On the Interaction of Neomycin with the Slow Vacuolar Channel ofArabidopsis thaliana

Abstract

This study investigates the interaction of the aminoglycoside antibiotic neomycin with the slow vacuolar (SV) channel in vacuoles from Arabidopsis thaliana mesophyll cells. Patch-clamp experiments in the excised patch configuration revealed a complex pattern of neomycin effects on the channel: applied at concentrations in the submicromolar to millimolar range neomycin (a) blocked macroscopic SV currents in a voltage- and concentration-dependent manner, (b) slowed down activation and deactivation kinetics of the channel, and most interestingly, (c) at concentrations above 10 μM, neomycin shifted the SV activation threshold towards negative membrane potentials, causing a two-phasic activation at high concentrations. Single channel experiments showed that neomycin causes these macroscopic effects by combining a decrease of the single channel conductance with a concomitant increase of the channel's open probability. Our results clearly demonstrate that the SV channel can be activated at physiologically relevant tonoplast potentials in the presence of an organic effector molecule. We therefore propose the existence of a cellular equivalent regulating the activity of the SV channel in vivo.

The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl−/H+ exchanger CLC‐7

“…This large organelle constitutes a convenient system for detailed patch‐clamp studies (Hedrich, 1995; Hedrich & Marten, 2006). In addition to macroscopic current recordings from the whole vacuole, many reports investigated single channel behaviour and modulation in excised vacuolar membrane patches, both in the cytosolic‐side‐out (Scholz‐Starke et al 2006) and vacuolar‐side‐out configurations (Pottosin & Martinez‐Estevez, 2003). Furthermore, fluorescent indicator dyes have been employed in combination with the patch‐clamp technique (Konrad & Hedrich, 2008; Gradogna et al 2009).…”

Plant Ion Channels: Gene Families, Physiology, and Functional Genomics Analyses

“…The voltage dependence of SV channels limits vacuolar Ca 2+ release, although recordings of deactivating (tail) currents show a clear Ca 2+ permeation toward the cytoplasmic membrane side (17, 180). For physiological Ca 2+ release (181), it has been proposed that the voltage dependence of SV channels would need to be shifted by modulators, as was recently found in Arabidopsis mesophyll vacuoles (182). An Arabidopsis TPC1 knockout mutant was defective in the inhibition of stomatal opening by external Ca 2+ (56).…”

Roles of ion channels and transporters in guard cell signal transduction

“…SV type vacuolar currents appear to be lost in mesophyll cells of knockout mutants of TPC1, a two-pore Ca 2+ -induced Ca 2+ release channel, suggesting that SV channels not only mediate K + flux but also function as conduits for Ca 2+ release [105]. However, whether a voltage-dependent shift of SV channel activity leads to Ca 2+ efflux in guard cells under physiological conditions is currently under debate [105,[149][150][151]. The release of Ca 2+ from the vacuole additionally involves several kinds of Ca 2+ -permeable channels that have been implicated by physiological experiments but not yet characterized at the molecular level, including InsP 3 -and InsP 6 -and cADPR-sensitive Ca 2+ -release channels [120].…”