Antonella Gradogna

Fluorescence combined with excised patch: measuring calcium currents in plant cation channels

Combined application of the patch-clamp technique and fura-2 fluorescence detection enables the study of study calcium fluxes or related increases in cytosolic calcium concentration. Here we used the excised patch configuration, focusing the photomultiplier on the tip of the recording pipette where the fluorescent dye was present (FLEP, fluorescence combined with excised patch). This configuration has several advantages, i.e. a lack of delay in loading the fluorophore, of interference by internal calcium buffers and of photobleaching, due to the quasi-infinite dye reservoir inside the pipette. Upon voltage stimulation of tonoplast patches, sustained and robust fluorescence signals indicated permeation of calcium through the slow vacuolar (SV) channel. Both SV currents and fluorescence signal changes were absent in the presence of SV channel inhibitors and in vacuoles from Arabidopsis tpc1 knockout plants that lack SV channel activity. The fractional calcium currents of this non-selective cation channel were voltage-dependent, and were approximately 10% of the total SV currents at elevated positive potentials. Interestingly, calcium permeation could be recorded as the same time as oppositely directed potassium fluxes. These events would have been impossible to detect using patch-clamp measurements alone. Thus, we propose use of the FLEP technique for the study of divalent ion-selective channels or transporters that may be difficult to access using conventional electrophysiological approaches.

A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels

The two human CLC Cl− channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca2+ and H+ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca2+]ext without full saturation up to 50 mM. However, in the absence of Ca2+, ClC-Ka currents were still 20% of currents in 10 mM [Ca2+]ext, demonstrating that Ca2+ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H+]ext with a practically complete block at pH 6. Ca2+ and H+ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca2+ assuming a 13-fold higher Ca2+ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca2+ and H+. A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca2+-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca2+. Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H+ block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H+ -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing conformational changes underlying the complex gating of CLC-K channels.

Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.

Targeting kidney CLC-K channels: pharmacological profile in a human cell line versus Xenopus oocytes.

CLC-K chloride channels play a crucial role in kidney physiology and genetic mutations, affecting their function are responsible for severe renal salt loss in humans. Thus, compounds that selectively bind to CLC-Ka and/or CLC-Kb channels and modulate their activity may have a significant therapeutic potential. Here, we compare the biophysical and pharmacological behaviors of human CLC-K channels expressed either in HEK293 cells or in Xenopus oocytes and we show that CLC-K channel properties are greatly influenced by the biochemical environment surrounding the channels. Indeed, in HEK293 cells the potentiating effect of niflumic acid (NFA) on CLC-Ka/barttin and CLC-Kb/barttin channels seems to be absent while the blocking efficacy of niflumic acid and benzofuran derivatives observed in oocytes is preserved. The NFA block does not seem to involve the accessory subunit barttin on CLC-K1 channels. In addition, the sensitivity of CLC-Ks to external Ca(2+) is reduced in HEK293 cells. Based on our findings, we propose that mammalian cell lines are a suitable expression system for the pharmacological profiling of CLC-Ks.

Dissecting a regulatory calcium-binding site of CLC-K kidney chloride channels

The kidney and inner ear CLC-K chloride channels, which are involved in salt absorption and endolymph production, are regulated by extracellular Ca2+ in the millimolar concentration range. Recently, Gradogna et al. (2010. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201010455) identified a pair of acidic residues (E261 and D278) located in the loop between helices I and J as forming a putative intersubunit Ca2+-binding site in hClC-Ka. In this study, we sought to explore the properties of the binding site in more detail. First, we verified that the site is conserved in hClC-Kb and rClC-K1. In addition, we could confer Ca2+ sensitivity to the Torpedo marmorata ClC-0 channel by exchanging its I–J loop with that from ClC-Ka, demonstrating a direct role of the loop in Ca2+ binding. Based on a structure of a bacterial CLC and a new sequence alignment, we built homology models of ClC-Ka. The models suggested additional amino acids involved in Ca2+ binding. Testing mutants of these residues, we could restrict the range of plausible models and positively identify two more residues (E259 and E281) involved in Ca2+ coordination. To investigate cation specificity, we applied extracellular Zn2+, Mg2+, Ba2+, Sr2+, and Mn2+. Zn2+ blocks ClC-Ka as well as its Ca2+-insensitive mutant, suggesting that Zn2+ binds to a different site. Mg2+ does not activate CLC-Ks, but the channels are activated by Ba2+, Sr2+, and Mn2+ with a rank order of potency of Ca2+ > Ba2+ > Sr2+ = Mn2+ for the human CLC-Ks. Dose–response analysis indicates that the less potent Ba2+ has a lower affinity rather than a lower efficacy. Interestingly, rClC-K1 shows an altered rank order (Ca2+ > Sr2+ >> Ba2+), but homology models suggest that residues outside the I–J loop are responsible for this difference. Our detailed characterization of the regulatory Ca2+-binding site provides a solid basis for the understanding of the physiological modulation of CLC-K channel function in the kidney and inner ear.

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 ∼1 mM. We attempted to identify the amino acids involved in the activation of ClC‐Ka by NFA.

Kidney CLC-K chloride channels inhibitors: structure-based studies and efficacy in hypertension and associated CLC-K polymorphisms.

