Arnaldo Ferroni

Functional characterization of the NCC27 nuclear protein in stable transfected CHO-K1 cells

NCC27 belongs to a family of small, highly conserved, organellar ion channel proteins. It is constitutively expressed by native CHO‐K1 and dominantly localized to the nucleus and nuclear membrane. When CHO‐K1 cells are transfected with NCC27‐expressing constructs, synthesized proteins spill over into the cytoplasm and ion channel activity can then be detected on the plasma as well as nuclear membrane. This provided a unique opportunity to directly compare electrophysiological characteristics of the one cloned channel, both on the nuclear and cytoplasmic membranes. At the same time, as NCC27 is unusually small for an ion channel protein, we wished to directly determine whether it is a membrane‐resident channel in its own right. In CHO‐K1 cells transfected with epitope‐tagged NCC27 constructs, we have demonstrated that the NCC27 conductance is chloride dependent and that the electrophysiological characteristics of the channels are essentially identical whether expressed on plasma or nuclear membranes. In addition, we show that a monoclonal antibody directed at an epitope tag added to NCC27 rapidly inhibits the ability of the expressed protein to conduct chloride, but only when the antibody has access to the tag epitope. By selectively tagging either the amino or carboxyl terminus of NCC27 and varying the side of the membrane from which we record channel activity, we have demonstrated conclusively that NCC27 is a transmembrane protein that directly forms part of the ion channel and, further, that the amino terminus projects outward and the carboxyl terminus inward. We conclude that despite its relatively small size, NCC27 must form an integral part of an ion channel complex.—Tonini, R., Ferroni, A., Valenzuela, S. M., Warton, K., Campbell, T. J., Breit, S. N., Mazzanti, M. Functional characterization of the NCC27 nuclear protein in stable transfected CHO‐K1 cells. FASEB J. 14, 1171–1178 (2000)

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimers disease, for example, is characterized by an accumulation of β-amyloid protein (Aβ) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Aβ and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Aβ stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Aβ, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-α (TNF-α). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Aβ-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-α release induced by Aβ stimulation. These results provide a direct link between Aβ-induced microglial activation and CLIC1 functional expression.

αLatrotoxin of the black widow spider venom opens a small, non-closing cation channel

alpha Latrotoxin, a presynaptically acting polypeptide neurotoxin, induces massive neurotransmitter release from both synapses of vertebrates and the neurosecretory cells of the line PC12, derived from a rat pheochromocytoma. Single PC12 cells, differentiated by treatment with nerve growth factor, were used to investigate by the patch-clamp technique i) the alterations of the resting cell conditions (membrane potential and resistance) and ii) the microscopic mechanism of the permeability changes that underly the response to alpha LTx. The toxin was found to open a channel, 15 pS in conductance, that is permeable to various cations (Na+, K+ and probably Ca2+) and has little tendency to close. This channel is different from the classical voltage- and receptor-operated channels present in PC12 cells, as well as from the large conductances induced by the toxin in artificial lipid membranes.

Cytoskeletal control of rectification and expression of four substates in cardiac inward rectifier K+ channels.

Cardiac inward rectifiers may have a three‐barrel channel structure, based on evidence for three substates in single‐channel recordings. However, some reports indicate four substates, a feature more compatible with the four‐subunit structure for which there is evidence in cloned voltage‐activated K+ channels. Here we show that although the fourth is easily missed, inward rectifier channels have four substates whose expression is controlled by intracellular Ca2+ ions. Fourth substate openings also appear after rectification loss in intracellular divalent cation‐free solution. We find that this process is accelerated by cylochalasin, a microfilament disrupter. Cylochalasin also abolishes Ca2+, but not Mg2+,‐induced rectification by restoring fourth sub‐state openings. Thus, cytoskeletal elements control Ca2+‐dependent substate expression and rectifica‐tion in native inwardly rectifying K+ channels.—Maz‐zanti, M., Assandri, R., Ferroni, A., DiFrancesco, D. Cytoskeletal control of rectification and expression of four substrates in cardiac inward rectifier K+ channels, FASEB J. 10, 357‐361 (1996)

