Nonselective Conductance Research Articles

The Effect of Cu2+ on Ion Transport Systems of the Plant Cell Plasmalemma.

Changes in plasmalemma permeability caused by excessive Cu2+ levels were examined in cells of a freshwater alga (Nitella flexilis) using a conventional microelectrode voltage-clamp technique. A rapid Cu2+-induced increase of plasmalemma conductance starting from 5 [mu]M Cu2+ was shown. Cu2+-induced plasmalemma conductance (ClGm) was nonselective and potential-independent, resembling the conductance of nonselective ionic leakage of the plasmalemma. The K+ channel conductance was shown to be unaltered by Cu2+, and a decrease in plasmalemma Cl- channel conductance at Cu2+ concentrations above 5 [mu]M was found. The depression of Cl- channels and ClGm were time-, dosage-, and Ca2+-dependent processes, revealing a great similarity in all parameters, with Ca2+ causing the preventive effect by shifting the effective Cu2+ concentrations to higher levels. This phenomenon may be explained by the same Cu2+-modified target on the plasmalemma both for ClGm and Cl- channel depression. In addition, a reversible, inhibitory effect of Cu2+ (>10 [mu]M) on the light-stimulated H+-ATPase electrogenic pump in the plasmalemma was demonstrated. This effect was Ca2+- independent, which made it possible to distinguish it from ClGm. Therefore, the Cu2+-induced dramatic alterations in plant cell plasmalemma permeability are caused mainly by nonselective conductance increases and electrogenic pump inhibition.

Properties of a slow nonselective cation conductance modulated by neurotensin and other neurotransmitters in midbrain dopaminergic neurons.

1. A widespread mechanism of slow excitation throughout the nervous system involves overlapping changes in nonselective ion conductance and K+ conductance. We used whole cell patch-clamp recording to characterize such a nonselective conductance induced by neurotensin (NT) and other neurotransmitters in immunocytochemically identified dopaminergic neurons cultured from the rat ventral tegmental area (VTA). 2. The NT-induced inward current consisted of an initial peak and later "hump." The response was blocked reversibly by the nonpeptide NT-receptor antagonist SR48692, suggesting that it resulted from activation of NT receptors. 3. The channel was almost equally permeable to Na+ and K+, as determined from the reversal potential shift upon switching from Na+- to K(+)-containing external solution. The permeability of Cs+ was similar to that of Na+, as determined from the zero-current equation and average reversal potential in the 75 mM Na+ solution. Cl- was not significantly permeable. 4. In Ca(2+)-free external solution, the NT-induced current showed a fourfold increase in amplitude, and in high Mg2+ (20 mM) external solution, the NT-induced current showed an 80% decrease in amplitude, suggesting that external Ca2+ and Mg2+ could block the nonselective conductance. 5. The NT response was unaffected by loading the neurons with either the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or with 1 mM ca2+. The nonselective conductance was therefore not Ca2+ activated. 6. Loading the neurons with cyclic GMP or cyclic AMP (each with the phosphodiesterase inhibitor isobutyl-methylxanthine) did not affect the NT response. The NT-induced nonselective conductance was therefore not cyclic nucleotide-activated. 7. The latency of the NT response was long (> or = 185 ms, average 406 ms, 30 degrees C), indicating that NT did not induce the conductance through ligand-gated channels. Thus, NT activated a slow nonselective cation conductance. 8. Neurokinin B, a metabotropic glutamate agonist, and muscarine elicited responses similar to the NT response. The NT response could be elicited after desensitizing the responses to these other neurotransmitters, indicating receptor specificity in the activation of the nonselective conductance.

Sodium channel heterogeneity in the apical membrane of porcine thyroid epithelial cells

Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and secrete Cl-. Two types of Na+ channels were found in cell-attached patches of apical membrane. A low conductance Na+ channel (conductance g = 4 picosiemens (pS)) remained open for seconds and showed a high selectivity for Na+ compared with K+. In contrast, a high conductance Na+ channel (g = 10 pS) flickered rapidly and had reduced selectivity. Both types of Na+ channel became more prevalent when the cells were exposed to Na(+)-free medium, though only the high conductance channel increased in prevalence on addition of prostaglandin E2, a stimulator of adenylate cyclase which increases Na+ absorption in this cultured epithelium. Two minority types of channel were also found: a non-selective small conductance cation channel which had been reported previously, and an intermediate conductance channel found only in Na(+)-free medium. It was concluded that passage of Na+ across the apical membrane of thyroid cells is mediated by typical epithelial Na+ channels, but that the two types of channel are differentially regulated.

A Nonselective High Conductance Channel in Bovine Pigmented Ciliary Epithelial Cells

An ion channel activated by hyperpolarization was identified in excised patches of bovine pigmented ciliary epithelial cells using the single channel patch clamp technique. In symmetrical NaGluconate, the channel had a maximum conductance of 285 pS. The channel was characterized by frequent flickery transitions between the fully open and closed levels. The channel did not discriminate very clearly between anions and cations; when the cytoplasmic face of excised patches was bathed in a dilute NaCl solution, the channel had a chloride-to-sodium permeability ratio (PCl/PNa) of 1.3. However, the channel showed a small anion selectivity (PCl/PNa = 3.7) when bathed in a concentrated NaCl solution. Gd3+ blocked the channel reversibly. Channel kinetics were characterised by slow (approximately min) voltage-dependent activation and inactivation rate constants. The channel was most active in the range-60 to -140 mV and showed a peak at -120 mV. A similar time- and voltage-dependent activation was also observed in cell-attached recordings. In conclusion, hyperpolarization of pigmented ciliary epithelial cell membrane patches activated a large conductance, nonselective ion channel. This combination of nonselectivity and hyperpolarizing activation is consistent with the involvement of this channel in ion loading from the blood into pigmented ciliary epithelial cells-the first phase in the secretion of aqueous humor.

