Nonspecific Cation Current Research Articles

Modulation of ion channels in rod photoreceptors by nitric oxide

Subcellular compartments in the outer retina of the larval tiger salamander were identified as likely sites of production of nitric oxide (NO), a recently recognized intercellular messenger. NADPH diaphorase histochemistry and NO synthase immunocytochemistry labeled photoreceptor ellipsoids and the distal regions of bipolar and glial cells apposing photoreceptor inner segments, suggesting a role for NO in visual processing in the outer retina. We investigated the actions of NO on several rod photoreceptor ion channels. Application of the NO-generating compound S-nitrosocysteine increased Ca 2+ channel current and a voltage-independent conductance, but had no affect on voltage-gated K + or nonspecific cation currents. Given the steep relation between voltage-dependent Ca 2+ influx and photoreceptor synaptic output, these results indicate that NO could modulate transmission of the photoresponse to second order cells.

Endothelin-1 inhibits inward rectifier potassium channels and activates nonspecific cation channels in cultured endothelial cells.

A predominant inward rectifier and a small outward potassium current were obtained in whole-cell patch-clamp recordings from cultured bovine pulmonary arterial endothelial cells. Application of endothelial-1 (ET-1; 10-100 nmol/l) inhibited the inward rectifier. Washout with bath solution did not recover the current decreased by ET-1. In cell-attached studies, ET-1 (1 nmol/l) inhibited single-channel activity of the inward rectifier and in some patches enhanced activity of the outward potassium current without change of conductance. A non-specific cation current which is permeable to calcium was identified in cell-attached patches in cultured human umbilical vein endothelial cells. ET-1 (1 nmol/l) increased activity of the nonspecific cation channel. ET-1 may increase calcium influx into endothelial cells and promote synthase and release of endothelium-derived factors.

Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells

The fractional contribution of Ca to current flow through neuronal-type nicotinic acetylcholine receptor channels was determined by quantitative fluorescence microfluorimetry using fura-2. The method, which has been applied already to several types of cells and channels is described in detail here. At -70 mV and 2 mM external Ca concentration it was found that Ca contributes 2.5% to the net current. The fractional contribution was found to be voltage dependent, increasing at negative potentials e-fold for a 110 mV potential difference. Total non-specific cation current was found to have a bell-shaped dependence on external Ca concentration peaking at 2 mM.

Activation of membrane outward currents by human low density lipoprotein in mouse peritoneal macrophages.

The aim of the present study was to search for electrophysiological effects of human lipoproteins on membrane currents in mouse peritoneal macrophages which had been cultured for 5 to 20 days. Whole-cell currents were recorded by using a voltage-clamp technique. Low density lipoprotein (LDL, 100 micrograms/ml) increased a slowly activating nonspecific cation current (iso) in the positive potential range to 244 +/- 23% of the reference (test potential + 55 mV, n = 13, P < 0.005). Augmentation of current resulted out of a negative shift of the activation curve along the voltage axis (-22 mV) and an increase of maximally available current. Furthermore, LDL increased a rapidly activating outward current (ifo) at test potentials positive to the potassium equilibrium potential. At +55 mV ifo-amplitude increased to 165 +/- 14% of reference (n = 16, P < 0.005). LDL-induced effects on ifo-current could be mimicked by application of the calcium ionophore A 23,187 (1 mumol/l) which led to an increase of ifo-current to 161 +/- 25% of the reference (test potential +55 mV, n = 11, P < 0.005). Acetylated-LDL (100 micrograms/ml, 5-15 min) produced no significant effect on the membrane currents under investigation.

Cytosolic Ca2+ spikes evoked by the thiol reagent thimerosal in both intact and internally perfused single pancreatic acinar cells.

