Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters

open18siA distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.openFesta M.; Minicozzi V.; Boccaccio A.; Lagostena L.; Gradogna A.; Qi T.; Costa A.; Larisch N.; Hamamoto S.; Pedrazzini E.; Milenkovic S.; Scholz-Starke J.; Ceccarelli M.; Vitale A.; Dietrich P.; Uozumi N.; Gambale F.; Carpaneto A.Festa, M.; Minicozzi, V.; Boccaccio, A.; Lagostena, L.; Gradogna, A.; Qi, T.; Costa, A.; Larisch, N.; Hamamoto, S.; Pedrazzini, E.; Milenkovic, S.; Scholz-Starke, J.; Ceccarelli, M.; Vitale, A.; Dietrich, P.; Uozumi, N.; Gambale, F.; Carpaneto, A

Kidins220/ARMS is an essential modulator of cardiovascular and nervous system development

The growth factor family of neurotrophins has major roles both inside and outside the nervous system. Here, we report a detailed histological analysis of key phenotypes generated by the ablation of the Kinase D interacting substrate of 220 kDa/Ankyrin repeat-rich membrane spanning (Kidins220/ARMS) protein, a membrane-anchored scaffold for the neurotrophin receptors Trk and p75NTR. Kidins220 is important for heart development, as shown by the severe defects in the outflow tract and ventricle wall formation displayed by the Kidins220 mutant mice. Kidins220 is also important for peripheral nervous system development, as the loss of Kidins220 in vivo caused extensive apoptosis of DRGs and other sensory ganglia. Moreover, the neuronal-specific deletion of this protein leads to early postnatal death, showing that Kidins220 also has a critical function in the postnatal brain

Stepping out of the shade: control of neuronal activity by the scaffold protein Kidins220/ARMS

The correct functioning of the nervous system depends on the exquisitely fine control of neuronal excitability and synaptic plasticity, which relies on an intricate network of protein-protein interactions and signaling that shapes neuronal homeostasis during development and in adulthood. In this complex scenario, Kinase D interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning (Kidins220/ARMS) acts as a multi-functional scaffold protein with preferential expression in the nervous system. Engaged in a plethora of interactions with membrane receptors, cytosolic signaling components and cytoskeletal proteins, Kidins220/ARMS is implicated in numerous cellular functions including neuronal survival, neurite outgrowth and maturation and neuronal activity, often in the context of neurotrophin (NT) signaling pathways. Recent studies have highlighted a number of cell- and context-specific roles for this protein in the control of synaptic transmission and neuronal excitability, which are at present far from being completely understood. In addition, some evidence has began to emerge, linking alterations of Kidins220 expression to the onset of various neurodegenerative diseases and neuropsychiatric disorders. In this review, we present a concise summary of our fragmentary knowledge of Kidins220/ARMS biological functions, focusing on the mechanism(s) by which it controls various aspects of neuronal activity. We have tried, where possible, to discuss the available evidence in the wider context of NT-mediated regulation, and to outline emerging roles of Kidins220/ARMS in human pathologies

Modulation of plant ion channels by oxidizing and reducing agents

Ion channels are proteins forming hydrophilic pathways through the membranes of all living organisms. They play important roles in the electrogenic transport of ions and metabolites. Because of biophysical properties such as high selectivity for the permeant ion, high turnover rate, and modulation by physico-chemical parameters (e.g., membrane potential, calcium concentration), they are involved in several physiological processes in plant cells (e.g., maintenance of the turgor pressure, stomatal movements, and nutrient absorption by the roots). As plants cannot move, plant metabolism must be flexible and dynamic, to cope with environmental changes, to compete with other living species and to prevent pathogen invasion. An example of this flexibility and dynamic behavior is represented by their handling of the so-called reactive oxygen species, inevitable by-products of aerobic metabolism. Plants cope with these species on one side avoiding their toxic effects, on the other utilizing them as signalling molecules and as a means of defence against pathogens. In this review, we present the state-of-the-art of the modulation of plant ion channels by oxidizing and reducing agents

