Biomedical subjects

Andrea Becchetti

Publications and source records attributed to Andrea Becchetti.

9 recordsLinked to original sources

PubMed2006

Complex functional interaction between integrin receptors and ion channels.

Integrin receptors mediate adhesion of the cell to the extracellular matrix and thereby regulate cell motility, proliferation, differentiation and apoptosis. These processes are frequently accompanied by alterations in ion flow. Recent evidence suggests that integrins can regulate ion channels and form macromolecular complexes, thus contributing to the localization of the channel onto the plasma membrane. The integrin-channel complex regulates downstream signaling proteins, such as tyrosine kinases and GTPases. This process could occur in plasma membrane microdomains, such as caveolae. It seems that ion channels sometimes transmit their signals through conformational coupling, instead of change in ion fluxes. Finally, the channel protein is not merely a final target, because it often feeds back by controlling integrin activation and/or expression. These findings have important implications for the physiology of normal and neoplastic cells and suggest interesting perspectives for studies of synaptic plasticity.

Animals↗

PubMed2006

Increased sensitivity of the neuronal nicotinic receptor alpha 2 subunit causes familial epilepsy with nocturnal wandering and ictal fear.

Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the alpha 4 and beta 2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular genetic study of a large pedigree segregating sleep-related epilepsy in which seizures are associated with fear sensation, tongue movements, and nocturnal wandering, closely resembling nightmares and sleep walking. We identified a new genetic locus for familial sleep-related focal epilepsy on chromosome 8p12.3-8q12.3. By sequencing the positional candidate neuronal cholinergic receptor alpha 2 subunit gene (CHRNA2), we detected a heterozygous missense mutation, I279N, in the first transmembrane domain that is crucial for receptor function. Whole-cell recordings of transiently transfected HEK293 cells expressing either the mutant or the wild-type receptor showed that the new CHRNA2 mutation markedly increases the receptor sensitivity to acetylcholine, therefore indicating that the nicotinic alpha 2 subunit alteration is the underlying cause. CHRNA2 is the third neuronal cholinergic receptor gene to be associated with familial sleep-related epilepsies. Compared with the CHRNA4 and CHRNB2 mutations reported elsewhere, CHRNA2 mutations cause a more complex and finalized ictal behavior.

Acetylcholine↗

PubMed2005

Expression pattern of the ether-a-go-go-related (ERG) family proteins in the adult mouse central nervous system: evidence for coassembly of different subunits.

Voltage-dependent K+ channels are the main determinants in controlling cellular excitability within the central nervous system. Among voltage-dependent K+ channels, the ERG subfamily is deeply involved in the control of cellular excitability, both in mammals and in invertebrates. ERG channels are encoded by different genes: the erg1 gene, which can generate two alternative transcripts (erg1a and erg1b), erg2 and erg3. The aim of the present study was to determine the expression pattern and cellular localization of ERG proteins (ERG1, ERG2, and ERG3) in the mouse CNS, differentiating, for the first time, the ERG1A and ERG1B isoforms. To this purpose, novel specific antibodies were raised against the various channel proteins and their specificity and immunoreactivity tested. It emerged that: 1) all the erg genes were indeed translated in neuronal tissue; 2) ERG proteins distribution in the mouse CNS often overlapped, and only in specific areas each ERG protein showed a distinct pattern of expression; and 3) ERG proteins were generally expressed in neuronal soma, but dendritic and/or white matter labeling could be detected in specific areas. The finding that ERG proteins often have an overlapping expression suggests that neuronal ERG currents could be determined, at least in part, by heterotetrameric ERG channels. This suggestion is demonstrated to occur for ERG1A/ERG1B by showing that the two isoforms coassemble in mouse brain.

Animals↗

PubMed2005

Human ether-a-go-go-related gene 1 channels are physically linked to beta1 integrins and modulate adhesion-dependent signaling.

Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adhesion. Additionally, integrins trigger multiple signaling pathways that are involved in cell migration, proliferation, survival, and differentiation. We previously demonstrated that the activation of integrins containing the beta(1) subunit leads to a selective increase in potassium currents carried by the human ether-a-go-go-related gene (hERG) channels in neuroblastoma and leukemia cells; this current activation modulates adhesion-dependent differentiation in these cells. We hypothesized that the cross-talk between integrins and hERG channels could be traced back to the assembly of a macromolecular signaling complex comprising the two proteins. We tested this hypothesis in both SH-SY5Y neuroblastoma cells and in human embryonic kidney 293 cells stably transfected with hERG1 and, therefore, expressing only the full-length hERG1 protein on the plasma membrane. The beta(1) integrin and hERG1 coprecipitate in these cells and colocalize in both intracellular and surface membrane compartments. The two proteins also coprecipitate with caveolin-1, suggesting the localization of the complex in lipid rafts/caveolae. hERG1-transfected cells undergo an activation of hERG currents after beta(1) integrin-mediated adhesion to fibronectin; concomitant with this activation, the focal adhesion kinase associates with the hERG1 protein and becomes tyrosine phosphorylated. Using hERG1-specific inhibitors, we show that the tyrosine phosphorylation of focal adhesion kinase is strictly dependent on hERG channel activity. Similarly, the activity of the small GTPase Rac1 turned out to be dependent on hERG currents. On the whole, these data indicate that the hERG1 protein associates with beta(1) integrins and modulates adhesion receptor signaling.

Cell Adhesion↗

PubMed2003

The NO pathway acts late during the fertilization response in sea urchin eggs.

Both the inositol 1,4,5-trisphosphate (InsP(3)) and ryanodine receptor pathways contribute to the Ca(2+) transient at fertilization in sea urchin eggs. To date, the precise contribution of each pathway has been difficult to ascertain. Evidence has accumulated to suggest that the InsP(3) receptor pathway has a primary role in causing Ca(2+) release and egg activation. However, this was recently called into question by a report implicating NO as the primary egg activator. In the present study we pursue the hypothesis that NO is a primary egg activator in sea urchin eggs and build on previous findings that an NO/cGMP/cyclic ADP-ribose (cADPR) pathway is active at fertilization in sea urchin eggs to define its role. Using a fluorescence indicator of NO levels, we have measured both NO and Ca(2+) at fertilization and establish that NO levels rise after, not before, the Ca(2+) wave is initiated and that this rise is Ca(2+)-dependent. By inhibiting the increase in NO at fertilization, we find not that the Ca(2+) transient is abolished but that the duration of the transient is significantly reduced. The latency and rise time of the transient are unaffected. This effect is mirrored by the inhibition of cGMP and cADPR signaling in sea urchin eggs at fertilization. We establish that cADPR is generated at fertilization, at a time comparable to the time of the rise in NO levels. We conclude that NO is unlikely to be a primary egg activator but, rather, acts after the initiation of the Ca(2+) wave to regulate the duration of the fertilization Ca(2+) transient.

Animals↗

PubMed2003

Developmentally regulated expression of the mouse homologues of the potassium channel encoding genes m-erg1, m-erg2 and m-erg3.

Deciphering the expression pattern of K+ channel encoding genes during development can help in the understanding of the establishment of cellular excitability and unravel the molecular mechanisms of neuromuscular diseases. We focused our attention on genes belonging to the erg family, which is deeply involved in the control of neuromuscular excitability in Drosophila flies and possibly other organisms. Both in situ hybridisation and RNase Protection Assay experiments were used to study the expression pattern of mouse (m)erg1, m-erg2 and m-erg3 genes during mouse embryo development, to allow the pattern to be compared with their expression in the adult. M-erg1 is first expressed in the heart and in the central nervous system (CNS) of embryonic day 9.5 (E9.5) embryos; the gene appears in ganglia of the peripheral nervous system (PNS) (dorsal root (DRG) and sympathetic (SCG) ganglia, mioenteric plexus), in the neural layer of retina, skeletal muscles, gonads and gut at E13.5. In the adult m-erg1 is expressed in the heart, various structures of the CNS, DRG and retina. M-erg2 is first expressed at E9.5 in the CNS, thereafter (E13.5) in the neural layer of retina, DRG, SCG, and in the atrium. In the adult the gene is present in some restricted areas of the CNS, retina and DRG. M-erg3 displayed an expression pattern partially overlapping that of m-erg1, with a transitory expression in the developing heart as well. A detailed study of the mouse adult brain showed a peculiar expression pattern of the three genes, sometimes overlapping in different encephalic areas.

Animals↗

PubMed2002

Cell cycle-dependent expression of HERG1 and HERG1B isoforms in tumor cells.

The role of K(+) channel activity during cell cycle progression has become a research topic of considerable interest. Blocking of K(+) channels inhibits the proliferation of many cell types, although the mechanism of this inhibition is unclear. There is speculation that K(+) channels differentially regulate the electrical potential of the plasma membrane (V(m)) during proliferation. We have demonstrated that in tumor cells the value of V(m) is clamped to rather depolarized values by K(+) channels belonging to the HERG family. We report here that tumor cell lines preferentially express the herg1 gene and a truncated, N-deleted form that corresponds to herg1b. This alternative transcript is also expressed in human primary acute myeloid leukemias. Both HERG1 and HERG1B proteins are expressed on the plasma membrane of tumor cells and can form heterotetramers. The expression of HERG protein isoforms is strongly cell cycle-dependent, accounting for variations in HERG currents along the mitotic cycle. Moreover, the blocking of HERG channels dramatically impairs cell growth of HERG-bearing tumor cells. These results suggest that modulated expression of different K(+) channels is the molecular basis of a novel mechanism regulating neoplastic cell proliferation.

Base Sequence↗

PubMed2002

The functional properties of the human ether-à-go-go-like (HELK2) K+ channel.

The voltage-dependent K+ channels belonging to the ether-à-go-go family (eag, erg, elk) are widely expressed in the mammalian CNS. Their neuronal function, however, is poorly understood. Among the elk clones, elk2 is the most abundantly expressed in the brain. We have characterized the human ELK2 channel (HELK2) expressed in mammalian cell lines. Moreover, we have detected helk2 mRNA and ELK2-like currents in freshly dissociated human astrocytoma cells. HELK2 was inhibited by Cs+ in a voltage-dependent way (Kd was 0.7 mm, at -120 mV). It was not affected by Way 123398 (5 micro m), dofetilide (10 micro m), quinidine (10 micro m), verapamil (20 micro m), haloperidol (2 micro m), astemizole (1 micro m), terfenadine (1 micro m) and hydroxyzine (30 micro m), compounds known to inhibit the biophysically related HERG channel. The crossover of the activation and inactivation curves produced a steady state 'window' current with a peak around -20 mV and considerably broader than it usually is in voltage-dependent channels, including HERG. Similar features were observed in the ELK2 clone from rat, in the same experimental conditions. Thus, ELK2 channels are active within a wide range of membrane potentials, both sub- and suprathreshold. Moreover, the kinetics of channel deactivation and removal of inactivation was about one order of magnitude quicker in HELK2, compared to HERG. Overall, these properties suggest that ELK2 channels are very effective at dampening the neuronal excitability, but less so at producing adaptation of action potential firing frequency. In addition, we suggest experimental ways to recognize HELK2 currents in vivo and raise the issue of the possible function of these channels in astrocytoma.

Action Potentials↗

PubMed1992

A Quantitative Description of Low- and High-threshold Ca2+ Spikes in Rat Sensory Neurons: A Perforated-patch Study.

Action potentials generated by voltage-dependent Ca2+ conductances were studied at 25 degrees C with the perforated-patch technique, in freshly dispersed adult rat sensory neurons perfused with Na-free solutions containing tetraethylammonium. Brief depolarizing currents from membrane potentials negative to - 75 mV always elicited long (> 100 ms) plateau spikes which had different thresholds in different neurons: a low threshold around - 60/- 50 mV and a high-threshold at - 30/- 20 mV. Stimulations from potentials positive to - 55 mV, on the contrary, elicited spikes originating only in the high threshold region and sensitive to 25 microM Cd2+, designated high-threshold spikes. In neurons which showed spikes with low threshold, addition of 25 microM Cd2+ disclosed a smaller and shorter regenerative response, the low-threshold spike. Moreover, the classical 'anode-break' stimulation from - 50/- 60 mV uncovered isolated low-threshold spikes, indicating a time- and voltage-dependent de-inactivating process. From the properties of the low (LVA) and high (HVA) voltage-activated Ca2+ currents, recorded under the same extracellular conditions, a Hodgkin - Huxley model was derived and used to reconstruct all the features of the recorded spikes. The model was also able to simulate experimental blocking of LVA channels by amiloride, modulation of HVA channels by baclofen and induced oscillatory firing. This agreement between the behaviour of recorded spikes and their mathematical description led us to conclude that the LVA and HVA Ca2+ currents underlie the low- and high-threshold Ca2+ spikes, respectively. Furthermore, our data suggest that complex behaviour known to be typical of central nervous system neurons is also present in sensory peripheral neurons.

Journal Article↗