Enzo Wanke | Università di Milano-Bicocca (original) (raw)

Papers by Enzo Wanke

Frontiers in cellular neuroscience, 2015

Frontiers in cellular neuroscience, 2015

Febs Letters, 1998

The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) which plays a fundamenta... more The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) which plays a fundamental role in heart excitability and in neurons by contributing to action potential repolarization and to spike-frequency adaptation, respectively. In this paper we show that IHERG, recorded in neuroblastoma cells and guinea-pig ventricular myocytes, was reversibly inhibited by the KATP channel blocker glibenclamide (IC50=74 μM). The voltage

Febs Letters, 1998

The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) which plays a fundamenta... more The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) which plays a fundamental role in heart excitability and in neurons by contributing to action potential repolarization and to spike-frequency adaptation, respectively. In this paper we show that IHERG, recorded in neuroblastoma cells and guinea-pig ventricular myocytes, was reversibly inhibited by the KATP channel blocker glibenclamide (IC50=74 μM). The voltage

The Journal of Physiology, 1997

1. The regular firing of a Hodgkin-Huxley neurone endowed with fast Nae and delayed K+ channels c... more 1. The regular firing of a Hodgkin-Huxley neurone endowed with fast Nae and delayed K+ channels can be converted into adapting firing by appending HERG (human eag-related gene) channels. 2. The computer model predictions were verified by studying the firing properties of F-11 DRG neurone x neuroblastoma hybrid cells induced to differentiate by long-term exposure to retinoic acid. These cells, which express HERG currents (IHERG), show clear spike-frequency adaptation of their firing when current clamped with long depolarizations. 3. In agreement with the prediction, the selective blocking of IHERG by class III antiarrhythmic drugs always led to the disappearance of the spike-frequency adaptation, and the conversion of adapting firing to regular firing. 4. It is proposed that, in addition to their role in the repolarization of the heart action potential, HERG channels may sustain a process of spike-frequency adaptation, and hence contribute to the control of burst duration in a way that is similar to that of the K+ currents,

The Journal of Physiology, 1997

1. The regular firing of a Hodgkin-Huxley neurone endowed with fast Nae and delayed K+ channels c... more 1. The regular firing of a Hodgkin-Huxley neurone endowed with fast Nae and delayed K+ channels can be converted into adapting firing by appending HERG (human eag-related gene) channels. 2. The computer model predictions were verified by studying the firing properties of F-11 DRG neurone x neuroblastoma hybrid cells induced to differentiate by long-term exposure to retinoic acid. These cells, which express HERG currents (IHERG), show clear spike-frequency adaptation of their firing when current clamped with long depolarizations. 3. In agreement with the prediction, the selective blocking of IHERG by class III antiarrhythmic drugs always led to the disappearance of the spike-frequency adaptation, and the conversion of adapting firing to regular firing. 4. It is proposed that, in addition to their role in the repolarization of the heart action potential, HERG channels may sustain a process of spike-frequency adaptation, and hence contribute to the control of burst duration in a way that is similar to that of the K+ currents,

European Journal of Biochemistry, 2004

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, clos... more Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.

Molecular Biology of the Cell, 2005

Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adh... more 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 ␤ 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 ␤ 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 ␤ 1 integrinmediated 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 ␤ 1 integrins and modulates adhesion receptor signaling.

Molecular Biology of the Cell, 2005

Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adh... more 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 ␤ 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 ␤ 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 ␤ 1 integrinmediated 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 ␤ 1 integrins and modulates adhesion receptor signaling.