A perspective on the slow vacuolar channel in vacuoles from higher plant cells

The physiological role of the slow vacuolar (SV) channel in vacuoles from higher plant cells, was analyzed. The SV channel was found to be dependent on gating and voltage, permeable to both monovalent and to divalent cations, and displayed a main conductance which depended on potassium concentration. The functional properties of the channel suggested an implication in a calcium-induced calcium-released mechanism based on the channel's calcium dependent activation and calcium permeability. Another hypotheses that was made was the channel's involvement in the homeostasis of cytosolic potassium on the basis of its gating properties, and its involvement in the Na+ storage inside the vacuole

Malate transport by the vacuolar AtALMT6 channel in guard cells is subject to multiple regulation

Gas exchange in plants is controlled by guard cells, specialized cells acting as turgor pressure-driven valves. Malate is one of the major anions accumulated inside the vacuole during stomatal opening counteracting the positive charge of potassium. AtALMT6, a member of the aluminum-activated malate transporter family, is expressed in guard cells of leaves and stems as well as in flower organs of Arabidopsis thaliana. An AtALMT6-GFP fusion protein was targeted to the vacuolar membrane both in transient and stable expression systems. Patch-clamp experiments on vacuoles isolated from AtALMT6-GFP over-expressing Arabidopsis plants revealed large inward-rectifying malate currents only in the presence of micromolar cytosolic calcium concentrations. Further analyses showed that vacuolar pH and cytosolic malate regulate the threshold of activation of AtALMT6-mediated currents. The interplay of these two factors determines the AtALMT6 function as a malate influx or efflux channel depending on the tonoplast potential. Guard cell vacuoles isolated from Atalmt6 knock-out plants displayed reduced malate currents compared with wild-type vacuoles. This reduction, however, was not accompanied by phenotypic differences in the stomatal movements in knock-out plants, probably because of functional redundancy of malate transporters in guard cell vacuoles

Beyond the patch-clamp resolution:functional activity of non-electrogenic vacuolar NHX proton/potassium antiporters and inhibition by phosphoinositides

reserved4We combined the patch-clamp technique with ratiometric fluorescence imaging using the proton-responsive BCECF dye as a luminal probe. Upon application of a steep cytosol-directed K+ gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, while no associated electrical currents were observed, in agreement with electroneutral cation/H+ exchange. Our data show that this acidification was generated by NHX antiport activity, since: it did not distinguish between K+ and Na+ ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants. Our data further show that NHX activity could be reversed, was voltage independent and specifically impaired by the low-abundance signaling lipid PI(3,5)P2 , which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. This work, therefore, represents a proof of principle that can be applied to the study of proton-dependent exchangers from plants and animals, which are barely detectable using conventional techniques.mixedGradogna, Antonella; Scholz-Starke, Joachim; Pardo, José M; Carpaneto, ArmandoGradogna, Antonella; Scholz-Starke, Joachim; Pardo, José M; Carpaneto, Armand

Response to cytosolic nickel of Slow Vacuolar channels in the hyperaccumulator plant Alyssum bertolonii

We applied the patch-clamp technique to investigate the transport properties of the Slow Vacuolar (SV) channel identified in leaf vacuoles of Alyssum bertolonii Desv., a nickel hyperaccumulator plant growing in serpentine soil of the northern Apennines (Italy). SV currents recorded in vacuoles from adult plants collected in their natural habitat showed high sensitivity towards cytosolic nickel. Dose-response analyses indicated halfmaximal current inhibition at submicromolar concentrations, i.e. up to three orders of magnitude lower than previously reported values from other plant species. The voltage-dependent increase of residual currents at saturating nickel concentrations could be interpreted as relief of channel block by nickel ermeation at high positive membrane potentials. Including young plants of A. bertolonii into the study, we found that SV channels from these plants did not display elevated nickel sensitivity. This difference may be related to age-dependent changes in nickel hyperaccumulation of A. bertolonii leaf cells