Study of TRP channels by automated patch clamp systems.

Ion channels are responsible for the permeation of ions across the membrane and their central role in cellular physiology is well established. Historically, the direct study of ion channels has been considered technically challenging. As such, a significant barrier to drug discovery for ion channels has been the low throughput of high quality electrophysiological data.

Classification of drugs based on properties of sodium channel inhibition: a comparative automated patch-clamp study.

Background: There 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.

Automated patch-clamp technique: increased throughput in functional characterization and in pharmacological screening of small-conductance Ca2+ release-activated Ca2+ channels.

The suitability of an automated patch clamp for the characterization and pharmacological screening of calcium release-activated calcium (CRAC) channels endogenously expressed in RBL-2H3 cells was explored with the QPatch system. CRAC currents (I( CRAC)) are small, and thus precise recordings require high signal-to-noise ratios obtained by high seal resistances. Automated whole-cell establishment resulted in membrane resistances of 1728 +/- 226 MOmega (n = 44).

Mechanism of Cl- selection by a glutamate-gated chloride (GluCl) receptor revealed through mutations in the selectivity filter.

To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClbeta receptor of Caenorhabditis elegans and the vertebrate cationic alpha7-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 GluClbeta 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 GluClbeta receptor's pore from anionic to cationic.

Pore conformations and gating mechanism of a Cys-loop receptor.

Neurons regulate the propagation of chemoelectric signals throughout the nervous system by opening and closing ion channels, a process known as gating. Here, histidine-based metal-binding sites were engineered along the intrinsic pore of a chimeric Cys-loop receptor to probe state-dependent Zn(2+)-channel interactions. Patterns of Zn(2+) ion binding within the pore reveal that, in the closed state, the five pore-lining segments adopt an oblique orientation relative to the axis of ion conduction and constrict into a physical gate at their intracellular end.

Transcription in neuromuscular junction formation: who turns on whom?

The neuromuscular junction is a particular advantageous synapse to investigate the molecular processes engaged in synaptogenesis. Both the motor nerve and the muscle cell contribute to the patterned distribution of a definite set of membrane proteins into two distinct regions, a sub-synaptic end plate zone and an extra-synaptic region. Accordingly, the transcription of acetylcholine receptors is highly compartmentalized being much higher in the sub-synaptic the in the extra-synaptic region.

Transcriptional regulation of the murine brca2 gene by CREB/ATF transcription factors.

The brca2 gene encodes a nuclear protein which is mainly involved in DNA repair and, when mutated, is responsible for some of the hereditary breast cancers. However, brca2 expression is also deregulated in sporadic breast tumors. In the mouse brca2 gene we had earlier identified a region of 148bp upstream of the transcription start site sufficient to activate its expression.

Phosphorylation-elicited quaternary changes of GA binding protein in transcriptional activation.

Enrichment of nicotinic acetylcholine receptors (nAChR) on the tip of the subjunctional folds of the postsynaptic membrane is a central event in the development of the vertebrate neuromuscular junction. This is attained, in part, through a selective transcription in the subsynaptic nuclei, and it has recently been shown that the GA binding protein (GABP) plays an important role in this compartmentalized expression. The neural factor heregulin (HRG) activates nAChR transcription in cultured cells by stimulating a signaling cascade of protein kinases.

Repair of 8-oxoG is slower in endogenous nuclear genes than in mitochondrial DNA and is without strand bias.

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).

Global genome repair of 8-oxoG in hamster cells requires a functional CSB gene product.

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 transcription-coupled repair (TCR) pathway of nucleotide excision repair (NER) that efficiently removes bulky lesions from the transcribed strand of RNA polymerase II transcribed genes.