Dynamic Ca2+-Induced Inward Rectification of K+ Current During the Ventricular Action Potential

Inward rectification, an important determinant of cell excitability, can result from channel blockade by intracellular cations, including Ca2+. However, mostly on the basis of indirect arguments, Ca2+-mediated rectification of inward rectifier K+ current (IK1) is claimed to play no role in the mammalian heart. The present study investigates Ca2+-mediated IK1 rectification during the mammalian ventricular action potential. Guinea pig ventricular myocytes were patch-clamped in the whole-cell configuration. The action potential waveform was recorded and then applied to reproduce normal excitation under voltage-clamp conditions. Subtraction currents obtained during blockade of K+ currents by either 1 mmol/L Ba2+ (IBa) or K+-free solution (I0K) were used to estimate IK1. Similar time courses were observed for IBa and I0K; both currents were strongly reduced during depolarization (inward rectification). Blockade of L-type Ca2+ current by dihydropyridines (DHPs) increased systolic IBa and I0K by 50.7% and 254.5%, respectively. beta-Adrenergic stimulation, when tested on I0K, had an opposite effect; ie, it reduced this current by 66.5%. Ryanodine, an inhibitor of sarcoplasmic Ca2+ release, increased systolic IBa by 47.7%, with effects similar to those of DHPs. Intracellular Ca2+ buffering (BAPTA-AM) increased systolic IBa by 87.7% and blunted the effect of DHPs. Thus, IK1 may be significantly reduced by physiological Ca2+ transients determined by both Ca2+ influx and release. Although Ca2+-induced effects may represent only a small fraction of total IK1 rectification, they are large enough to affect excitability and repolarization. They may also contribute to facilitation of early afterdepolarizations by conditions increasing Ca2+ influx.

Muscarinic Regulation of Ca2+ Currents in Rat Sensory Neurons: Channel and Receptor Types, Dose ‐ response Relationships and Cross‐talk Pathways

We studied, in rat sensory neurons, the modulation of high voltage‐activated Ca2+ currents (ICa mediated by the pertussis toxin‐sensitive activation of muscarinic receptors, which were found to be of subtypes M2, or M4. Muscarine reversibly blocked somatic Ca2+ spikes but strong predepolarizations only partially relieved the inhibited Ca2+ current. On the other hand, the putative coupling messenger could not rapidly diffuse towards channels whose activity was recorded from a macro‐patch. The perforated patch technique virtually prevented the response rundown present during whole‐cell experiments. Both ω‐conotoxin GVIA (ω‐CgTx)‐sensitive channels and ω‐CgTx‐ and dihydropyridine‐resistant channels are coupled to the muscarinic receptor, but not the L‐channel. When measured in the same neuron, dose ‐ response relationships for the first and subsequent agonist applications differed; maximal inhibition, the reciprocal of half‐maximal concentration and the Hill coefficient were always highest in the first trial. Muscarine and oxotremorine exhibited monotone dose ‐ response curves, but oxotremorine‐M showed non‐linear relationships which became monotonic when cells were intracellularly perfused with inhibitors of protein kinase A (PKA) and C (PKC), suggesting that either PKA or receptor‐induced PKC could phosphorylate and thus inactivate G‐proteins or other unknown proteins involved in inhibitory muscarinic actions on ICa. In summary, these data provide a preliminary pharmacological characterization of the muscarinic inhibition of the Ca2+ channels in sensory neurons, with implications about agonist specificity and the interplay between signalling pathways.

Expression of Ras-GRF in the SK-N-BE neuroblastoma accelerates retinoic-acid-induced neuronal differentiation and increases the functional expression of the IRK1 potassium channel