Brain Research, 1995

In in vitro slices prepared from rat sensorimotor cortex, intraceilular recordings were obtained ... more In in vitro slices prepared from rat sensorimotor cortex, intraceilular recordings were obtained from 107 layer V pyramidal neurons, subsequently injected with biocytin for morphological reconstruction. Of the 107 neurons, 59 (55.1%) were identified as adapting (45) or non-adapting (13) regular spiking neurons (RS), and 48 (44.9%) as intrinsically bursting (IB) neurons discharging with an initial cluster of action potentials, which tended to recur rhythmically in a subset of 19 cells. The block of IAR by extracellular Cs + did not affect burst generation, but enhanced the tendency to reburst in IB neurons. A similar effect was induced by other procedures affecting K+-dependent post-burst hyperpolarization. In IB neurons Ca 2+ spikes had a longer decay time than in RS neurons, however selective blockers of both low and high threshold Ca a+ conductances failed to impair bursting activity. On the contrary, the perfusion of the slices with 0.5-1 /zM qTX suppressed bursting behaviour in a critical time interval preceding the complete block of Na +-dependent action potentials. It is concluded that the persistent Na + current INA P is the most important intrinsic factor for the typical firing properties of IB neurons, while Ca ~+ and K + conductances appear to contribute towards shaping bursts and controlling their recurrence rate. The morphology, connectivity and physiological properties of adapting and non-adapting RS neurons are particularly suited to the processing of respectively phasic and tonic inputs, whereas the properties of IB neurons are consistent with their suggested role in cortical rhythmogenesis and in the pathophysiological synchronized activities underlying epileptogenesis.

European Journal of Neuroscience, 2003

In this report we studied and compared the biochemical and the electrophysiological characteristi... more In this report we studied and compared the biochemical and the electrophysiological characteristics of two cell lines (GT1-7 and GN11) of immortalized mouse LHRH-expressing neurons and the correlation with their maturational stage and migratory activity. In fact, previous results indicated that GN11, but not GT1-7, cells exhibit an elevated motility in vitro. The results show that the two cell lines differ in terms of immunoreactivity for tyrosine hydroxylase and nestin as well as of production and release of 3,4-dihydroxyphenylalanine (DOPA) and of intracellular distribution and release of the LHRH. Patch-clamp recordings in GN11 cells, reveal the presence of a single inward recti®er K current indicative of an immature neuronal phenotype (neither ®ring nor electrical activity). In contrast, as known from previous studies, GT1-7 cells show the characteristics of mature LHRH neurons with a high electrical activity characterized by spontaneous ®ring and excitatory postsynaptic potentials. K -induced depolarization induces in GT1-7 cells, but not in GN11 cells, a strong increase in the release of LHRH in the culture medium. However, depolarization of GN11 cells signi®cantly decreases their chemomigratory response. In conclusion, these results indicate that GT1-7 and GN11 cells show different biochemical and electrophysiological characteristics and are representative of mature and immature LHRH neurons, respectively. The early stage of maturation of GN11 cells, as well as the low electrical activity detected in these cells, appears to correlate with their migratory activity in vitro.

Cancer Research, 2007

We worked out an experimental protocol able to purge the stem cell compartment of the SH-SY5Y neu... more We worked out an experimental protocol able to purge the stem cell compartment of the SH-SY5Y neuroblastoma clone. This protocol was based on the prolonged treatment of the wild-type cell population with either hypoxia or the antiblastic etoposide. Cell fate was monitored by immunocytochemical and electrophysiologic (patch-clamp) techniques. Both treatments produced the progressive disappearance of neuronal type (N) cells (which constitute the bulk of the tumor), leaving space for a special category of epithelial-like substrate-adherent cells (S 0 ). The latter represent a minimal cell component of the untreated population and are endowed with immunocytochemical markers (p75, c-kit, and CD133) and the electrophysiologic ''nude'' profile, typical of the neural crest stem cells. S 0 cells displayed a highly clonogenic potency and a substantial plasticity, generating both the N component and an alternative subpopulation terminally committed to the fibromuscular lineage. Unlike the N component, this lineage was highly insensitive to the apoptotic activity of hypoxia and etoposide and developed only when the neuronal option was abolished. Under these conditions, the fibromuscular progeny of S 0 expanded and progressed up to the exhaustion of the staminal compartment and to the extinction of the tumor. When combined, hypoxia and etoposide cooperated in abolishing the N cell generation and promoting the conversion of the tumor described. This synergy might mirror a natural condition in the ischemic areas occurring in cancer. These results have relevant implications for the understanding of the documented tendency of neuroblastomas to regress from a malignant to a benign phenotype, either spontaneously or on antiblastic treatment.

