Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction

Neuronal voltage-gated potassium channels, K_V7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule K_V7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. K_V7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual K_V7 isoforms was investigated in human detrusor tissue using a panel of K_V7 openers with distinct activity profiles among K_V7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric K_V7.4 did not reduce detrusor contraction. This may suggest that the homomeric K_V7.4 channel plays a less significant role in bladder contraction and further investigation is needed.     

Primary Cilium-Mediated Retinal Pigment Epithelium Maturation Is Disrupted in Ciliopathy Patient Cells

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Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair

DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.     

Potential novel candidate polymorphisms identified in genome-wide association study for breast cancer susceptibility

Previous genome-wide association studies (GWAS) have shown several risk alleles to be associated with breast cancer. However, the variants identified so far contribute to only a small proportion of disease risk. The objective of our GWAS was to identify additional novel breast cancer susceptibility variants and to replicate these findings in an independent cohort. We performed a two-stage association study in a cohort of 3,064 women from Alberta, Canada. In Stage I, we interrogated 906,600 single nucleotide polymorphisms (SNPs) on Affymetrix SNP 6.0 arrays using 348 breast cancer cases and 348 controls. We used single-locus association tests to determine statistical significance for the observed differences in allele frequencies between cases and controls. In Stage II, we attempted to replicate 35 significant markers identified in Stage I in an independent study of 1,153 cases and 1,215 controls. Genotyping of Stage II samples was done using Sequenom Mass-ARRAY iPlex platform. Six loci from four different gene regions (chromosomes 4, 5, 16 and 19) showed statistically significant differences between cases and controls in both Stage I and Stage II testing, and also in joint analysis. The identified variants were from EDNRA, ROPN1L, C16orf61 and ZNF577 gene regions. The presented joint analyses from the two-stage study design were not significant after genome-wide correction. The SNPs identified in this study may serve as potential candidate loci for breast cancer risk in a further replication study in Stage III from Alberta population or independent validation in Caucasian cohorts elsewhere.     

Mechanisms of atrial-selective block of Na⁺ channels by ranolazine: II. Insights from a mathematical model

Block of Na(+) channel conductance by ranolazine displays marked atrial selectivity that is an order of magnitude higher that of other class I antiarrhythmic drugs. Here, we present a Markovian model of the Na(+) channel gating, which includes activation-inactivation coupling, aimed at elucidating the mechanisms underlying this potent atrial selectivity of ranolazine. The model incorporates experimentally observed differences between atrial and ventricular Na(+) channel gating, including a more negative position of the steady-state inactivation curve in atrial versus ventricular cells. The model assumes that ranolazine requires a hydrophilic access pathway to the channel binding site, which is modulated by both activation and inactivation gates of the channel. Kinetic rate constants were obtained using guarded receptor analysis of the use-dependent block of the fast Na(+) current (I(Na)). The model successfully reproduces all experimentally observed phenomena, including the shift of channel availability, the sensitivity of block to holding or diastolic potential, and the preferential block of slow versus fast I(Na.) Using atrial and ventricular action potential-shaped voltage pulses, the model confirms significantly greater use-dependent block of peak I(Na) in atrial versus ventricular cells. The model highlights the importance of action potential prolongation and of a steeper voltage dependence of the time constant of unbinding of ranolazine from the atrial Na(+) channel in the development of use-dependent I(Na) block. Our model predictions indicate that differences in channel gating properties as well as action potential morphology between atrial and ventricular cells contribute equally to the atrial selectivity of ranolazine. The model indicates that the steep voltage dependence of ranolazine interaction with the Na(+) channel at negative potentials underlies the mechanism of the predominant block of I(Na) in atrial cells by ranolazine.     

AFP-Pred: A random forest approach for predicting antifreeze proteins from sequence-derived properties

Some creatures living in extremely low temperatures can produce some special materials called “antifreeze proteins” (AFPs), which can prevent the cell and body fluids from freezing. AFPs are present in vertebrates, invertebrates, plants, bacteria, fungi, etc. Although AFPs have a common function, they show a high degree of diversity in sequences and structures. Therefore, sequence similarity based search methods often fails to predict AFPs from sequence databases. In this work, we report a random forest approach “AFP-Pred” for the prediction of antifreeze proteins from protein sequence. AFP-Pred was trained on the dataset containing 300 AFPs and 300 non-AFPs and tested on the dataset containing 181 AFPs and 9193 non-AFPs. AFP-Pred achieved 81.33% accuracy from training and 83.38% from testing. The performance of AFP-Pred was compared with BLAST and HMM. High prediction accuracy and successful of prediction of hypothetical proteins suggests that AFP-Pred can be a useful approach to identify antifreeze proteins from sequence information, irrespective of their sequence similarity.     

Insights into the Role of the Peroxisomal Ubiquitination Machinery in Pex13p Degradation in the Yeast Hansenula polymorpha

The import of matrix proteins into peroxisomes in yeast requires the action of the ubiquitin-conjugating enzyme Pex4p and a complex consisting of the ubiquitin E3 ligases Pex2p, Pex10p and Pex12p. Together, this peroxisomal ubiquitination machinery is thought to ubiquitinate the cycling receptor protein Pex5p and members of the Pex20p family of co-receptors, a modification that is required for receptor recycling. However, recent reports have demonstrated that this machinery plays a role in additional peroxisome-associated processes. Hence, our understanding of the function of these proteins in peroxisome biology is still incomplete. Here, we identify a role for the peroxisomal ubiquitination machinery in the degradation of the peroxisomal membrane protein Pex13p. Our data demonstrate that Pex13p levels build up in cells lacking members of this machinery and also establish that Pex13p undergoes rapid degradation in wild-type cells. Furthermore, we show that Pex13p is ubiquitinated in wild-type cells and also establish that Pex13p ubiquitination is reduced in cells lacking a functional peroxisomal E3 ligase complex. Finally, deletion of PEX2 causes Pex13p to build up at the peroxisomal membrane. Taken together, our data provide further evidence that the role of the peroxisomal ubiquitination machinery in peroxisome biology goes much deeper than receptor recycling alone.