Ras‐GRF, a neuron‐specific Ras exchange factor of the central nervous system, was transfected in the SK‐N‐BE neuroblastoma cell line and stable clones were obtained. When exposed to retinoic acid, these clones showed a remarkable enhancement of Ras‐GRF expression with a concomitant high increase in the level of active (GTP‐bound) Ras already after 24 h of treatment. In the presence of retinoic acid, the transfected cells stopped growing and acquired a differentiated neuronal‐like phenotype more rapidly than the parental ones. Cells expressing Ras‐GRF also exhibited a more hyperpolarized membrane potential. Moreover, treatment with retinoic acid led to the appearance of an inward rectifying potassium channel with electrophysiological properties similar to IRK1. This current was present in a large number of cells expressing Ras‐GRF, while only a small percentage of parental cells exhibited this current. However, Northern analysis with a murine cDNA probe indicated that IRK1 mRNA was induced by retinoic acid at a similar level in both kinds of cells. Brief treatment with a specific inhibitor of the mitogen‐activated protein kinase (MAPK) pathway reduced the number of transfected cells showing IRK1 activity. These findings suggest that activation of the Ras pathway accelerates neuronal differentiation of this cell line. In addition, our results suggest that Ras‐GRF and/or Ras‐pathway may have a modulatory effect on IRK1 channel activity.

Three types of ion channels are present on the plasma membrane of Friend erythroleukemia cells

In Friend murine erythroleukemia cells the presence of ion channels was investigated with the patch-clamp technique. During the first 48 hours after cell seeding, three types of ion channels, with the following order of membrane density, were found: i) a Ca2+-dependent K+ channel, fully activated at a cytosolic Ca2+ concentration of 10(-6) M and moderately activated at 10(-7)M; ii) a monovalent cation channel non voltage-activated, with an open-close kinetics dependent on the pressure gradient across the patch; iii) a chloride channel with a slow open-close kinetics. The latter two channels were labile and did not survive during intracellular perfusion. The membrane potential of the leukemia cells was not constant, but underwent large (tens of millivolts) fluctuations due to the opening of a few channels. The average resting membrane potential recorded in this study agrees with that measured in these cells by means of the accumulation ratio of the lipophilic cation Tetraphenylphosphonium.

Direct inhibition of the pacemaker (If) current in rabbit sinoatrial node cells by genistein

Genistein is a tyrosine kinase inhibitor which interferes with the activity of several ionic channels either by altering modulatory phosphorylating processes or by direct binding. In whole‐cell conditions, genistein induces a partial inhibition of the pacemaker (If) current recorded in cardiac sinoatrial and ventricular myocytes. We investigated the mechanism of action of genistein (50 μM) on the If current in whole‐cell, cell‐attached, and inside‐out configurations, and the measured fractional inhibitions were similar: 26.6, 27.2, and 33.6%, respectively. When ATP was removed from the whole‐cell pipette solution no differences were revealed in the effect of the drug when compared to metabolically active cells. Genistein fully maintained its blocking ability even when herbimycin, a tyrosine kinase inhibitor, was added to the whole‐cell ATP‐free pipette solution. Genistein‐induced block was independent of the gating state of the channel and did not display voltage or current dependence; this independence distinguishes genistein from all other f‐channel blockers. When inside‐out experiments were performed to test for a direct interaction with the channel, genistein, superfused on the intracellular side of the membrane, decreased the maximal If conductance, and slightly shifted the current–activation curve to the left. Furthermore, the effect of genistein was independent of cAMP modulation. We conclude that, in addition to its tyrosine kinase‐inhibitory properties, genistein also blocks If by directly interacting with the channel, and thus cannot be considered a valuable pharmacological tool to investigate phosphorylation‐dependent modulatory pathways of the If current and of cardiac rhythm.

Two high voltage-activated calcium currents are present in isolation in adult rat spinal neurons

In neurons enzymatically isolated from adult rat dorsal root ganglia and used during the following 24 hours, the Ca2+ currents were investigated with the whole-cell patch-clamp technique. In contrast to the neonatal neurons, the salient feature of these adult neurons is the well separated (in the voltage-range) activation and inactivation properties of each recorded current. The low-threshold T-, the high-threshold inactivating N-, and the long-lasting L-currents have a threshold for activation at -60, -45 and -10 mV, and a 50% inactivation at -75, -45 and -5 mV respectively. The N and L currents were poorly affected by 100 microM Ni, a known blocker of T channels and completely blocked by 100 microM Cd2+. Frequently we could find neurons with only one type of current present. We conclude that adult sensory neurons are a better preparation for studying, in isolation, the physiological relevance of the three types of Ca2+ channels.