Electrophysiology and fluorescence to investigate cation channels and transporters in isolated plant vacuoles

Abstract

The plant vacuole plays a fundamental role in cell homeostasis. The successful application of patch-clamp technique on isolated vacuoles allows the determination of the functional characteristics of tonoplast ion channels and transporters. The parallel use of a sensor-based fluorescence approach capable of detecting changes in calcium and proton concentrations opens up new possibilities for investigation. In excised patch, the presence of fura-2 in the vacuolar solution reveals the direct permeation of calcium in plant TPC channels. In whole-vacuole, the activity of non-electrogenic NHX potassium proton antiporters can be measured by using the proton sensitive dye BCECF loaded in the vacuolar lumen by the patch pipette. Both vacuolar NHXs and CLCa (chloride/nitrate antiporter) are inhibited by the phosphoinositide PI(3,5)P2, suggesting a coordinated role of these proteins in salt accumulation. Increased knowledge in the molecular mechanisms of vacuolar ion channels and transporters has the potential to improve our understanding on how plants cope with a rapidly changing environment.

Tonoplast cytochrome b561 is a transmembrane ascorbate‐dependent monodehydroascorbate reductase: functional characterization of electron currents in plant vacuoles

“…Mesophyll protoplasts were isolated from well‐expanded leaves from 7‐wk‐old plants and transiently transformed using the polyethylene glycol method as described elsewhere (Yoo et al ., 2007; Festa et al ., 2022; Gradogna & Carpaneto, 2022). Confocal microscopy analysis of vacuoles expressing the CYB561A‐EGFP protein was performed using a Leica SP2 (http://www.leica-microsystems.com) laser‐scanning confocal imaging system.…”

TPC1-Type Channels in Physcomitrium patens: Interaction between EF-Hands and Ca2+

“…While two paralogs of this channel class are known in the human genome (TPC1s and TPC2s) [1], only TPC1-type channels exist in plants [2]. They are the molecular basis of the well-characterized non-selective slow vacuolar (SV) channels [3][4][5][6]. The prototype for studies on TPCs in plants is AtTPC1, the TPC1 from the model plant Arabidopsis thaliana [3,[7][8][9][10].…”

“…Topology of a plant Two-Pore Cation Channel 1 (TPC1). A TPC1-subunit is built of two concatenated copies of a structural 6-TM motif, each with six transmembrane domains (1)(2)(3)(4)(5)(6). The cytosolic linker between them contains two EF-hands, which sense the cytosolic Ca 2+ concentration.…”