The small DNA virus proteins E1A and E1B from human Adenovirus, E6 and E7 from human papillomavirus, and large T and small T antigens from SV40, are multifaceted molecular tools that can carry out an impressive number of tasks in the host cell. These viral factors, collectively termed 'oncoproteins' for their ability to induce cancer, can be viewed as paradigmatic oncogenic factors which can disrupt checkpoint controls at multiple levels -they interfere with both 'gatekeeper' cellular functions, including major control pathways of cell cycle and apoptosis, and with 'caretaker' functions, thereby inducing mitotic abnormalities and increasing genomic instability. Both E1A and E7 have been recently found to interact physically with the Ran GTPase. This interaction is key in uncoupling the centrosome cycle from the cell cycle, highlighting a direct link between viral infection and the induction of genomic instability. Further expanding our current knowledge in this field will be crucial to elucidate viral strategies leading to cellular transformation and cancer progression, as well as design novel preventive or therapeutic approaches to human cancer.
A cDNA coding for the human neuronal nicotinic ␣7 receptor subunit with Leu-248 mutated to threonine was expressed in Xenopus oocytes. When activated by acetylcholine (AcCho), the receptors expressed generated currents that had low desensitization, linear current-voltage relation, and high apparent affinity for both AcCho and nicotine. These characteristics are similar to those already described for the chick threonine-for-leucine-247 ␣7 nicotinic AcCho receptor (nAcChoR) mutant (L247T␣7). These properties were all substantially maintained when the human L248T␣7 mutant was transiently expressed in human Bosc 23 cells. Simultaneous whole-cell clamp and fluorescence measurements with the Ca 2؉ indicator dye Fura-2 showed that nicotine induced a Ca 2؉ influx in standard 2 mM Ca 2؉ solution. The average fractional Ca 2؉ current flowing through L248T␣7 nAcChoRs was 6.7%, which is larger than that flowing through muscle ␣ ␦ nAcChoRs (4.1%). The relative Ca 2؉ permeability, determined in oocytes in the absence of Cl ؊ , was measured from the shift in reversal potential caused by increasing the external Ca 2؉ concentration from 1 to 10 mM. The human wild-type ␣7 nAcChoR was found to be more permeable than the L248T␣7 mutant to Ca 2؉ . Our findings indicate that the Ca 2؉ permeability of the homomeric ␣7 nAcChoR is larger than that of the heteromeric neuronal nicotinic receptors studied to date and is possibly similar to that of the N-methyl-D-aspartate subtype of brain glutamate receptors. F or many years, it has been known that Ca 2ϩ crosses the postjunctional membrane after activation of nicotinic acetylcholine receptors (nAcChoRs; refs. 1-5), and it is now well established that Ca 2ϩ may permeate the pore of various types of neurotransmitter-gated receptors in the brain. Nevertheless, direct determinations of the transmitter-activated Ca 2ϩ inflow lacked until the Ca 2ϩ entry into the cells was monitored by a combination of electrophysiological and optical techniques with Ca 2ϩ sensitive dyes (6-12). While trying to measure Ca 2ϩ influx through nicotinic receptors, we and others found that the adult muscle nAcChoR channel is much more permeable than the embryonic muscle nAcChoR (11, 13) but less permeable than some neuronal nAcChoR channels (7,11,14).The ␣7 nAcChoR subunit is expressed throughout the brain; however, it is still not entirely clear whether it is assembled into a full homomeric receptor, because it occurs in heterologous cell expression systems, or whether it is also made into heteromeric ␣7-containing receptors of uncertain stoichiometry, as has been suggested for the chick nervous system (15-18). The ␣7 nAcChoR is located at the presynaptic site (18) and, to a minor extent, possibly also at postsynaptic sites (19,20). Furthermore, ␣7 nAcChoRs are believed to be involved in a variety of neurological disorders (21) and have the largest Ca 2ϩ permeability among the family of nAcChoRs (18,(22)(23)(24). For these reasons, we thought it of interest to measure directly the Ca 2ϩ permeability of the homomeri...
A cDNA coding for the human neuronal nicotinic ␣7 receptor subunit with Leu-248 mutated to threonine was expressed in Xenopus oocytes. When activated by acetylcholine (AcCho), the receptors expressed generated currents that had low desensitization, linear current-voltage relation, and high apparent affinity for both AcCho and nicotine. These characteristics are similar to those already described for the chick threonine-for-leucine-247 ␣7 nicotinic AcCho receptor (nAcChoR) mutant (L247T␣7). These properties were all substantially maintained when the human L248T␣7 mutant was transiently expressed in human Bosc 23 cells. Simultaneous whole-cell clamp and fluorescence measurements with the Ca 2؉ indicator dye Fura-2 showed that nicotine induced a Ca 2؉ influx in standard 2 mM Ca 2؉ solution. The average fractional Ca 2؉ current flowing through L248T␣7 nAcChoRs was 6.7%, which is larger than that flowing through muscle ␣␦ nAcChoRs (4.1%). The relative Ca 2؉ permeability, determined in oocytes in the absence of Cl ؊ , was measured from the shift in reversal potential caused by increasing the external Ca 2؉ concentration from 1 to 10 mM. The human wild-type ␣7 nAcChoR was found to be more permeable than the L248T␣7 mutant to Ca 2؉ . Our findings indicate that the Ca 2؉ permeability of the homomeric ␣7 nAcChoR is larger than that of the heteromeric neuronal nicotinic receptors studied to date and is possibly similar to that of the N-methyl-D-aspartate subtype of brain glutamate receptors. sensitive dyes (6-12). While trying to measure Ca 2ϩ influx through nicotinic receptors, we and others found that the adult muscle nAcChoR channel is much more permeable than the embryonic muscle nAcChoR (11, 13) but less permeable than some neuronal nAcChoR channels (7,11,14).The ␣7 nAcChoR subunit is expressed throughout the brain; however, it is still not entirely clear whether it is assembled into a full homomeric receptor, because it occurs in heterologous cell expression systems, or whether it is also made into heteromeric ␣7-containing receptors of uncertain stoichiometry, as has been suggested for the chick nervous system (15-18). The ␣7 nAcChoR is located at the presynaptic site (18) and, to a minor extent, possibly also at postsynaptic sites (19,20). Furthermore, ␣7 nAcChoRs are believed to be involved in a variety of neurological disorders (21) and have the largest Ca 2ϩ permeability among the family of nAcChoRs (18,(22)(23)(24). For these reasons, we thought it of interest to measure directly the Ca 2ϩ permeability of the homomeric ␣7 nAcChoRs. Specifically, using combined patch-clamp techniques and Fura-2 fluorescence measurements, we determined the Ca 2ϩ influx through agonistactivated ␣7 receptors in which Leu-248 was mutated into threonine (L248T␣7). Our experiments focused on the human L248T␣7 mutant because of its relatively large transient expression in Bosc 23 human cells, and because the mutant receptors desensitize much less than the wild-type (WT)␣7 nAcChoRs, which desensitize so fast that it becomes very diffic...
Electrophysiological, biochemical, and Ca2+ imaging studies of cultured mouse myotubes were used to investigate whether the neurotransmitter acetylcholine causes an increase in intracellular Ca2+ concentration ([Ca2+J1) through activation of a second messenger system. Bath applications of acetylcholine to myotubes (i) elicited a significant membrane current even in a Na+-free Ca2+ medium, when the current was carried mainly by calcium ions; (ii) caused a rapid and transient cytosolic accumulation of inositol 1,4,5-trisphosphate; (iii) evoked a conspicuous a-bungarotoxinsensitive long-lasting [Ca2+]i enhancement even in the presence of Cd2+; and (iv) transiently increased [Ca211, when cells were equilibrated in a Ca2+-free atropine-containing medium. We propose that, in addition to opening ion channels, the nicotinic action of acetylcholine on the muscle cell membrane increases It has been recently reported that the neurotransmitters acetylcholine (AcCho) in muscle cells and glutamate in nerve cells and oocytes stimulate the turnover of phosphatidylinositol (Ptdlns) (1-4). This event has been shown to evoke a conspicuous Ca2" release from specialized intracellular stores (5, 6). However, the question remains whether the related PtdIns stimulation is derived from the coupling of the transmitter receptor to the PtdIns second messenger system (7) or is a consequence of transmitter-induced increase in cell membrane permeability to Ca2" (8).To examine this question, we have studied inositol phosphate metabolism and determined the cytosolic calcium level in mouse aneural cultured myotubes exposed to AcCho. Here we present evidence that the nicotinic action of AcCho Electrophysiology. Experiments were performed at room temperature (23-25°C) on 2-day-old replicating satellite cells or on 4-to 6-day-old cultured myotubes. Membrane currents were recorded using the whole-cell configuration of the patch-clamp method as described (10) and digitized at 100 Hz. Cells were equilibrated in a normal external solution (NES) containing 140 mM NaCl, 2.8 mM KCl, 2 mM CaC12, 2 mM MgCl2, 10 mM glucose, and 10 mM Hepes-NaOH (pH 7.3). Ca2+-free medium was obtained by replacing Ca2" with Mg2+ (4 mM). High-Ca2+ Na+-free medium contained 20 mM CaCl2, 2.8 mM KCI, 220 mM sucrose, and 5 mM HepesCsOH (pH 7.3) (280 milliosmolar). The whole-cell recording pipette solution consisted of 120 mM CsCl, 20 mM tetraethylammonium chloride, 2 mM MgCl2, 1 mM CaC12, 11 mM EGTA, 2 mM ATP, and 10 mM Hepes-CsOH (pH 7.3). Cells were continuously superfused with saline solutions applied by a gravity-driven perfusion system. Two distinct perfusion lines contained either the external solution alone or with 10-30 AM AcCho. Solutions were applied individually and in succession through a double-barreled pipette with a tip diameter of 80-100 ,m positioned -200 ,um from the cell, such that the whole myotube was superfused.Determination of Ptdlns Turnover. PtdIns turnover in the myotubes was determined by established procedures (11). Differentiated 5-day-old m...
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