Andrea Becchetti

Increased Sensitivity of the Neuronal Nicotinic Receptor α2 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.

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.

HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias and regulate cell proliferation of normal and leukemic hemopoietic progenitors.

An important target in the understanding of the pathogenesis of acute myeloid leukemias (AML) relies on deciphering the molecular features of normal and leukemic hemopoietic progenitors. In particular, the analysis of the mechanisms involved in the regulation of cell proliferation is decisive for the establishment of new targeted therapies. To gain further insight into this topic we report herein a novel approach by analyzing the role of HERG K+ channels in the regulation of hemopoietic cell proliferation. These channels, encoded by the human ether-a-gò-gò-related gene (herg), belong to a family of K+ channels, whose role in oncogenesis has been recently demonstrated. We report here that herg is switched off in normal peripheral blood mononuclear cells (PBMNC) as well as in circulating CD34+ cells, however, it is rapidly turned on in the latter upon induction of the mitotic cycle. Moreover, hergappears to be constitutively activated in leukemic cell lines as well as in the majority of circulating blasts from primary AML. Evidence is also provided that HERG channel activity regulates cell proliferation in stimulated CD34+ as well as in blast cells from AML patients. These results open new perspectives on the pathogenetic role of HERG K+ channels in leukemias.

HERG potassium channels are more frequently expressed in human endometrial cancer as compared to non-cancerous endometrium

HERG K+channels, besides contributing to regulate cardiac and neuronal excitability, are preferentially expressed in tumour cell lines of different histogenesis, where their role in the development and maintenance of the neoplastic phenotype is under study. We show here that both herg gene and HERG protein are expressed with high frequency in primary human endometrial cancers, as compared to normal and hyperplastic endometrium. RT-PCR and immunohistochemistry, using specific anti-HERG antibodies developed in our laboratory, were applied to tissue specimens obtained from 18 endometrial cancers and 11 non-cancerous endometrial tissues. herg RNA and HERG protein are expressed in 67% and 82%, respectively, of cancerous, while in only 18% of non-cancerous tissues. In particular, no expression was found in endometrial hyperplasia. Moreover, electrophysiological experiments confirmed the presence of functioning HERG channels on the plasma membrane of tumour cells. On the whole, these data are the first demonstration of the presence of HERG channels in primary human neoplasias, and could candidate HERG as a potential tool capable of marking cancerous versus hyperplastic endometrial growth.

hERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines

Recent studies have led to considerable advancement in our understanding of the molecular mechanisms that underlie the relentless cell growth and invasiveness of human gliomas. Partial understanding of these mechanisms has (1) improved the classification for gliomas, by identifying prognostic subgroups, and (2) pointed to novel potential therapeutic targets. Some classes of ion channels have turned out to be involved in the pathogenesis and malignancy of gliomas. We studied the expression and properties of K+ channels in primary cultures obtained from surgical specimens: human ether a gò-gò related (hERG)1 voltage-dependent K+ channels, which have been found to be overexpressed in various human cancers, and human ether a gò-gò-like 2 channels, that share many of hERG1s biophysical features. The expression pattern of these two channels was compared to that of the classical inward rectifying K+ channels, IRK, that are widely expressed in astrocytic cells and classically considered a marker of astrocytic differentiation. In our study, hERG1 was found to be specifically overexpressed in high-grade astrocytomas, that is, glioblastoma multiforme (GBM). In addition, we present evidence that, in GBM cell lines, hERG1 channel activity actively contributes to malignancy by promoting vascular endothelial growth factor secretion, thus stimulating the neoangiogenesis typical of high-grade gliomas. Our data provide important confirmation for studies proposing the hERG1 channel as a molecular marker of tumour progression and a possible target for novel anticancer therapies.

Chemotherapy resistance in acute lymphoblastic leukemia requires hERG1 channels and is overcome by hERG1 blockers

Bone marrow mesenchymal cells (MSCs) can protect leukemic cells from chemotherapy, thus increasing their survival rate. We studied the potential molecular mechanisms underlying this effect in acute lymphoblastic leukemia (ALL) cells. Coculture of ALL cells with MSCs induced on the lymphoblast plasma membrane the expression of a signaling complex formed by hERG1 (human ether-à-go-go-related gene 1) channels, the β(1)-integrin subunit, and the chemokine receptor CXC chemokine receptor-4. The assembly of such a protein complex activated both the extracellular signal-related kinase 1/2 (ERK1/2) and the phosphoinositide 3-kinase (PI3K)/Akt prosurvival signaling pathways. At the same time, ALL cells became markedly resistant to chemotherapy-induced apoptosis. hERG1 channel function appeared to be important for both the initiation of prosurvival signals and the development of drug resistance, because specific channel blockers decreased the protective effect of MSCs. NOD/SCID mice engrafted with ALL cells and treated with channel blockers showed reduced leukemic infiltration and had higher survival rates. Moreover, hERG1 blockade enhanced the therapeutic effect produced by corticosteroids. Our findings provide a rationale for clinical testing of hERG1 blockers in the context of antileukemic therapy for patients with ALL.