Cytosolic calcium signals evoked by the sulphydryl-group-oxidising agent, thimerosal, have been investigated in acutely isolated pancreatic acinar cells. Two techniques were employed for the assessment of the cytosolic free-calcium concentration ([Ca2+]i): measurement of calcium-dependent chloride and non-specific cation currents (whole-cell patch-clamp recording) and microfluorimetry (fura-2). Thimerosal (0.5-100 microM) evoked repetitive spikes in both chloride and cation currents as seen by patch-clamp recording, and in [Ca2+]i as seen by microfluorimetry, with a latency of 1-3 min. The response increased in magnitude over time and was not reversed on removal of thimerosal. The thimerosal-induced spikes were reversibly blocked by 2 mM dithiothreitol and by 20 mM caffeine. Inclusion of heparin (200 micrograms/ml) in the pipette solution blocked the thimerosal-induced spikes. The calcium spikes continued after the removal of extracellular calcium; however, low concentrations of thimerosal (0.5-5 microM) were unable to initiate a current response in the absence of external calcium. High concentrations of thimerosal (50-100 microM) could initiate spikes without extracellular calcium. Thimerosal, at concentrations that failed to produce an independent effect, potentiated the acetylcholine-evoked oscillations in [Ca2+]i. We conclude that thimerosal is able to mobilise calcium from an intracellular store; the blockade by heparin may indicate that thimerosal exerts an action on the inositol trisphosphate pathway. The dependence on extracellular calcium for initiation, but not for continuation of the thimerosal-induced calcium spikes suggests that thimerosal may have the additional effect of inhibiting the plasma membrane calcium ATPase.

Metabotropic glutamate receptor agonists potentiate a slow afterdepolarization in CNS neurons.

We have previously reported that, in the rat dorsolateral septal nucleus (DLSN), metabotropic glutamate receptor (met-GluR) agonists evoked a slow depolarization accompanied by an increase in membrane conductance and burst firing. We have speculated that the burst firing elicited by met-GluR agonists may be due to activation or enhancement of a non-specific cation current, which exists in some DLSN neurons. Now we report that a slow afterdepolarization (sADP) mediated by a non-specific cation current was potentiated by both 1S,3R-ACPD and quisqualate. In addition, met-GluR agonists unmask a sADP in DLSN neurons which did not show a sADP under control conditions. Our data suggest that a non-specific cation current can be potentiated by activation of the met-GluR.

Activation of beta-adrenergic receptor induces Na +-dependent inward currents in acutely dissociated motoneurons of bullfrog spinal cord

Effects of adrenergic drugs on single motoneurons acutely dissociated from the lumbar enlargement of adult bullfrogs were examined. The dissociated large cells were identified as motoneurons by retrograde labeling with a fluorescent dye. Adrenaline caused membrane depolarization with a decrease in input resistance. Under whole-cell voltage clamp conditions at a holding potential of −70 mV, adrenergic drugs induced inward currents in a dose-dependent manner. Adrenaline was more potent than noradrenaline. Under K +-free conditions, adrenaline (10 −6−10 −5 M) induced inward currents which were blocked by propanolol (10 −6 M) but not by phentolamine (10 −5 M). CoCl 2 (1 mM) did not affect the currents. Substitution of choline + in the recording solution for Na + abolished the currents, but tetrodotoxin (TTX, 10 −6 M) had no effect on them. The adrenaline-induced currents exhibited a characteristic voltage-dependency: the conductance became large at hyperpolarized membrane potential (−150 to −30 mV) and approached zero at the depolarized membrane potential (> −30 mV), but was never reversed up to 30 mV, suggesting that the currents are different from non-specific cation currents. Substitution of isethionate − for Cl − in the recording solution had no effect on the voltage-dependency of the adrenaline-induced currents, whereas substitution of choline + for Na + apparently attenuated the voltage-dependency of the currents. These results indicate that adrenaline induced Na +-dependent inward currents through activation of β-adrenergic receptors in bullfrog motoneurons.

