Characterization of a ligand binding site in the human transient receptor potential ankyrin 1 pore.

The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule.

First and second generation γ-secretase modulators (GSMs) modulate amyloid-ß (Aß) peptide production through different mechanisms.

γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-ß (Aß) peptides. The Aß42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs), such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide, have been suggested to modulate the Alzheimer-related Aß production by targeting the APP. Here, we describe novel GSMs that are selective for Aß modulation and do not impair processing of Notch, EphB2, or EphA4. The GSMs modulate Aß both in cell and cell-free systems as well as lower amyloidogenic Aß42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM, E2012, but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a (3)H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined, these data provide compelling evidence of a growing class of in vivo active GSMs, which are selective for Aß modulation and have a different mechanism of action compared with the original class of GSMs described.

SCN10A Mutation in a Patient with Erythromelalgia Enhances C-Fiber Activity Dependent Slowing.

Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient's peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences.

Tau-tubulin kinase 1 expression, phosphorylation and co-localization with phospho-Ser422 tau in the Alzheimer's disease brain.

Recent reports have implicated tau-tubulin kinase 1 (TTBK1) in the pathological phosphorylation of tau that occurs in Alzheimer's disease (AD). The present study was undertaken to provide an extensive characterization of TTBK1 mRNA and protein expression in human brain from AD cases and non-demented controls so as to better understand the disease relevance of this novel kinase. In situ hybridization and immunohistochemistry revealed abundant expression of TTBK1 in the somatodendritic compartment of cortical and hippocampal neurons of both AD cases and controls. TTBK1 immunoreactivity appeared to vary with the level of phospho-tau staining, and was strong in the somatodendritic compartment of apparently healthy hippocampal neurons as well as in pre-tangle neurons where it co-localized with diffuse phospho-Ser422 tau staining. Ser422 was confirmed as a TTBK1 substrate in vitro, and an antibody towards the site, in addition to labeling AT8-positive neurofibrillary tangles (NFTs), neuritic plaques and neuropil threads, also labeled a small population of neurons that were unlabeled with AT8. These data suggest a role for TTBK1 in pre-tangle formation prior to the formation of fibrillar tau and strengthen the idea that tau is phosphorylated at Ser422 at an early/intermediate stage in NFT formation.