Integrins and Ion Channels in Cell Migration: Implications for Neuronal Development, Wound Healing and Metastatic Spread

Cells migration is necessary for proper embryonic development and adult tissue remodeling. Its mechanisms determine the physiopathology of processes such as neuronal targeting, inflammation, wound healing and metastatic spread. Crawling of cells onto solid surfaces requires a controlled sequence of cell protrusions and retractions that mainly depends on sophisticated regulation of the actin cytoskeleton, although the contribution of microtubules should not be neglected. This process is triggered and modulated by a combination of diffusible and fixed environmental signals. External cues are sensed and integrated by membrane receptors, including integrins, which transduce these signals into cellular signaling pathways, often centered on the small GTPase proteins belonging to the Rho family. These pathways regulate the coordinated cytoskeletal rearrangements necessary for proper timing of adhesion, contraction and detachement at the front and rear side of cells finding their way through the extracellular spaces. The overall process involves continuous modulation of cell motility, shape and volume, in which ion channels play major roles. In particular, Ca2+ signals have both global and local regulatory effects on cell motility, because they target the contractile proteins as well as many regulatory proteins. After reviewing the fundamental mechanisms of eukaryotic cell migration onto solid substrates, we briefly describe how integrin receptors and ion channels are involved in cell movement. We next examine a few processes in which these mechanisms have been studied in depth. We thus illustrate how integrins and K+ channels control cell volume and migration, how intracellular Ca2+ homeostasis affects the motility of neuronal growth cones and what is known about the ion channel roles in epithelial cell migration. These mechanisms are implicated in a variety of pathological processes, such as the disruption of neural circuits and wound healing. Finally, we describe the interaction between neoplastic cells and their local environment and how derangement of adhesion can lead to metastatic spread. It is likely that the cellular mechanisms controlled by integrin receptors, ion channels or both participate in the entire metastatic process. Until now, however, evidence is limited to a few steps of the metastatic cascade, such as brain tumor invasiveness.

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. J. Comp. Neurol. 491:157–174, 2005.

Response to fibronectin–integrin interaction in leukaemia cells: delayed enhancing of a K+ current

In murine erythroleukaemia cells, the response of ion channels was followed before and after contact with fibronectin-coated latex microspheres. Patch-clamp experiments in ‘whole-cell’ and in ‘cell-attached’ configurations showed that cell adhesion to fibronectin promoted plasma membrane hyperpolarization mediated by activation of potassium channels that were indistinguishable from calcium-dependent potassium channels K(Ca) in these cells. K+ current increase began in 5-6 min and was completed about 10 min after the first contact. The timecourse of this process recorded from ‘whole-cell’ was very similar to that followed in intact cells by observing the increase of single channel currents. The open probability of single channels in the patch increased after contact, revealing that this activation is propagated at distance from the adhesion site. The slow onset of the effect suggests the presence of a complex regulatory pathway between fibronectin-integrin binding and activation of potassium channels. Decreasing cytoplasmic free Ca2+ concentration to pCa 9 diminished, but did not inhibit, the response. The current induced by fibronectin was not blocked by apamin, α-charybdotoxin or glibenclamide, but was abolished by high concentrations of tetraethylammonium (TEA). These data suggest for the first time the existence of a specific regulative connection between integrin receptors and ionic channels.

New Insights into the Regulation of Ion Channels by Integrins

By controlling cell adhesion to the extracellular matrix, integrin receptors regulate processes as diverse as cell migration, proliferation, differentiation, apoptosis, and synaptic stability. Because the underlying mechanisms are generally accompanied by changes in transmembrane ion flow, a complex interplay occurs between integrins, ion channels, and other membrane transporters. This reciprocal interaction regulates bidirectional signal transduction across the cell surface and may take place at all levels of control, from transcription to direct conformational coupling. In particular, it is becoming increasingly clear that integrin receptors form macromolecular complexes with ion channels. Besides contributing to the membrane localization of the channel protein, the integrin/channel complex can regulate a variety of downstream signaling pathways, centered on regulatory proteins like tyrosine kinases and small GTPases. In turn, the channel protein usually controls integrin activation and expression. We review some recent advances in the field, with special emphasis on hematology and neuroscience. Some oncological implications are also discussed.