Correlations of Specific Ionic Effects using Ion Channels and Surface Charge Measurements

poster abstractSpecific ionic effects, as captured in the Hofmeister series, have been observed in many biological phenomena including protein folding and aggregation and lipid bilayer interactions. Previously we have shown that the Hofmeister effect is present in the activity of gramicidin A channels. In particular, measurements of channel open lifetime and conductance in potassium salts clearly show the existence of two distinct ionic classes that could be identified as kosmotropic and chaotropic. To further investigate this behavior, we have measured the zeta potential of diphytanoyl phosphatidylcholine (DPhPC) liposomes in salt solutions. We observe that anions alter the surface charge of the liposomes depending on the classification of the anion as kosmotropic or chaotropic. Chaotropic anions (SCN-, ClO4-) decrease the surface charge of the liposomes while kosmotropic anions (Cl-, H2PO4-, SO42-) have the opposite effect. These results correlate with our previous studies of cation conductance through gramicidin A channels adding new insight into ionic interactions at the lipid-water interface

The correlation of two different real-time PCR devices for the analysis of CYP2C19 pharmacogenetic results

CYP2C19 is a highly polymorphic gene responsible for the metabolism of commonly used drugs. CYP2C19*1, the wild-type allele, is associated with normal enzyme activity, whereas CYP2C19*2 and CYP2C19*17 lead to null and increased enzyme activity, respectively. The use of different instruments to perform the same pharmacogenetic tests should not affect the reliability of the results reported to clinicians, as required by the ISO 15189 standard. Genotyping assays allowed for the identification of gene variants corresponding to the CYP2C19*2 and CYP2C19*17 haplotypes in 44 selected samples. Each sample was analyzed in duplicate using the Thermo Fisher Taqman Drug Metabolism probes CYP2C19*2: c_25986767_70 (rs4244285) and CYP2C19*17: c_469857_10 (rs12248560). The experiments were performed on two widely used types of real-time PCR analyzers: ABI PRSIM™7500 and QuantStudioTM12KFlex (both from Applied Biosystems, Thermofisher). The data were analyzed in a Thermo Fisher Cloud facility. The analysis was performed independently by two qualified professionals. Both different instruments and analysts’ interpretations were consistent in identifying the native homozygous, heterozygous, and mutant homozygous variants for CYP2C19*2 and CYP2C19*17. The results provided by both the primary and backup analyzers showed a perfect correlation. This would allow for the use of the backup analyzer in case the main one is not available

Magic Angle Spinning Nuclear Magnetic Resonance Characterization of Voltage-Dependent Anion Channel Gating in Two-Dimensional Lipid Crystalline Bilayers

National Institutes of Health (U.S.) (EB001960)National Institutes of Health (EB002026

Magic Angle Spinning Nuclear Magnetic Resonance Characterization of Voltage-Dependent Anion Channel Gating in Two-Dimensional Lipid Crystalline Bilayers

The N-terminus of the voltage-dependent anion channel (VDAC) has been proposed to contain the mechanistically important gating helices that modulate channel opening and closing. In this study, we utilize magic angle spinning nuclear magnetic resonance (MAS NMR) to determine the location and structure of the N-terminus for functional channels in lipid bilayers by measuring long-range 13C–13C distances between residues in the N-terminus and other domains of VDAC reconstituted into DMPC lipid bilayers. Our structural studies show that the distance between A14 Cβ in the N-terminal helix and S193 Cβ is ∼4–6 Å. Furthermore, VDAC phosphorylation by a mitochondrial kinase at residue S193 has been claimed to delay mitochondrial cell death by causing a conformational change that closes the channel, and a VDAC-Ser193Glu mutant has been reported to show properties very similar to those of phosphorylated VDAC in a cellular context. We expressed VDAC-S193E and reconstituted it into DMPC lipid bilayers. Two-dimensional 13C–13C correlation experiments showed chemical shift perturbations for residues located in the N-terminus, indicating possible structural perturbations to that region. However, electrophysiological data recorded on VDAC-S193E showed that channel characteristics were identical to those of wild type samples, indicating that phosphorylation of S193 does not directly affect channel gating. The combination of NMR and electrophysiological results allows us to discuss the validity of proposed gating models

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 EGFreceptor. 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

Pharmacoepigenomic Interventions as Novel Potential Treatments for Alzheimer’s and Parkinson’s Diseases

Cerebrovascular and neurodegenerative disorders affect one billion people around the world and result from a combination of genomic, epigenomic, metabolic, and environmental factors. Diagnosis at late stages of disease progression, limited knowledge of gene biomarkers and molecular mechanisms of the pathology, and conventional compounds based on symptomatic rather than mechanistic features, determine the lack of success of current treatments, including current FDA-approved conventional drugs. The epigenetic approach opens new avenues for the detection of early presymptomatic pathological events that would allow the implementation of novel strategies in order to stop or delay the pathological process. The reversibility and potential restoring of epigenetic aberrations along with their potential use as targets for pharmacological and dietary interventions sited the use of epidrugs as potential novel candidates for successful treatments of multifactorial disorders involving neurodegeneration. This manuscript includes a description of the most relevant epigenetic mechanisms involved in the most prevalent neurodegenerative disorders worldwide, as well as the main potential epigenetic-based compounds under investigation for treatment of those disorders and their limitations

Regulation of Bax mitochondrial localization by Bcl-2 and Bcl-x(L): Keep your friends close but your enemies closer.

International audienceBax-induced mitochondrial outer membrane permeabilization (MOMP) is considered as one of the key control switches of apoptosis. MOMP requires Bax relocation to and insertion into the outer mitochondrial membrane to oligomerize and form pores allowing the release of apoptogenic factors such as cytochrome c. Even if these essential steps are now well-defined, it is necessary to better understand the molecular changes underlying the switch between inactive Bax and active (pore-forming) Bax. One of the ongoing issues is to determine whether Bax mitochondrial translocation is a critical step in the control of Bax activation or if this control is carried by latter regulatory steps. In this focus article we discuss recent data suggesting that although Bcl-2 and Bcl-x(L) block the MOMP, they can also regulate the mitochondrial Bax content. A new model in which Bax inhibition by Bcl-x(L) occurs at the immediate proximity of the outer mitochondrial membrane is also discussed. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy