Lack of apoptosis leads to cellular senescence and tumorigenesis in Drosophila epithelial cells.

Programmed cell death (apoptosis) is a homeostasis program of animal tissues designed to remove cells that are unwanted or are damaged by physiological insults. To assess the functional role of apoptosis, we have studied the consequences of subjecting Drosophila epithelial cells defective in apoptosis to stress or genetic perturbations that normally cause massive cell death. We find that many of those cells acquire persistent activity of the JNK pathway, which drives them into senescent status, characterized by arrest of cell division, cell hypertrophy, Senescent Associated ß-gal activity (SA-ß-gal), reactive oxygen species (ROS) production, Senescent Associated Secretory Phenotype (SASP) and migratory behaviour. We have identified two classes of senescent cells in the wing disc: 1) those that localize to the appendage part of the disc, express the upd, wg and dpp signalling genes and generate tumour overgrowths, and 2) those located in the thoracic region do not express wg and dpp nor they induce tumour overgrowths. Whether to become tumorigenic or non-tumorigenic depends on the original identity of the cell prior to the transformation. We also find that the p53 gene contributes to senescence by enhancing the activity of JNK.

The Trans Golgi Region is a Labile Intracellular Ca2+ Store Sensitive to Emetine.

The Golgi apparatus (GA) is a bona fide Ca2+ store; however, there is a lack of GA-specific Ca2+ mobilizing agents. Here, we report that emetine specifically releases Ca2+ from GA in HeLa and HL-1 atrial myocytes. Additionally, it has become evident that the trans-Golgi is a labile Ca2+ store that requires a continuous source of Ca2+ from either the external milieu or from the ER, to enable it to produce a detectable transient increase in cytosolic Ca2+. Our data indicates that the emetine-sensitive Ca2+ mobilizing mechanism is different from the two classical Ca2+ release mechanisms, i.e. IP3 and ryanodine receptors. This newly discovered ability of emetine to release Ca2+ from the GA may explain why chronic consumption of ipecac syrup has muscle side effects.

Functional histamine H3 and adenosine A2A receptor heteromers in recombinant cells and rat striatum.

In the striatum, histamine H3 receptors (H3Rs) are co-expressed with adenosine A2A receptors (A2ARs) in the cortico-striatal glutamatergic afferents and the GABAergic medium-sized spiny neurons that originate the indirect pathway of the basal ganglia. This location allows H3Rs and A2ARs to regulate the striatal GABAergic and glutamatergic transmission. However, whether these receptors can physically interact has not yet been assessed. To test this hypothesis, a heteromer-selective in vitro assay was used to detect functional complementation between a chimeric A2AR302-Gαqi4 and wild-type H3Rs in transfected HEK-293T cells. H3R activation with the agonist RAMH resulted in Ca2+ mobilization (pEC50 7.31 ±â€¯0.23; maximal stimulation, Emax 449 ±â€¯25% of basal) indicative of receptor heterodimerization. Functional H3R-A2AR heteromers were confirmed by co-immunoprecipitation and observations of differential cAMP signaling when both receptors were co-expressed in the same cells. In membranes from rat striatal synaptosomes, H3R activation decreased A2AR affinity for the agonist CGS-21680 (pKi values 8.10 ±â€¯0.04 and 7.70 ±â€¯0.04). Moreover, H3Rs and A2ARs co-immunoprecipitated in protein extracts from striatal synaptosomes. These results support the existence of a H3R-A2AR heteromer with possible physiological implications for the modulation of the intra-striatal transmission.

Differential homologous desensitization of the human histamine H3 receptors of 445 and 365 amino acids expressed in CHO-K1 cells.

Histamine H3 receptors (H3Rs) signal through Gαi/o proteins and are found in neuronal cells as auto- and hetero-receptors. Alternative splicing of the human H3R (hH3R) originates 20 isoforms, and the mRNAs of two receptors of 445 and 365 amino acids (hH3R445 and hH3R365) are widely expressed in the human brain. We previously showed that the hH3R445 stably expressed in CHO-K1 cells experiences homologous desensitization. The hH3R365 lacks 80 residues in the third intracellular loop, and in this work we therefore studied whether this isoform also experiences homologous desensitization and the possible differences with the hH3R445. In clones of CHO-K1 cells stably expressing similar receptor levels (211 ± 12 and 199 ± 16 fmol/mg protein for hH3R445 and hH3R365, respectively), there were no differences in receptor affinity for selective H3R ligands or for agonist-induced [35S]-GTPγS binding to membranes and inhibition of forskolin-stimulated cAMP accumulation in intact cells. For both cell clones, pre-incubation with the H3R agonist RAMH (1 µM) resulted in functional receptor desensitization, as indicated by cAMP accumulation assays, and loss of receptors from the cell surface and reduced affinity for the agonist immepip in cell membranes, evaluated by radioligand binding. However, functional desensitization differed in the maximal extent (96 ± 15% and 58 ± 8% for hH3R445 and hH3R365, respectively) and the length of pre-exposure required to reach the maximum desensitization (60 and 30 min, respectively). Furthermore, the isoforms differed in their recovery from desensitization. These results indicate that the hH3R365 experiences homologous desensitization, but that the process differs between the isoforms in time-course, magnitude and re-sensitization.

The naturally occurring mutation Y197C does not affect the expression or signaling of the human histamine H3 receptor.

There is evidence for genetic polymorphism within the human histamine H3 receptor (hH3R), and a Tyr to Cys exchange at position 197 (Y197C), located in the amino terminus of the fifth transmembrane domain, has been reported. In this work we compared the expression and the pharmacological and signaling properties of wild-type (hH3RWT) and mutant (hH3RY197C) receptors transiently expressed in CHO-K1 cells. The hH3RY197C cDNA was created by overlap extension PCR amplification. Receptor expression and affinity were assessed by N-α-[methyl-3H]-histamine binding to cell membranes and intact cells. Receptor function was evaluated by stimulation of [35S]-GTPγS binding to cell membranes and by inhibition of forskolin-induced cAMP accumulation in intact cells. The hH3RWT and hH3RY197C were expressed at similar levels (761±68 and 663±66fmol/mg protein for membranes, and 13,434±1533 and 15,894±1884 receptors per cell, respectively). There were no significant differences in the affinities for H3R agonists or antagonists/inverse agonists between the hH3RWT and hH3RY197C, and the H3R agonist RAMH was similarly efficacious and potent to stimulate [35S]-GTPγS binding and to inhibit forskolin-induced cAMP accumulation. These results indicate that the Y197C mutation does not affect the expression, ligand affinity or signaling of the human H3 receptor.

Large area graphene nanomesh: an artificial platform for edge-electrochemical biosensing at the sub-attomolar level.

Recent advances in large area graphene growth have led to tremendous applications in a variety of areas. The graphene nanomesh with its tunable band-gap is of great interest for both fundamental research, to explore the effect of edges on both the 2D electrical conduction and its electrochemical behavior, and applications such as nanoelectronic devices or highly sensitive biosensors. Here, we report on the fabrication of a large surface graphene nanomesh by nanoimprint lithography (NIL) to produce controlled artificial edges. The electrochemical response of this high quality single graphene layer imprinted nanomesh shows an enhancement in capacitance associated with faster electron transfer which can be attributed to the high density of edges. The electrochemical performances of this nanomesh graphene platform have been also studied for label-free DNA detection from Hepatitis C virus as a model. We demonstrate that such a nanomesh platform allows direct detection at the sub-attomolar level with more than 90% of molecules located on the imprinted artificial edges. Such a graphene nanomesh electrode will find useful future applications in the field of biosensing.

A single-point mutation (Ala280Val) in the third intracellular loop alters the signalling properties of the human histamine H3 receptor stably expressed in CHO-K1 cells.

BACKGROUND AND PURPOSE: An alanine to valine exchange at amino acid position 280 (A280V) in the third intracellular loop of the human histamine H3 receptor was first identified in a patient suffering from Shy-Drager syndrome and later reported as a risk factor for migraine. Here, we have compared the pharmacological and signalling properties of wild-type (hH3 R(WT)) and A280V mutant (hH3 R(A280V)) receptors stably expressed in CHO-K1 cells. EXPERIMENTAL APPROACH: The hH3 R(A280V) cDNA was created by overlapping extension PCR amplification. Receptor expression and affinity were assessed by radioligand (N-α-[methyl-³H]-histamine) binding to cell membranes, and receptor function by the inhibition of forskolin-induced cAMP accumulation and stimulation of ERK1/2 phosphorylation in intact cells, as well as stimulation of [³5S]-GTPγS binding to cell membranes. KEY RESULTS: Both receptors were expressed at similar levels with no significant differences in their affinities for H3 receptor ligands. Upon activation the hH3 RWT was significantly more efficacious to inhibit forskolin-induced cAMP accumulation and to stimulate [³5S]-GTPγS binding, with no difference in pEC50 estimates. The hH3 RWT was also more efficacious to stimulate ERK1/2 phosphorylation, but this difference was not significant. The inverse agonist ciproxifan was more efficacious at hH3 RWT to reduce [³5S]-GTPγS binding but, for both receptors, failed to enhance forskolin-induced cAMP accumulation. CONCLUSIONS AND IMPLICATIONS: The A280V mutation reduces the signalling efficacy of the human H3 receptor. This effect may be relevant to the pathophysiology of disorders associated with the mutation.

Orientation of the calcium channel beta relative to the alpha(1)2.2 subunit is critical for its regulation of channel activity.

BACKGROUND: The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure. METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity.

The I-II loop controls plasma membrane expression and gating of Ca(v)3.2 T-type Ca2+ channels: a paradigm for childhood absence epilepsy mutations.

Calcium currents via low-voltage-activated T-type channels mediate burst firing, particularly in thalamic neurons. Considerable evidence supports the hypothesis that overactive T-channels may contribute to thalamocortical dysrhythmia, including absence epilepsy. Single nucleotide polymorphisms in one of the T-channel genes (CACNA1H, which encodes Ca(v)3.2) are associated with childhood absence epilepsy in a Chinese population. Because only a fraction of these polymorphisms are predicted to increase channel activity and neuronal firing, we hypothesized that other channel properties may be affected. Here we describe that all the polymorphisms clustered in the intracellular loop connecting repeats I and II (I-II loop) increase the surface expression of extracellularly tagged Ca(v)3.2 channels. The functional domains within the I-II loop were then mapped by deletion analysis. The first 62 amino acids of the loop (post IS6) are involved in regulating the voltage dependence of channel gating and inactivation. Similarly, the last 15 amino acids of the loop (pre IIS1) are involved in channel inactivation. In contrast, the central region of I-II loop regulates surface expression, with no significant effect on channel biophysics. Electrophysiology, luminometry, fluorescence-activated cell sorting measurements, and confocal microscopy studies demonstrate that deletion of this central region leads to enhanced surface expression of channels from intracellular compartments to the plasma membrane. These results provide novel insights into how CACNA1H polymorphisms may contribute to Ca(v)3.2 channel overactivity and consequently to absence epilepsy and establish the I-II loop as an important regulator of Ca(v)3.2 channel function and expression.

Transfer of beta subunit regulation from high to low voltage-gated Ca2+ channels.

High voltage-activated Ca(2+) channel expression and gating is controlled by their beta subunits. Although the sites of interaction are known at the atomic level, how beta modulates gating remains to be determined. Using a chimeric approach, beta subunit regulation was conferred to a low voltage-activated channel. Regulation was dependent on a rigid linker connecting the alpha(1) interaction domain to IS6. Chimeric channels also revealed a role for IS6 in channel gating. Taken together, these results support a direct coupling model where beta subunits alter movements in IS6 that occur as the channel transits between closed, open, and inactivated states.

Functional impact of alternative splicing of human T-type Cav3.3 calcium channels.

Low-voltage-activated T-type (Cav3) Ca2+ channels produce low-threshold spikes that trigger burst firing in many neurons. The CACNA1I gene encodes the Cav3.3 isoform, which activates and inactivates much more slowly than the other Cav3 channels. These distinctive kinetic features, along with its brain-region-specific expression, suggest that Cav3.3 channels endow neurons with the ability to generate long-lasting bursts of firing. The human CACNA1I gene contains two regions of alternative splicing: variable inclusion of exon 9 and an alternative acceptor site within exon 33, which leads to deletion of 13 amino acids (Delta33). The goal of this study is to determine the functional consequences of these variations in the full-length channel. The cDNA encoding these regions were cloned using RT-PCR from human brain, and currents were recorded by whole cell patch clamp. Introduction of the Delta33 deletion slowed the rate of channel opening. Addition of exon 9 had little effect on kinetics, whereas its addition to Delta33 channels unexpectedly slowed both activation and inactivation kinetics. Modeling of neuronal firing showed that exon 9 or Delta33 alone reduced burst firing, whereas the combination enhanced firing. The major conclusions of this study are that the intracellular regions after repeats I and IV play a role in channel gating, that their effects are interdependent, suggesting a direct interaction, and that splice variation of Cav3.3 channels provides a mechanism for fine-tuning the latency and duration of low-threshold spikes.

Cloning of a novel one-repeat calcium channel-like gene.

We describe the cloning of a cDNA from a human testis library that encodes a novel protein with similarity to one repeat of voltage-gated Ca(2+) channels (Ca(v)). Northern and dot blot analyses indicate that the novel Ca(v)-like gene is expressed predominantly in testis and at lower levels in many other tissues. Heterologous expression of the Ca(v)-like protein did not lead to the induction of any detectable ionic current and failed to modify intracellular Ca(2+) concentrations. Similar one-repeat Ca(v)-like proteins have been cloned from Bacillus, Mus, and Homo, and appear to encode ion channels involved in renal function, axis determination, and sperm motility.

Alternative splicing of the rat Ca(v)3.3 T-type calcium channel gene produces variants with distinct functional properties(1).

Molecular diversity in T-type Ca(2+) channels is produced by expression of three genes, and alternative splicing of those genes. Prompted by differences noted between rat and human Ca(v)3.3 sequences, we searched for splice variants. We cloned six variants, which are produced by splicing at exon 33 and exon 34. Expression of the variants differed between brain regions. The electrophysiological properties of the variants displayed similar voltage-dependent gating, but differed in their kinetic properties. The functional impact of splicing was inter-related, suggesting an interaction. We conclude that alternative splicing of the Ca(v)3.3 gene produces channels with distinct properties.

Cloning and expression of the human T-type channel Ca(v)3.3: insights into prepulse facilitation.

The full-length human Ca(v)3.3 (alpha(1I)) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3' untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5 mM Ca(2+) as charge carrier. From a holding potential of -100 mV, step depolarizations elicited inward currents with an apparent threshold of -70 mV, a peak of -30 mV, and reversed at +40 mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Ca(v)3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Ca(v)3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Ca(v)3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100 mV, 200 ms) increased the fraction of channels that gated at low voltages. In contrast, human Ca(v)3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Ca(v)3.3 is similar to high voltage-activated Ca(2+) channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity.