytoplasmic calcium concentration, while the activity of the FV1 channel
did not increase appreciably by changing the calcium concentration in the range from 6 μm to 1 mm. All the channels identified showed a linear current-voltage characteristic in the range ±100 mV and at least the three most
conductive ones displayed potassium selectivity properties. Substitution of potassium with tetramethylammonium (TMA) on the
cytosolic side demonstrated that both the SV and FV1 channels are impermeable to TMA influx into the vacuole and support the
potassium selectivity properties of these two channels. Moreover, the single channel conductances of all the channels identified
increased as a function of the potassium concentration and reached a maximum conductivity at K+] ∼0.5 m. This behavior can be explained by a multi-ion occupancy single-file permeation mechanism.
Received: 26 December 1995/Revised: 10 July 1996 |
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Keywords: | : Ion channels — Vacuole — Sugar beet — Multi-ion pore — Single-file diffusion |
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