Brain Research, 1996

Pyramidal neurons were acutely isolated from neocortex slices of 14-to 20-day-old rats and patch-... more Pyramidal neurons were acutely isolated from neocortex slices of 14-to 20-day-old rats and patch-clamped under physiological conditions. Current-clamp recordings revealed firing patterns corresponding to those previously reported in slices as regular spiking (RS) and intrinsically bursting (IB), i.e., single action potentials (AP), trains of regular spikes and bursts with depolarizing after-potentials (DAP). In IB neurons, intracellular perfusion with KF blocked the high-voltage-activated Ca 2+ and the Ca2+-dependent K ÷ currents, revealing APs with a 10-30 ms shoulder at -35 mV (shoulder AP), which was the supporting plateau of the intraburst spikes. The use of the A channel blocker, 4-aminopyridine, caused a three-fold reduction in the AP repolarizing rate. A study of the de-and repolarizing rates modulating the spike shape (shoulder AP, burst or single APs) suggested that the percentage of available A channels could play a crucial role in burst formation. Blockade of the residual T-type Ca 2+ current by Ni 2+ did not inhibit the AP shoulder, whereas it was completely and reversibly inhibited by 30 nM TTX, which did not affect AP amplitude, The AP rising rate was only halved by 100 nM TTX. The data concerning the A channel-mediated burst formation and the role of the TTX-sensitive conductance have been successfully simulated in a model cell. We suggest that bursting is an intrinsic property of the membrane of neocortex neurons, and is sustained by TTX-sensitive slowly inactivating and/or persistent Na + conductances.

Brain Research, 1995

In in vitro slices prepared from rat sensorimotor cortex, intracellular recordings were obtained ... more In in vitro slices prepared from rat sensorimotor cortex, intracellular recordings were obtained from 107 layer V pyramidal neurons, subsequently injected with biocytin for morphological reconstruction. Of the 107 neurons, 59 (55.1%) were identified as adapting (45) or non-adapting (13) regular spiking neurons (RS), and 48 (44.9%) as intrinsically bursting (IB) neurons discharging with an initial cluster of action potentials, which tended to recur rhythmically in a subset of 19 cells. The block of IAR by extracellular Cs+ did not affect burst generation, but enhanced the tendency to reburst in IB neurons. A similar effect was induced by other procedures affecting K(+)-dependent post-burst hyperpolarization. In IB neurons Ca2+ spikes had a longer decay time than in RS neurons, however selective blockers of both low and high threshold Ca2+ conductances failed to impair bursting activity. On the contrary, the perfusion of the slices with 0.5-1 microM TTX suppressed bursting behaviour in a critical time interval preceding the complete block of Na(+)-dependent action potentials. It is concluded that the persistent Na+ current INAP is the most important intrinsic factor for the typical firing properties of IB neurons, while Ca2+ and K+ conductances appear to contribute towards shaping bursts and controlling their recurrence rate. The morphology, connectivity and physiological properties of adapting and non-adapting RS neurons are particularly suited to the processing of respectively phasic and tonic inputs, whereas the properties of IB neurons are consistent with their suggested role in cortical rhythmogenesis and in the pathophysiological synchronized activities underlying epileptogenesis.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006

The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal acci... more The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal accidents, but nothing is known about its venom components. This communication reports the separation of at least 57 fractions from the venom by high performance liquid chromatography. From these, at least 104 distinct molecular weight compounds were identified by mass spectrometry analysis. The complete amino acid sequences of three peptides were determined and the partial sequences of three others were also identified. Electrophysiological experiments conducted with ion-channels expressed heterologously on Sf9 cells showed the presence of a potent Shaker B K + -channel blocker. This peptide (trivial name Tpa1) contains 23 amino acid residues closely packed by three disulfide bridges with a molecular mass of 2457 atomic mass units. It is the third member of the sub-family 13, for which the systematic name is proposed to be a-KTx13.3. The mice assay showed clearly the presence of toxic peptides to mammals. One of them named Tpa2, containing 65 amino acid residues with molecular mass of 7522.5 atomic mass units, is stabilized by four disulfide bridges. It was shown to modify the Na + -currents of F-11 and TE671 cells in culture, similar to the beta scorpion toxins. These results demonstrate the presence of toxic peptides in the venom of T. pachyurus and confirm that accidents with this species of scorpion should be considered an important human hazard in Colombia. D

Frontiers in Neural Circuits, 2014

† These authors have contributed equally to this work.

Frontiers in Neural Circuits, 2014

† These authors have contributed equally to this work.

Toxicon, 2007

Crotamine is a peptide toxin from the venom of the rattlesnake Crotalus durissus terrificus that ... more Crotamine is a peptide toxin from the venom of the rattlesnake Crotalus durissus terrificus that induces a typical hindlimb paralysis of unknown nature. Hind limbs have a predominance of fast-twitching muscles that bear a higher density of sodium channels believed until now to be the primary target of crotamine. Hypothetically, this makes these muscles more sensitive to crotamine and would explain such hind-limb paralysis. To challenge this hypothesis, we performed concentration vs. response curves on fast (extensor digitorum longus (EDL)) and slow (soleus) muscles of adult male rats. Crotamine was tested on various human Na + channel isoforms (Na v 1.1-Na v 1.6 a-subunits) expressed in HEK293 cells in patch-clamp experiments, as well as in acutely dissociated dorsal root ganglion (DRG) neurons. Also, the behavioral effects of crotamine intoxication were compared with those of a muscle-selective sodium channel antagonist m-CgTx-GIIIA, and other sodium-acting toxins such as tetrodotoxin aand b-pompilidotoxins, sea anemone toxin BcIII, spider toxin Tx2-6. Results pointed out that EDL was more susceptible to crotamine than soleus under direct electrical stimulation. Surprisingly, electrophysiological experiments in human Na v 1.1 to Na v 1.6 Na + channels failed to show any significant change in channel characteristics, in a clear contrast with former studies. DRG neurons did not respond to crotamine. The behavioral effects of the toxins were described in detail and showed remarkable differences. We conclude that, although differences in the physiology of fast and slow muscles may cause the typical crotamine syndrome, sodium channels are not the primary target of crotamine and therefore, the real mechanism of action of this toxin is still unknown. r

Frontiers in cellular neuroscience, 2015

Frontiers in cellular neuroscience, 2015

Febs Letters, 1998

The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) which plays a fundamenta... more The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) which plays a fundamental role in heart excitability and in neurons by contributing to action potential repolarization and to spike-frequency adaptation, respectively. In this paper we show that IHERG, recorded in neuroblastoma cells and guinea-pig ventricular myocytes, was reversibly inhibited by the KATP channel blocker glibenclamide (IC50=74 μM). The voltage

Febs Letters, 1998

The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) which plays a fundamenta... more The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) which plays a fundamental role in heart excitability and in neurons by contributing to action potential repolarization and to spike-frequency adaptation, respectively. In this paper we show that IHERG, recorded in neuroblastoma cells and guinea-pig ventricular myocytes, was reversibly inhibited by the KATP channel blocker glibenclamide (IC50=74 μM). The voltage

The Journal of Physiology, 1997

1. The regular firing of a Hodgkin-Huxley neurone endowed with fast Nae and delayed K+ channels c... more 1. The regular firing of a Hodgkin-Huxley neurone endowed with fast Nae and delayed K+ channels can be converted into adapting firing by appending HERG (human eag-related gene) channels. 2. The computer model predictions were verified by studying the firing properties of F-11 DRG neurone x neuroblastoma hybrid cells induced to differentiate by long-term exposure to retinoic acid. These cells, which express HERG currents (IHERG), show clear spike-frequency adaptation of their firing when current clamped with long depolarizations. 3. In agreement with the prediction, the selective blocking of IHERG by class III antiarrhythmic drugs always led to the disappearance of the spike-frequency adaptation, and the conversion of adapting firing to regular firing. 4. It is proposed that, in addition to their role in the repolarization of the heart action potential, HERG channels may sustain a process of spike-frequency adaptation, and hence contribute to the control of burst duration in a way that is similar to that of the K+ currents,

The Journal of Physiology, 1997

1. The regular firing of a Hodgkin-Huxley neurone endowed with fast Nae and delayed K+ channels c... more 1. The regular firing of a Hodgkin-Huxley neurone endowed with fast Nae and delayed K+ channels can be converted into adapting firing by appending HERG (human eag-related gene) channels. 2. The computer model predictions were verified by studying the firing properties of F-11 DRG neurone x neuroblastoma hybrid cells induced to differentiate by long-term exposure to retinoic acid. These cells, which express HERG currents (IHERG), show clear spike-frequency adaptation of their firing when current clamped with long depolarizations. 3. In agreement with the prediction, the selective blocking of IHERG by class III antiarrhythmic drugs always led to the disappearance of the spike-frequency adaptation, and the conversion of adapting firing to regular firing. 4. It is proposed that, in addition to their role in the repolarization of the heart action potential, HERG channels may sustain a process of spike-frequency adaptation, and hence contribute to the control of burst duration in a way that is similar to that of the K+ currents,

European Journal of Biochemistry, 2004

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, clos... more Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.

Molecular Biology of the Cell, 2005

Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adh... more 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 ␤ 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 ␤ 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 ␤ 1 integrinmediated 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 ␤ 1 integrins and modulates adhesion receptor signaling.

Molecular Biology of the Cell, 2005

Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adh... more 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 ␤ 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 ␤ 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 ␤ 1 integrinmediated 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 ␤ 1 integrins and modulates adhesion receptor signaling.

Brain Research, 1995

In in vitro slices prepared from rat sensorimotor cortex, intraceilular recordings were obtained ... more In in vitro slices prepared from rat sensorimotor cortex, intraceilular recordings were obtained from 107 layer V pyramidal neurons, subsequently injected with biocytin for morphological reconstruction. Of the 107 neurons, 59 (55.1%) were identified as adapting (45) or non-adapting (13) regular spiking neurons (RS), and 48 (44.9%) as intrinsically bursting (IB) neurons discharging with an initial cluster of action potentials, which tended to recur rhythmically in a subset of 19 cells. The block of IAR by extracellular Cs + did not affect burst generation, but enhanced the tendency to reburst in IB neurons. A similar effect was induced by other procedures affecting K+-dependent post-burst hyperpolarization. In IB neurons Ca 2+ spikes had a longer decay time than in RS neurons, however selective blockers of both low and high threshold Ca a+ conductances failed to impair bursting activity. On the contrary, the perfusion of the slices with 0.5-1 /zM qTX suppressed bursting behaviour in a critical time interval preceding the complete block of Na +-dependent action potentials. It is concluded that the persistent Na + current INA P is the most important intrinsic factor for the typical firing properties of IB neurons, while Ca ~+ and K + conductances appear to contribute towards shaping bursts and controlling their recurrence rate. The morphology, connectivity and physiological properties of adapting and non-adapting RS neurons are particularly suited to the processing of respectively phasic and tonic inputs, whereas the properties of IB neurons are consistent with their suggested role in cortical rhythmogenesis and in the pathophysiological synchronized activities underlying epileptogenesis.

European Journal of Neuroscience, 2003

In this report we studied and compared the biochemical and the electrophysiological characteristi... more In this report we studied and compared the biochemical and the electrophysiological characteristics of two cell lines (GT1-7 and GN11) of immortalized mouse LHRH-expressing neurons and the correlation with their maturational stage and migratory activity. In fact, previous results indicated that GN11, but not GT1-7, cells exhibit an elevated motility in vitro. The results show that the two cell lines differ in terms of immunoreactivity for tyrosine hydroxylase and nestin as well as of production and release of 3,4-dihydroxyphenylalanine (DOPA) and of intracellular distribution and release of the LHRH. Patch-clamp recordings in GN11 cells, reveal the presence of a single inward recti®er K current indicative of an immature neuronal phenotype (neither ®ring nor electrical activity). In contrast, as known from previous studies, GT1-7 cells show the characteristics of mature LHRH neurons with a high electrical activity characterized by spontaneous ®ring and excitatory postsynaptic potentials. K -induced depolarization induces in GT1-7 cells, but not in GN11 cells, a strong increase in the release of LHRH in the culture medium. However, depolarization of GN11 cells signi®cantly decreases their chemomigratory response. In conclusion, these results indicate that GT1-7 and GN11 cells show different biochemical and electrophysiological characteristics and are representative of mature and immature LHRH neurons, respectively. The early stage of maturation of GN11 cells, as well as the low electrical activity detected in these cells, appears to correlate with their migratory activity in vitro.

Cancer Research, 2007

We worked out an experimental protocol able to purge the stem cell compartment of the SH-SY5Y neu... more We worked out an experimental protocol able to purge the stem cell compartment of the SH-SY5Y neuroblastoma clone. This protocol was based on the prolonged treatment of the wild-type cell population with either hypoxia or the antiblastic etoposide. Cell fate was monitored by immunocytochemical and electrophysiologic (patch-clamp) techniques. Both treatments produced the progressive disappearance of neuronal type (N) cells (which constitute the bulk of the tumor), leaving space for a special category of epithelial-like substrate-adherent cells (S 0 ). The latter represent a minimal cell component of the untreated population and are endowed with immunocytochemical markers (p75, c-kit, and CD133) and the electrophysiologic ''nude'' profile, typical of the neural crest stem cells. S 0 cells displayed a highly clonogenic potency and a substantial plasticity, generating both the N component and an alternative subpopulation terminally committed to the fibromuscular lineage. Unlike the N component, this lineage was highly insensitive to the apoptotic activity of hypoxia and etoposide and developed only when the neuronal option was abolished. Under these conditions, the fibromuscular progeny of S 0 expanded and progressed up to the exhaustion of the staminal compartment and to the extinction of the tumor. When combined, hypoxia and etoposide cooperated in abolishing the N cell generation and promoting the conversion of the tumor described. This synergy might mirror a natural condition in the ischemic areas occurring in cancer. These results have relevant implications for the understanding of the documented tendency of neuroblastomas to regress from a malignant to a benign phenotype, either spontaneously or on antiblastic treatment.

Brain Research, 1996

Pyramidal neurons were acutely isolated from neocortex slices of 14-to 20-day-old rats and patch-... more Pyramidal neurons were acutely isolated from neocortex slices of 14-to 20-day-old rats and patch-clamped under physiological conditions. Current-clamp recordings revealed firing patterns corresponding to those previously reported in slices as regular spiking (RS) and intrinsically bursting (IB), i.e., single action potentials (AP), trains of regular spikes and bursts with depolarizing after-potentials (DAP). In IB neurons, intracellular perfusion with KF blocked the high-voltage-activated Ca 2+ and the Ca2+-dependent K ÷ currents, revealing APs with a 10-30 ms shoulder at -35 mV (shoulder AP), which was the supporting plateau of the intraburst spikes. The use of the A channel blocker, 4-aminopyridine, caused a three-fold reduction in the AP repolarizing rate. A study of the de-and repolarizing rates modulating the spike shape (shoulder AP, burst or single APs) suggested that the percentage of available A channels could play a crucial role in burst formation. Blockade of the residual T-type Ca 2+ current by Ni 2+ did not inhibit the AP shoulder, whereas it was completely and reversibly inhibited by 30 nM TTX, which did not affect AP amplitude, The AP rising rate was only halved by 100 nM TTX. The data concerning the A channel-mediated burst formation and the role of the TTX-sensitive conductance have been successfully simulated in a model cell. We suggest that bursting is an intrinsic property of the membrane of neocortex neurons, and is sustained by TTX-sensitive slowly inactivating and/or persistent Na + conductances.

Brain Research, 1995

In in vitro slices prepared from rat sensorimotor cortex, intracellular recordings were obtained ... more In in vitro slices prepared from rat sensorimotor cortex, intracellular recordings were obtained from 107 layer V pyramidal neurons, subsequently injected with biocytin for morphological reconstruction. Of the 107 neurons, 59 (55.1%) were identified as adapting (45) or non-adapting (13) regular spiking neurons (RS), and 48 (44.9%) as intrinsically bursting (IB) neurons discharging with an initial cluster of action potentials, which tended to recur rhythmically in a subset of 19 cells. The block of IAR by extracellular Cs+ did not affect burst generation, but enhanced the tendency to reburst in IB neurons. A similar effect was induced by other procedures affecting K(+)-dependent post-burst hyperpolarization. In IB neurons Ca2+ spikes had a longer decay time than in RS neurons, however selective blockers of both low and high threshold Ca2+ conductances failed to impair bursting activity. On the contrary, the perfusion of the slices with 0.5-1 microM TTX suppressed bursting behaviour in a critical time interval preceding the complete block of Na(+)-dependent action potentials. It is concluded that the persistent Na+ current INAP is the most important intrinsic factor for the typical firing properties of IB neurons, while Ca2+ and K+ conductances appear to contribute towards shaping bursts and controlling their recurrence rate. The morphology, connectivity and physiological properties of adapting and non-adapting RS neurons are particularly suited to the processing of respectively phasic and tonic inputs, whereas the properties of IB neurons are consistent with their suggested role in cortical rhythmogenesis and in the pathophysiological synchronized activities underlying epileptogenesis.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006

The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal acci... more The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal accidents, but nothing is known about its venom components. This communication reports the separation of at least 57 fractions from the venom by high performance liquid chromatography. From these, at least 104 distinct molecular weight compounds were identified by mass spectrometry analysis. The complete amino acid sequences of three peptides were determined and the partial sequences of three others were also identified. Electrophysiological experiments conducted with ion-channels expressed heterologously on Sf9 cells showed the presence of a potent Shaker B K + -channel blocker. This peptide (trivial name Tpa1) contains 23 amino acid residues closely packed by three disulfide bridges with a molecular mass of 2457 atomic mass units. It is the third member of the sub-family 13, for which the systematic name is proposed to be a-KTx13.3. The mice assay showed clearly the presence of toxic peptides to mammals. One of them named Tpa2, containing 65 amino acid residues with molecular mass of 7522.5 atomic mass units, is stabilized by four disulfide bridges. It was shown to modify the Na + -currents of F-11 and TE671 cells in culture, similar to the beta scorpion toxins. These results demonstrate the presence of toxic peptides in the venom of T. pachyurus and confirm that accidents with this species of scorpion should be considered an important human hazard in Colombia. D

Frontiers in Neural Circuits, 2014

† These authors have contributed equally to this work.

Frontiers in Neural Circuits, 2014

† These authors have contributed equally to this work.

Toxicon, 2007

Crotamine is a peptide toxin from the venom of the rattlesnake Crotalus durissus terrificus that ... more Crotamine is a peptide toxin from the venom of the rattlesnake Crotalus durissus terrificus that induces a typical hindlimb paralysis of unknown nature. Hind limbs have a predominance of fast-twitching muscles that bear a higher density of sodium channels believed until now to be the primary target of crotamine. Hypothetically, this makes these muscles more sensitive to crotamine and would explain such hind-limb paralysis. To challenge this hypothesis, we performed concentration vs. response curves on fast (extensor digitorum longus (EDL)) and slow (soleus) muscles of adult male rats. Crotamine was tested on various human Na + channel isoforms (Na v 1.1-Na v 1.6 a-subunits) expressed in HEK293 cells in patch-clamp experiments, as well as in acutely dissociated dorsal root ganglion (DRG) neurons. Also, the behavioral effects of crotamine intoxication were compared with those of a muscle-selective sodium channel antagonist m-CgTx-GIIIA, and other sodium-acting toxins such as tetrodotoxin aand b-pompilidotoxins, sea anemone toxin BcIII, spider toxin Tx2-6. Results pointed out that EDL was more susceptible to crotamine than soleus under direct electrical stimulation. Surprisingly, electrophysiological experiments in human Na v 1.1 to Na v 1.6 Na + channels failed to show any significant change in channel characteristics, in a clear contrast with former studies. DRG neurons did not respond to crotamine. The behavioral effects of the toxins were described in detail and showed remarkable differences. We conclude that, although differences in the physiology of fast and slow muscles may cause the typical crotamine syndrome, sodium channels are not the primary target of crotamine and therefore, the real mechanism of action of this toxin is still unknown. r