Objective: Alterations in the handling of renal salt reabsorption may contribute to interindividual differences in blood pressure regulation and susceptibility to hypertension. CLC-K chloride channels and their accessory subunit barttin play a pivotal role in kidney by controlling chloride and water absorption. Compounds selective for CLC-Ks, such as the benzofuran derivative MT-189, may have a significant therapeutic potential. Here, we assessed the feasibility of using CLC-K blockers in hypertension and aimed at enhancing drug inhibitory affinity. Methods and results: We demonstrated that acute in-vivo administration of MT-189 to spontaneously hypertensive rats (SHR) caused a reduction of blood pressure and defined the CLC-K/barttin gene expression pattern in kidney of SHR in comparison with normotensive Wistar–Kyoto rats. Based on MT-189, we designed and tested a new series of benzofuran derivatives on CLC-K chloride channels heterologously expressed in HEK293 cells. These studies enabled us to elucidate the causative molecular relationship for obtaining the most potent and selective inhibitor (SRA-36) described so far, with an IC50 of 6.6 ± 1 &mgr;mol/l. The biophysical and pharmacological characterization of A447T CLC-Ka and Y315F CLC-Ka, both polymorphisms associated with hypertension, showed that SRA-36 is an efficacious inhibitor of the chloride currents sustained by these polymorphisms. Molecular docking studies allowed hypothesizing an inhibition mechanism for the considered ligands, laying the foundations for the rational design of new and more effective CLC-K inhibitors. Conclusion: The SRA-36 molecule represents a new potential therapeutic option for hypertension.

Molecular Pharmacology of Kidney and Inner Ear CLC-K Chloride Channels

CLC-K channels belong to the CLC gene family, which comprises both Cl− channels and Cl−/H+ antiporters. They form homodimers which additionally co-assemble with the small protein barttin. In the kidney, they are involved in NaCl reabsorption; in the inner ear they are important for endolymph production. Mutations in CLC-Kb lead to renal salt loss (Bartters syndrome); mutations in barttin lead additionally to deafness. CLC-K channels are interesting potential drug targets. CLC-K channel blockers have potential as alternative diuretics, whereas CLC-K activators could be used for the treatment of patients with Bartters syndrome. Several small organic acids inhibit CLC-K channels from the outside by binding to a site in the external vestibule of the ion conducting pore. Benzofuran derivatives with affinities better than 10 μM have been discovered. Niflumic acid (NFA) exhibits a complex interaction with CLC-K channels. Below ∼1 mM, NFA activates CLC-Ka, whereas at higher concentrations NFA inhibits channel activity. The co-planarity of the rings of the NFA molecule is essential for its activating action. Mutagenesis has led to the identification of potential regions of the channel that interact with NFA. CLC-K channels are also modulated by pH and [Ca2+]ext. The inhibition at low pH has been shown to be mediated by a His-residue at the beginning of helix Q, the penultimate transmembrane helix. Two acidic residues from opposite subunits form two symmetrically related intersubunit Ca2+ binding sites, whose occupation increases channel activity. The relatively high affinity CLC-K blockers may already serve as leads for the development of useful drugs. On the other hand, the CLC-K potentiator NFA has a quite low affinity, and, being a non-steroidal anti-inflammatory drug, can be expected to exert significant side effects. More specific and more potent activators will be needed and it will be important to understand the molecular mechanisms that underlie NFA activation.

Subunit‐dependent oxidative stress sensitivity of LRRC8 volume‐regulated anion channels

Swelling‐activated anion currents are modulated by oxidative conditions, but it is unknown if oxidation acts directly on the LRRC8 channel‐forming proteins or on regulatory factors. We found that LRRC8A–LRRC8E heteromeric channels are dramatically activated by oxidation of intracellular cysteines, whereas LRRC8A–LRRC8C and LRRC8A–LRRC8D heteromers are inhibited by oxidation. Volume‐regulated anion currents in Jurkat T lymphocytes were inhibited by oxidation, in agreement with a low expression of the LRRC8E subunit in these cells. Our results show that LRRC8 channel proteins are directly modulated by oxidation in a subunit‐specific manner.

Alkaline pH Block of CLC-K Kidney Chloride Channels Mediated by a Pore Lysine Residue

CLC-K chloride channels are expressed in the kidney and the inner ear, where they are involved in NaCl reabsorption and endolymph production, respectively. These channels require the beta subunit barttin for proper function. Mutations in ClC-Kb and barttin, lead to Bartters syndrome. Block of CLC-K channels by acid pH was described in a previous work, and we had identified His-497 as being responsible for the acidic block of CLC-K channels. Here, we show that ClC-K currents are blocked also by alkaline pH with an apparent pK value of ∼8.7 for ClC-K1. Using noise analysis, we demonstrate that alkaline block is mediated by an allosteric reduction of the open probability. By an extensive mutagenic screen we identified K165, a highly conserved residue in the extracellular vestibule of the channel, as the major element responsible for the alkaline pH modulation. Deprotonation of K165 underlies the alkaline block. However, MTS modification of the K165C mutant demonstrated that not only the charge but also the chemical and sterical properties of lysine 165 are determinants of CLC-K gating.