Héctor Gaitán-Peñas

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.

Megalencephalic Leukoencephalopathy with subcortical Cysts protein 1 regulates glial surface localization of GLIALCAM from fish to humans

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by myelin vacuolization and caused by mutations in MLC1 or GLIALCAM. Patients with recessive mutations in either MLC1 or GLIALCAM show the same clinical phenotype. It has been shown that GLIALCAM is necessary for the correct targeting of MLC1 to the membrane at cell junctions, but its own localization was independent of MLC1 in vitro. However, recent studies in Mlc1(-/-) mice have shown that GlialCAM is mislocalized in glial cells. In order to investigate whether the relationship between Mlc1 and GlialCAM is species-specific, we first identified MLC-related genes in zebrafish and generated an mlc1(-/-) zebrafish. We have characterized mlc1(-/-) zebrafish both functionally and histologically and compared the phenotype with that of the Mlc1(-/-) mice. In mlc1(-/-) zebrafish, as in Mlc1(-/-) mice, Glialcam is mislocalized. Re-examination of a brain biopsy from an MLC patient indicates that GLIALCAM is also mislocalized in Bergmann glia in the cerebellum. In vitro, impaired localization of GlialCAM was observed in astrocyte cultures from Mlc1(-/-) mouse only in the presence of elevated potassium levels, which mimics neuronal activity. In summary, here we demonstrate an evolutionary conserved role for MLC1 in regulating glial surface levels of GLIALCAM, and this interrelationship explains why patients with mutations in either gene (MLC1 or GLIALCAM) share the same clinical phenotype.

Identification and characterization of the zebrafish ClC-2 chloride channel orthologs.

ClC-2 is a Cl− channel that belongs to the CLC family of chloride channel/transport proteins. ClC-2 molecular role is not clear, and Clcn2 knockout mice develop blindness, sterility, and leukodystrophy by unknown reasons. ClC-2 is associated in the brain with the adhesion molecule GlialCAM, which is defective in a type of leukodystrophy, involving ClC-2 in the homeostasis of myelin. To get more insight into the functions of ClC-2, we have identified in this work the three ClC-2 orthologs in zebrafish. clcn2a and clcn2b resulted from the teleost-specific whole genome duplication, while clcn2c arose from a gene duplication from clcn2b. The expression patterns in adult tissues and embryos of zebrafish clcn2 paralogs support their subfunctionalization after the duplications, with clcn2a being enriched in excitable tissues and clcn2c in ionocytes. All three zebrafish clc-2 proteins interact with human GLIALCAM, that is able to target them to cell junctions, as it does with mammalian ClC-2. We could detect clc-2a and clc-2b inward rectified chloride currents with different voltage-dependence and kinetics in Xenopus oocytes, while clc-2c remained inactive. Interestingly, GlialCAM proteins did not modify clc-2b inward rectification. Then, our work extends the repertoire of ClC-2 proteins and provides new tools for structure-function and physiology studies.

Cisplatin activates volume sensitive LRRC8 channel mediated currents in Xenopus oocytes.

ABSTRACT LRRC8 proteins have been shown to underlie the ubiquitous volume regulated anion channel (VRAC). VRAC channels are composed of the LRRC8A subunit and at least one among the LRRC8B-E subunits. In addition to their role in volume regulation, LRRC8 proteins have been implicated in the uptake of chemotherapeutic agents. We had found that LRRC8 channels can be conveniently expressed in Xenopus oocytes, a system without endogenous VRAC activity. The fusion with fluorescent proteins yielded constitutive activity for A/C, A/D and A/E heteromers. Here we tested the effect of the anticancer drug cisplatin on LRRC8A-VFP/8E-mCherry and LRRC8A-VFP/8D-mCherry co-expressing oocytes. Incubation with cisplatin dramatically activated currents for both subunit combinations, confirming that VRAC channels provide an uptake pathway for cisplatin and that intracellular cisplatin accumulation strongly activates the channels. Thus, specific activators of LRRC8 proteins might be useful tools to counteract chemotherapeutic drug resistance.

Identification and Functional Characterization of CLCN1 Mutations Found in Nondystrophic Myotonia Patients

Mutations in the gene coding for the skeletal muscle Cl− channel (CLCN1) lead to dominant or recessive myotonia. Here, we identified and characterized CLCN1 mutations in Costa Rican patients, who had been clinically diagnosed with myotonic dystrophy type 1 but who were negative for DM1 mutations. CLCN1 mutations c.501C>G, p.F167L and c.1235A>C, p.Q412P appeared to have recessive inheritance but patients had atypical clinical phenotypes; c.313C>T, p.R105C was found in combination with c.501C>G, p.F167L in an apparently recessive family and the c.461A>G, p.Q154R variant was associated with a less clear clinical picture. In Xenopus oocytes, none of the mutations exhibited alterations of fast or slow gating parameters or single channel conductance, and mutations p.R105C, p.Q154R, and p.F167L were indistinguishable from wild‐type (WT). p.Q412P displayed a dramatically reduced current density, surface expression and exerted no dominant negative effect in the context of the homodimeric channel. Fluorescently tagged constructs revealed that p.Q412P is expressed inefficiently. Our study confirms p.F167L and p.R105C as myotonia mutations in the Costa Rican population, whereas p.Q154R may be a benign variant. p.Q412P most likely induces a severe folding defect, explaining the lack of dominance in patients and expression systems, but has WT properties once expressed in the plasma membrane.

Leukoencephalopathy‐causing CLCN2 mutations are associated with impaired Cl− channel function and trafficking

Characterisation of most mutations found in CLCN2 in patients with CC2L leukodystrophy show that they cause a reduction in function of the chloride channel ClC‐2. GlialCAM, a regulatory subunit of ClC‐2 in glial cells and involved in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), increases the activity of a ClC‐2 mutant by affecting ClC‐2 gating and by stabilising the mutant at the plasma membrane. The stabilisation of ClC‐2 at the plasma membrane by GlialCAM depends on its localisation at cell–cell junctions. The membrane protein MLC1, which is defective in MLC, also contributes to the stabilisation of ClC‐2 at the plasma membrane, providing further support for the view that GlialCAM, MLC1 and ClC‐2 form a protein complex in glial cells.

Expression of LRRC8/VRAC Currents in Xenopus Oocytes: Advantages and Caveats

Volume-regulated anion channels (VRACs) play a role in controlling cell volume by opening upon cell swelling. Apart from controlling cell volume, their function is important in many other physiological processes, such as transport of metabolites or drugs, and extracellular signal transduction. VRACs are formed by heteromers of the pannexin homologous protein LRRC8A (also named Swell1) with other LRRC8 members (B, C, D, and E). LRRC8 proteins are difficult to study, since they are expressed in all cells of our body, and the channel stoichiometry can be changed by overexpression, resulting in non-functional heteromers. Two different strategies have been developed to overcome this issue: complementation by transient transfection of LRRC8 genome-edited cell lines, and reconstitution in lipid bilayers. Alternatively, we have used Xenopus oocytes as a simple system to study LRRC8 proteins. Here, we have reviewed all previous experiments that have been performed with VRAC and LRRC8 proteins in Xenopus oocytes. We also discuss future strategies that may be used to perform structure-function analysis of the VRAC in oocytes and other systems, in order to understand its role in controlling multiple physiological functions.

Megalencephalic leukoencephalopathy with subcortical cysts: A personal biochemical retrospective

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-, GlialCAM- and Clcn2-knockout mice or Mlc1-knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGF receptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients.

GlialCAM/MLC1 modulates LRRC8/VRAC currents in an indirect manner: Implications for megalencephalic leukoencephalopathy

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes. Previous work indicated that chloride currents mediated by the volume-regulated anion channel (VRAC) and ClC-2 channels were affected in astrocytes deficient in either Mlc1 or Glialcam. ClC-2 forms a ternary complex with GlialCAM and MLC1. LRRC8 proteins have been identified recently as the molecular components of VRAC, but the relationship between MLC and LRRC8 proteins is unknown. Here, we first demonstrate that LRRC8 and MLC1 are functionally linked, as MLC1 cannot potentiate VRAC currents when LRRC8A, the main subunit of VRAC, is knocked down. We determine that LRRC8A and MLC1 do not co-localize or interact and, in Xenopus oocytes, MLC1 does not potentiate LRRC8-mediated VRAC currents, indicating that VRAC modulation in astrocytes by MLC1 may be indirect. Investigating the mechanism of modulation, we find that a lack of MLC1 does not influence either mRNA or total and plasma membrane protein levels of LRRC8A; and neither does it affect LRRC8A subcellular localization. In agreement with recent results that indicated that overexpression of MLC1 decreases the phosphorylation of extracellular signal-regulated kinases (ERK), we find that astrocytes lacking MLC1 show an increase in ERK phosphorylation. In astrocytes with reduced or increased levels of MLC1 we observe changes in the phosphorylation state of the VRAC subunit LRRC8C. Our results thus reinforce previous suggestions that indicated that GlialCAM/MLC1 might modify signal transduction pathways that influence the activity of different proteins, such as VRAC.

CLCN1 Myotonia congenita mutation with a variable pattern of inheritance suggests a novel mechanism of dominant myotonia: Short Reports

Introduction: Mutations in CLCN1 cause recessive or dominant forms of myotonia congenita (MC). Some mutations have been found to exhibit both patterns of inheritance but the mechanism explaining this behavior is unknown. Methods: A known recessive missense mutation, A493E, was identified in a family with dominant MC. The mutant p.A493E alone or in co‐expression with wild‐type (WT) ClC‐1 was expressed in Xenopus oocytes. Currents were measured and biochemical assays were performed. Results: The mutant showed no significant activity and reduced total and plasma membrane (PM) protein levels. Co‐expression with the mutant reduced the activity and PM levels of an engineered lower expression variant of ClC‐1, whereas no effect was observed on a higher expression variant. Discussion: Our results suggest that the dominant effect of some CLCN1 mutations showing recessive or dominant inheritance patterns may be due to a dose‐dependent defect in PM delivery of the WT channel. Muscle Nerve 58: 157–160, 2018