The Plant Vacuole as Heterologous System to Characterize the Functional Properties of TPC Channels.

Human TPC channels are an emerging family of intracellular proteins fundamental for cell physiology and involved in various severe pathologies. Their localization in the membranes of endo-lysosomes, intracellular compartments of submicrometric dimensions, makes their study difficult with usual electrophysiological techniques. In this work, we show how the plant vacuole, a versatile organelle that can occupy up to 90% of the volume in mature plant cells, can be used as a heterologous system of expression for functional characterization. For this purpose, the use of vacuoles isolated from mesophyll cells of the Arabidopsis thaliana mutant lacking the endogenous TPC avoids unwanted interferences. The patch-clamp technique can be successfully applied to plant vacuoles in all different configuration modes; of note, the whole-vacuole configuration allows to study channel modulation by cytosolic factors. The combination of patch-clamp with fluorescence techniques, for example, by using fluorescent probes sensitive to specific ions of interest, represents a useful extension to investigate the selectivity properties of the channels. Therefore, the plant vacuole, similar to Xenopus oocytes for ion channels and transporters localized in the plasma membrane, has the capability to become a model system for functional studies on intracellular ion channels and transporters.

A perspective on the modulation of plant and animal two pore channels (TPCs) by the flavonoid naringenin.

The inhibitory effect of the flavonoid naringenin on plant and human Two-Pore Channels (TPCs) was assessed by means of electrophysiological measurements. By acting on human TPC2, naringenin, was able to dampen intracellular calcium responses to VEGF in cultured human endothelial cells and to impair angiogenic activity in VEGF-containing matrigel plugs implanted in mice. Molecular docking predicts selective binding sites for naringenin in the TPC structure, thus suggesting a specific interaction between the flavonoid and the channel.

Identification of sites responsible for the potentiating effect of niflumic acid on ClC-Ka kidney chloride channels.

BACKGROUND AND PURPOSE: ClC-K kidney Cl(-) channels are important for renal and inner ear transepithelial Cl(-) transport, and are potentially interesting pharmacological targets. They are modulated by niflumic acid (NFA), a non-steroidal anti-inflammatory drug, in a biphasic way: NFA activates ClC-Ka at low concentrations, but blocks the channel above approximately 1 mM. We attempted to identify the amino acids involved in the activation of ClC-Ka by NFA. EXPERIMENTAL APPROACH: We used site-directed mutagenesis and two-electrode voltage clamp analysis of wild-type and mutant channels expressed in Xenopus oocytes. Guided by the crystal structure of a bacterial CLC homolog, we screened 97 ClC-Ka mutations for alterations of NFA effects. KEY RESULTS: Mutations of five residues significantly reduced the potentiating effect of NFA. Two of these (G167A and F213A) drastically altered general gating properties and are unlikely to be involved in NFA binding. The three remaining mutants (L155A, G345S and A349E) severely impaired or abolished NFA potentiation. CONCLUSIONS AND IMPLICATIONS: The three key residues identified (L155, G345, A349) are localized in two different protein regions that, based on the crystal structure of bacterial CLC homologs, are expected to be exposed to the extracellular side of the channel, relatively close to each other, and are thus good candidates for being part of the potentiating NFA binding site. Alternatively, the protein region identified mediates conformational changes following NFA binding. Our results are an important step towards the development of ClC-Ka activators for treating Bartter syndrome types III and IV with residual channel activity.