SUMMARY1. Individual cells were isolated from the sino-atrial node area of the rabbit heart using an enzyme medium containing collagenase and elastase. After enzymatic treatment the cells were placed in normal Tyrode solution, where beating resumed in a fraction of them.2. Isolated cells were studied in the whole cell configuration. Action potentials as well as membrane currents under voltage-clamp conditions were similar to those in multicellular preparations.3. Pulses to voltages more negative than about -50 mV caused activation of the hyperpolarizing-activated current, if. Investigation of the properties of this current was carried out under conditions that limited the influence of other current systems during voltage clamp.4. The if current activation range usually extended approximately from -50 to -100 mV, but varied from cell to cell. In several cases, pulsing to the region of -40 mV elicited a sizeable if. Both current activation and deactivation during voltage steps had S-shaped time courses. A high variability was however observed in the sigmoidal behaviour of if kinetics. 5. Plots of the fully-activated current-voltage (I-V) relation in different extracellular Na and K concentrations showed that both ions carry the current if. While changes in the external Na concentration caused the current I-V relation to undergo simple shifts along the voltage axis, changes in extracellular K concentration were also associated with changes in its slope. Again, a large variability was observed in the increase of I-V slope on raising the external K concentration.6. The current if was strongly depressed by Cs, and the block induced by 5 mM-Cs was markedly voltage dependent.

Sympathetic neurons dissociated from the superior cervical ganglion of 2-day-old rats were studied by whole-cell patch clamp and by fura-2 measurements of the cytosolic free Ca2+ concentration, [Ca21],.Step depolarizations in the presence of tetrodotoxin and hexamethonium triggered two Ca2+ currents that differed in the voltage dependence of activation and kinetics of inactivation. These currents resemble the L and N currents previously described in chicken sensory neurons [Nowycky, M. C., Fox, A. P. & Tsien, R. W. (1985) Nature (London) 316, 440442]. Treatment with acetylcholine resulted in the rapid (within seconds), selective, and reversible inhibition of the rapidly inactivated, N-type current, whereas the long-lasting L-type current remained unaffected. The high sensitivity to blocker drugs (atropine, pirenzepine) indicated that this effect of acetylcholine was due to a muscarinic M1 receptor. Intracellular perfusion with nonhydrolyzable guanine nucleotide analogs or pretreatment of the neurons with pertussis toxin had profound effects on the Ca2+ current modulation. Guanosine 5'-[y-thio]triphosphate caused the disappearance of the N-type current (an effect akin to that of acetylcholine, but irreversible), whereas guanosine 5'-[fithio]diphosphate and pertussis toxin pretreatment prevented the acetylcholine-induced inhibition. In contrast, cAMP, applied intracellularly together with 3-isobutyl-1-methylxanthine, as well as activators and inhibitors of protein kinase C, were without effect. Acetylcholine caused shortening of action potentials in neurons treated with tetraethylammonium to partially block K+ channels. Moreover, when applied to neurons loaded with the fluorescent indicator fura-2, acetylcholine failed to appreciably modify [Ca2+]1 at rest but caused a partial blunting of the initial [Ca2+], peak induced by depolarization with high K+. This effect was blocked by muscarinic antagonists and pertussis toxin and was unaffected by protein kinase activators. Thus, muscarinic modulation of the N-type Ca2+ channels appears to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein and independent of both cAMP-dependent protein kinase and protein kinase C.In various cellular systems the function of voltage-gated Ca2l channels is known to be modulated by intracellular events [phosphorylations by cAMP-and cGMP-dependent protein kinases (1-3), and protein kinase C (4, 5)] triggered by the activation of the receptors for various neurotransmitters [e.g., norepinephrine, y-aminobutyric acid, serotonin, adenosine, and acetylcholine (AcCho) (6-13)]. Studies carried out during the last few years have shown that Ca2+ channels are heterogeneous. In sensory neurons of the chicken dorsal root ganglion (DRG), three types of channels (L, N, and T) have been identified that differ in their unitary conductance, voltage dependence ofactivation, and kinetics of inactivation (14). Because of their different properties, these channels may play different physiological roles, and it is therefore imp...

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimer's disease, for example, is characterized by an accumulation of ␤-amyloid protein (A␤) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although A␤ 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 A␤ 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 A␤, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-␣ (TNF-␣). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(ϩ)-[ (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 A␤-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-␣ release induced by A␤ stimulation. These results provide a direct link between A␤-induced microglial activation and CLIC1 functional expression.

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.

Abstract-Inward rectification, an important determinant of cell excitability, can result from channel blockade by intracellular cations, including Ca 2ϩ . However, mostly on the basis of indirect arguments, Ca 2ϩ -mediated rectification of inward rectifier K ϩ current (I K1 ) is claimed to play no role in the mammalian heart. The present study investigates Ca 2ϩ -mediated I K1 rectification during the mammalian ventricular action potential. Guinea pig ventricular myocytes were patch-clamped in the whole-cell configuration. The aim of the present study was to reconsider the role for Ca 2ϩ -induced I K1 rectification in mammalian ventricular myocytes by adopting experimental conditions preserving cell integrity and simulating, as closely as possible, physiological electrical activity. The evidence obtained suggests that the transient rise in subsarcolemmal Ca 2ϩ , occurring during the plateau phase of the action potential as a consequence of both influx and release from the sarcoplasmic reticulum, may significantly contribute to I K1 rectification. Materials and Methods Cell IsolationGuinea pig ventricular myocytes were isolated by using a coronary perfusion method similar to the one described by Levi and Alloatti. 9 In brief, guinea pigs weighing 200 to 300 g were anesthetized by exposure to a cotton wad soaked in tribromoethanol solution (200 mg of tribromoethanol in 10 mL of ether), killed through cervical dislocation, and exsanguinated. Hearts were quickly removed, and the ascending aorta was connected to the outlet of a Langendorff column, perfused with Tyrode's solution (37°C) containing (mmol/L) NaCl 154, KCl 4, CaCl 2 2, MgCl 2 1, HEPES-NaOH 5, and D-glucose 5.5, adjusted to pH 7.35, and equilibrated with 100% O 2 . Perfusion with Tyrode's solution was maintained until vigorous mechanical activity resumed and blood was completely removed. The heart was then perfused, until arrest occurred, with a nominally Ca 2ϩ -free solution containing (mmol/L) NaCl 33.5, KCl 10, Dglucose 22, sucrose 132, KH 2 PO 4 1, MgSO 4 5, HEPES KOH 10, and taurine 50, adjusted to pH 7.3, followed by the same solution to which 140 U/mL collagenase (Sigma type V), 2.5 U/mL trypsin