Simulation of the bursting activity of neuron R15 in Aplysia: role of ionic currents, calcium balance, and modulatory transmitters

1. An equivalent circuit model of the R15 bursting neuron in Aplysia has been combined with a fluid compartment model, resulting in a model that incorporates descriptions of most of the membrane ion channels that are known to exist in the somata of R15, as well as providing a Ca2+ balance on the cell. 2. A voltage-activated, calcium-inactivated Ca2+ current (denoted the slow inward current ISI) was sufficient to produce bursting activity without invoking any other calcium-dependent currents (such as a nonspecific cation current, INS, or a calcium-activated K+ current, IK,Ca). Furthermore, many characteristics of a typical R15 burst could be simulated, such as a parabolic variation in interspike interval, the depolarizing afterpotential (DAP), and the progressive decrease in the undershoots of spikes during a burst. 3. The dynamic activity of R15 was analyzed by separately characterizing two different temporal domains; the fast dynamics associated with action potentials and the slow dynamics associated with low-amplitude oscillations lasting tens of seconds ("slow waves"). The slow dynamics were isolated by setting the Na+ conductance (gNa) to zero and then studied by the use of a system of equations reduced to two variables: intracellular concentration of Ca2+ and membrane potential. The fixed point of the system was located at the intersection of the nullclines for these two variables. A stability analysis of the fixed point was then used to determine whether a given set of parameters would produce slow-wave activity. 4. If the reduced model predicted slow-wave oscillations for a given set of parameters with gNa set to zero, then bursting activity was observed for the same set of parameters in the full model with gNa reset to its control value. However, for certain sets of parameters with gNa at its usual value, the full model exhibited bursting activity because of a slow oscillation produced by the activation of INS by action potentials. This oscillation resulted from an interaction between the fast and slow dynamics that the reduced model alone could not predict and was not observed when gNa was subsequently set to zero. If gNS was also set to zero, this discrepancy disappeared.(ABSTRACT TRUNCATED AT 400 WORDS)

Persistent pulsatile release of glutamate induced by N-methyl-D-aspartate in neonatal rat hippocampal neurones.

1. Intracellular recordings were made from CA3 hippocampal neurones in vitro, during the first ten days of postnatal life and in adulthood. 2. Repeated (three to six) applications of N-methyl-D-aspartate (NMDA), in the presence of tetrodotoxin (TTX, 1-3 microM) and K+ channel blockers (tetraethylammonium chloride or bromide (TEA), 10 mM, and Cs+, 2 mM; or 4-aminopyridine (4-AP), 30-50 microM, and Cs+, 2 mM) induced in neonatal but not in adult neurones, periodic inward currents (PICs) which persisted for several hours after the last application of NMDA. 3. PICs which were due to non-specific cation currents had a frequency of 0.10 +/- 0.04 Hz, and an amplitude of 1.1 +/- 0.28 nA at holding potentials between -40 and -50 mV. The amplitude was a linear function of the membrane potential over the range -70 to +20 mV. They reversed polarity at 4.1 +/- 9.8 mV. 4. K+ channel blockers alone failed to induce PICs. Repeated (three to six) brief applications of high (12 mM) K+ medium also induced PICs. The frequency and amplitude of K(+)-induced PICs were however considerably reduced by concomitant applications of the NMDA receptor antagonist D,L-3-[( +/- )-2-carboxypiperazin-4-yl-]propyl-1-phosphonic acid (CPP, 20 microM). PICs could be induced also by caffeine (1 mM) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 200 microM), TTX, TEA and Cs+. 5. Intracellular injection of the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) did not prevent the induction of PICs by NMDA. However PICs were blocked by removal of the external calcium and by the calcium antagonists cobalt (2 mM) and cadmium (50 microM). 6. In spite of blockade of propagated synaptic activity by TTX, PICs were synchronous in a pair of intracellularly recorded cells. They were also synchronous with extracellular spikes recorded by electrodes located into stratum pyramidal or stratum radiatum. 7. Once established, PICs were unaffected by NMDA receptor antagonists D(-)2-amino-5-phosphonovaleric acid (AP-5, 50 microM), CPP (20 microM) and the NMDA channel blocker ketamine (10 microM). They were reversibly blocked by the broad spectrum excitatory amino acid antagonist kynurenic acid (1 mM) and by the selective non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM). 8. It is concluded that PICs are generated in neonatal neurones by a synchronous, pulsatile release of glutamate from presynaptic nerve terminals, secondary to oscillations in intracellular calcium.

Tetrabutylammonium induces a voltage-dependent block of kainate current in Xenopus oocytes injected with rat brain mRNA

Following injection of rat striatal and cerebrellar mRNA, Xenopus oocytes were voltage clamped and current responses to the excitatory amino acid receptor agonist, kainate, were recorded. This nonspecific cationic current is carried principally by Na+ and K+ and reverses polarity at a membrane potential of approximately -5 mV. When the membrane potential was voltage clamped to -60 mV, bath-applied tetrabutylammonium (0.1-30 mM) produced a rapid, concentration dependent and reversible block of kainate-induced inward current with an IC50 of 1.3 mM. Tetraalkylammonium derivatives having shorter chains (methyl, ethyl, and propyl) were relatively ineffective blockers. Longer alkyl chain derivatives (pentyl, hexyl, and heptyl) were more potent than tetrabutylammonium but limited in their usefulness by their toxicity. The antagonism of kainate-induced current by tetrabutylammonium displayed apparently uncompetitive kinetics, in contrast with the competitive antagonism by gamma-D-glutamylaminomethylsulfonate. The block by tetrabutylammonium was strongly voltage dependent; an e-fold change in IC50 was observed for a 27 mV change in holding potential. Replacement of the Na+ in the medium with a more permeant cation (NH4+), a less permeant cation (tetramethylammonium), or an uncharged solute (mannitol) had little effect on the block of kainate-induced current by tetrabutylammonium. The rates of association and dissociation of tetrabutylammonium with the kainate receptor-channel are clearly rapid. These observations suggest that tetrabutylammonium enters and blocks the kainate receptor-associated cation selective channel. Tetrabutylammonium appears to traverse 80-90% of the membrane electrical field to reach a relatively low-affinity binding site that may simply be a narrowing of the channel.

Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons.

We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins.

Sodium-calcium exchange in guinea-pig cardiac cells: exchange current and changes in intracellular Ca2+.

1. Membrane currents and changes in [Ca2+]i attributable to the operation of an electrogenic Na-Ca exchange mechanism were recorded in single isolated guinea-pig ventricular myocytes under voltage clamp and internal perfusion with the Ca2+ indicator Fura-2. 2. Ionic currents that interfere with the measurement of Na-Ca exchange current were blocked through the use of caesium (Cs+), verapamil and tetrodotoxin (TTX). Entry of Ca2+ through surface membrane Ca2+ channels and release of Ca2+ from sarcoplasmic reticulum were blocked with verapamil and ryanodine, respectively. 3. In the presence of the blockers listed above, depolarization to positive membrane potentials elicited slow increases in [Ca2+]i and, after an instantaneous increase, a declining outward current. Repolarization elicited a decline in [Ca2+]i and, after an instantaneous increase, a declining inward current. The changes in [Ca2+]i and a major component of the current were abolished by nickel ions (Ni2+; 5 mM). 4. The reversal potential of the current abolished by Ni2+ (Ni2+-sensitive current) was determined at different levels of [Ca2+]i by ramp repolarizations from +80 to -80 mV (1-5 mV/ms). The reversal potential of the current increased linearly with log [Ca2+]i. As a result of the foregoing and other data, the Ni2+-sensitive current was taken to be Na-Ca exchange current (INaCa). 5. The relation between INaCa and [Ca2+]i (less than 1 microM) at constant voltage over the range of -80 to +60 mV was approximately linear. No evidence of saturation could be found; small deviations from linearity at high [Ca2+]i were in the direction expected for a minor contribution from Ca2+-activated non-specific cation current (Ehara, Noma & Ono, 1988). 6. When measured at the same [Ca2+]i, the peak INaCa upon repolarization to -80 to -140 mV seemed to approach a limiting value at very negative potentials. 7. Over the range of +40 to +160 mV INaCa (measured soon after depolarization and thus at the same [Ca2+]i) increased exponentially with clamp-pulse potential. These pulses (to potentials up to +160 mV) elicited a slow rise in [Ca2+]i with the peak at the end of the pulse also increasing exponentially with pulse potential. 8. Inward membrane currents with properties similar to those described above were also recorded in association with physiological [Ca2+]i transients, when Ca2+ channels and the sarcoplasmic reticulum were not blocked. 9. Some of the results are not consistent with certain predictions of a sequential step model, or with those of a simultaneous step model in which the internal binding site for Ca2+ is saturated, or with those of a model based only on thermodynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

"Caged calcium" in Aplysia pacemaker neurons. Characterization of calcium-activated potassium and nonspecific cation currents.

We have studied calcium-activated potassium current, IK(Ca), and calcium-activated nonspecific cation current, INS(Ca), in Aplysia bursting pacemaker neurons, using photolysis of a calcium chelator (nitr-5 or nitr-7) to release "caged calcium" intracellularly. A computer model of nitr photolysis, multiple buffer equilibration, and active calcium extrusion was developed to predict volume-average and front-surface calcium concentration transients. Changes in arsenazo III absorbance were used to measure calcium concentration changes caused by nitr photolysis in microcuvettes. Our model predicted the calcium increments caused by successive flashes, and their dependence on calcium loading, nitr concentration, and light intensity. Flashes also triggered the predicted calcium concentration jumps in neurons filled with nitr-arsenazo III mixtures. In physiological experiments, calcium-activated currents were recorded under voltage clamp in response to flashes of different intensity. Both IK(Ca) and INS(Ca) depended linearly without saturation upon calcium concentration jumps of 0.1-20 microM. Peak membrane currents in neurons exposed to repeated flashes first increased and then declined much like the arsenazo III absorbance changes in vitro, which also indicates a first-order calcium activation. Each flash-evoked current rose rapidly to a peak and decayed to half in 3-12 s. Our model mimicked this behavior when it included diffusion of calcium and nitr perpendicular to the surface of the neuron facing the flashlamp. Na/Ca exchange extruding about 1 pmol of calcium per square centimeter per second per micromolar free calcium appeared to speed the decline of calcium-activated membrane currents. Over a range of different membrane potentials, IK(Ca) and INS(Ca) decayed at similar rates, indicating similar calcium stoichiometries independent of voltage. IK(Ca), but not INS(Ca), relaxes exponentially to a different level when the voltage is suddenly changed. We have estimated voltage-dependent rate constants for a one-step first-order reaction scheme of the activation of IK(Ca) by calcium. After a depolarizing pulse, INS(Ca) decays at a rate that is well predicted by a model of diffusion of calcium away from the inner membrane surface after it has entered the cell, with active extrusion by surface pumps and uptake into organelles. IK(Ca) decays somewhat faster than INS(Ca) after a depolarization, because of its voltage-dependent relaxation combined with the decay of submembrane calcium. The interplay of these two currents accounts for the calcium-dependent outward-inward tail current sequence after a depolarization, and the corresponding afterpotentials after a burst

Control of cytoplasmic calcium with photolabile tetracarboxylate 2-nitrobenzhydrol chelators

This paper introduces nitr-2, a new Ca2+ chelator designed to release Ca2+ upon illumination with near UV (300-400 nm) light. Before illumination nitr-2 has Ca2+ dissociation constants of 160 and 630 nM in 0.1 and 0.3 M ionic strength respectively; after photoconversion to a nitrosobenzophenone the values shift to 7 and 18 microM, high enough to liberate substantial amounts of Ca2+ under intracellular conditions. The speed of release is limited by a dark reaction with rate constant 5 s-1. Aplysia central neurons injected with nitr-2 and exposed to UV light exhibit two separate Ca2+-dependent membrane currents: one carried by potassium ions and one a nonspecific cation current. A quantitative estimate of the spatial distribution of intracellular [Ca2+] changes in large cells filled with a high concentration of nitr-2 and exposed to an intense UV flash is offered.