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ion channel ͉ membrane protein ͉ structure ͉ acetylcholine

DNA is vulnerable to the attack of certain oxygen radicals and one of the major DNA lesions formed is 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic lesion that can mispair with adenine. The repair of 8-oxoG was studied by measuring the gene specific removal of 8-oxoG after treatment of Chinese hamster ovary (CHO) fibroblasts with the photosensitizer Ro19-8022. This compound introduces 8-oxoG lesions, which can then be detected with the Escherichia coli formamidopyrimidine DNA glycosylase (FPG). In this report we present gene specific repair analysis of endogenous genes situated in different important cellular regions and also the first analysis of strand specific DNA repair of 8-oxoG in an endogenous gene. We were not able to detect any preferential repair of transcribed genes compared to non-transcribed regions and we did not detect any strand-bias in the repair of the housekeeping gene, dihydrofolate reductase (DHFR). In vivo, mitochondrial DNA is highly exposed to reactive oxygen species (ROS), and we find that the repair of 8-oxoG is more efficient in the mitochondrial DNA than in the nuclear DNA.

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein.The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and genespecific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV-or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways. INTRODUCTIONCockayne syndrome (CS) is an autosomal recessive human disorder with diverse clinical symptoms that include severe mental and physical growth retardation, microcephaly, progressive neurological and retinal degeneration, skeletal abnormalities, and a hypersensitivity to sunlight (Friedberg, 1996). Genetic analysis of fused heterodikaryons have identified two complementation groups involved in CS, designated CSA and CSB (Tanaka et al., 1981;Lehmann, 1982). CS cells demonstrate a reduced rate of nucleotide excision repair (NER) of active genes and, more specifically, of the transcribed strand of such genes (Venema et al., 1990;van Hoffen et al., 1993;Evans and Bohr, 1994). However, no defect in global NER is observed in CS. Defective transcription-coupled repair (TCR) in CS cells is found not only after UV exposure but also after exposure to certain forms of oxidative stress, suggesting that TCR is responsible for processing at least some types of oxidative DNA damage as well (Leadon and Cooper, 1993;Cooper and Leadon, 1994;Cooper et al., 1997).A characteristic feature of CS cells is the lack of recovery of RNA synthesis...

BackgroundThere is only one established drug binding site on sodium channels. However, drug binding of sodium channels shows extreme promiscuity: ∼25% of investigated drugs have been found to potently inhibit sodium channels. The structural diversity of these molecules suggests that they may not share the binding site, and/or the mode of action. Our goal was to attempt classification of sodium channel inhibitors by measuring multiple properties of inhibition in electrophysiology experiments. We also aimed to investigate if different properties of inhibition correlate with specific chemical properties of the compounds.Methodology/Principal FindingsA comparative electrophysiological study of 35 compounds, including classic sodium channel inhibitors (anticonvulsants, antiarrhythmics and local anesthetics), as well as antidepressants, antipsychotics and neuroprotective agents, was carried out using rNav1.2 expressing HEK-293 cells and the QPatch automatic patch-clamp instrument. In the multi-dimensional space defined by the eight properties of inhibition (resting and inactivated affinity, potency, reversibility, time constants of onset and offset, use-dependence and state-dependence), at least three distinct types of inhibition could be identified; these probably reflect distinct modes of action. The compounds were clustered similarly in the multi-dimensional space defined by relevant chemical properties, including measures of lipophilicity, aromaticity, molecular size, polarity and electric charge. Drugs of the same therapeutic indication typically belonged to the same type. We identified chemical properties, which were important in determining specific properties of inhibition. State-dependence correlated with lipophilicity, the ratio of the neutral form of molecules, and aromaticity: We noticed that the highly state dependent inhibitors had at least two aromatic rings, logP>4.0, and pKa<8.0.Conclusions/SignificanceThe correlations of inhibition properties both with chemical properties and therapeutic profiles would not have been evident through the sole determination of IC50; therefore, recording multiple properties of inhibition may allow improved prediction of therapeutic usefulness.

To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluCl␤ receptor of Caenorhabditis elegans and the vertebrate cationic ␣7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluCl␤ receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluCl␤ receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the P Cl /P Na ratios, but the GluCl␤ receptor's mutants retained high capacity to select Cl The invertebrate GluCl 2 receptor channels are pentameric transmembrane receptors belonging to a wide superfamily of Cys-loop receptors activated by various neurotransmitters such as acetylcholine (ACh), serotonin (5-hydroxtryptamine, 5HT), ␥-aminobutyric acid (GABA), Gly, Glu, or histamine (Fig. 1A) (1-8). This superfamily consists of cationic channels permeable to Na ϩ , K ϩ , and, in many subunit combinations, to Ca 2ϩ ions, as well as of anionic channels selective to Cl Ϫ ions (reviewed by Keramidas et al. (9)). Structural similarities shared by Cys-loop receptors enabled swapping of pore sequences between cationic and anionic channels so as to assess the involvement of specific amino acids in ion charge selectivity. It was previously shown that concomitant replacement of the residues at positions Ϫ2Ј, Ϫ1Ј, and 13Ј ( Fig. 1, B and C) of cationic receptors by the residues found at the homologous positions of anionic receptors, and vice versa, lead to conversion of ion charge selectivity (10 -13).Further mutagenesis studies led to the recognition that the different capacities of cationic versus anionic Cys-loop receptors to distinguish between the charge of ions rely on the differences in the amino acid composition at positions Ϫ1Ј and Ϫ2Ј (Fig. 1C) (13-16). The conserved pore-facing Glu residue at position Ϫ1Ј of cationic Cys-loop receptors was further inferred to form, around the axis of ion conduction, a negatively charged ring that plays the key role in cationic selectivity by interacting with cations and repulsing anions (12,13,15,16). Conversely, a conserved arginine at position 0Ј of anionic Cys-loop receptors was inferred to interact with anions and repulse cations. A basic residue at position 0Ј is also typical of all cationic Cys-loop receptors (Fig. 1C and the ligand-gated ion channels data base), but it was suggested that local conformational differences in the M1-M2 connecting segment (M1-M2 loop) orient this basic residue to the pore lumen only in anionic Cys-loop receptors (9, 15). These local conformational differences have been attributed to a proline residue, which is present exclusively at pos...

Cockayne syndrome (CS) is an autosomal recessive human disease characterized by UV-sensitivity as well as neurological and developmental abnormalities. Two complementation groups have been established, designated CS-A and CS-B. Traditionally, CSA and CSB have been ascribed a function in the transcriptioncoupled repair (TCR) pathway of nucleotide excision repair (NER) that e ciently removes bulky lesions from the transcribed strand of RNA polymerase II transcribed genes. To assess the role of the CSB protein in the repair of the highly mutagenic base lesion 7,8-dihydro-8-oxoguanine (8-oxoG), we have investigated the removal of this lesion using an in vitro incision approach with cell extracts as well as an in vivo approach with a modi®ed protocol of the gene-speci®c repair assay, which allows the measurement of base lesion repair in intragenomic sequences. Our results demonstrate that the integrity of the CSB protein is pivotal for processes leading to incision at the site of 8-oxoG and that the global genome repair (GGR) of this lesion requires a functional CSB gene product in vivo.

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