Shrinkage activates a nonselective conductance: involvement of a Walker-motif protein and PKC.

The ability of all cells to maintain their volume during an osmotic challenge is dependent on the regulated movement of salt and water across the plasma membrane. We demonstrate the phosphorylation-dependent gating of a nonselective conductance in Caco-2 cells during cellular shrinkage. Intracellular application of exogenous purified rat brain protein kinase C (PKC) resulted in the activation of a current similar to that activated during shrinkage with a Na(+)-to-Cl- permeability ratio of approximately 1.7:1. To prevent possible PKC- and/or shrinkage-dependent activation of cystic fibrosis transmembrane regulator (CFTR), which is expressed at high levels in Caco-2 cells, a functional anti-peptide antibody, anti-CFTR505-511, was introduced into the cells via the patch pipette. Anti-CFTR505-511, which is directed against the Walker motif in the first nucleotide binding fold of CFTR, prevented the PKC/shrink-age current activation. The peptide CFTR505-511 also induced current inhibition, suggesting the possible involvement of a regulatory element in close proximity to the channel that shares sequence homology with the first nucleotide binding fold of CFTR and whose binding to the channel is required for channel gating.

A novel action of quinine and quinidine on the membrane conductance of neurons from the vertebrate retina.

The cinchona alkaloids quinine and quinidine have been shown to block a broad range of voltage-gated membrane conductances in a variety of excitable tissues. Using the whole-cell version of the patch clamp technique, we examined the effects of these compounds on voltage-dependent currents from horizontal cells dissociated enzymatically from the all-rod retina of the skate. We report here a novel and unexpected action of quinine and quinidine on isolated horizontal cells. In addition to blocking several of the voltage-activated currents of these cells, the introduction of the alkaloids evoked a large outward current when the cells were held at depolarized potentials. Using tail current analysis, the reversal potential of the outward current was close to O mV, and the current was markedly suppressed by extracellularly applied cobalt, acetate, and halothane. Depolarization in the presence of quinine also permitted entry into the cells of extracellularly applied Lucifer yellow (MW = 443 D), whereas a 3-kD fluorescein-dextran complex was excluded. These findings suggest that the large, apparently nonselective conductance induced by quinine and quinidine results from the opening of hemi-gap junctional channels.

The isoquinoline derivative LOE 908 selectively blocks vasopressin-activated nonselective cation currents in A7r5 aortic smooth muscle cells.

The effect of (R,S)-(3,4-dihydro 6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl- N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908), a cation channel blocker in HL-60 promyeloblasts, was studied in the A7r5 smooth muscle cell line from rat thoracic aorta, using the whole-cell patch-clamp technique. At a holding potential of -60 mV, application of vasopressin induced a nonselective cation conductance in voltage-clamped A7r5 cells. The current-voltage relation was linear, and currents reversed close to 0 mV regardless of the chloride gradient. The activation of the nonselective cation conductance by vasopressin was not affected by dialysing cells with Ca(2+)-free internal solution. LOE 908 blocked this current in a concentration-dependent manner with an IC50 of 560 nM, whereas dihydropyridine-sensitive Ba2+ current through voltage-dependent Ca2+ channels was blocked with an IC50 of 28 microM. Another organic blocker of receptor-mediated Ca2+ entry, 1-beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl-1H-imidazole hydrochloride (SK&F 96365), blocked both, the vasopressin-induced nonselective conductance and the voltage-activated Ba2+ current with similar IC50 values of 13 microM and 8 microM, respectively. The rank order of potency of inorganic blockers on the vasopressin-induced inward current was Gd3+ > La3+ > Cd2+. Vasopressin-induced non-selective cation current was also observed in pertussis toxin-pretreated A7r5 cells but was completely abolished after infusion of the GDP analogue, guanosine 5'-O-[3-thio]diphosphate, from the patch pipette. Furthermore, vasopressin induced a transient outward current, suggesting a Ca(2+)-activated K(+)-current, which overlapped with the nonselective cation conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbachol Potentiates Q Current and Activates a Calcium‐dependent Non‐specific Conductance in Rat Hippocampus In Vitro

Intracellular recordings were made from CA1 neurons in rat hippocampal slices maintained in vitro. When Na+ currents were blocked with tetrodotoxin and K+ conductances known to be sensitive to suppression by muscarinic agonists were blocked by 2 mM Ba2+, CA1 cells were depolarized by carbachol (3-10 microM) with an attendant conductance increase, whereas prior to Ba2+ the agonist produced a decrease or no change in conductance. Under voltage clamp at approximately -60 mV and in the presence of tetrodotoxin and Ba2+, carbachol (3-10 microM) induced a variable-latency biphasic inward current of up to 380 pA associated with a conductance increase of approximately 50%. The first phase was associated with an increase (more than 2-fold) of the Cs(+)-sensitive, hyperpolarization-activated cationic current, IQ. Carbachol also accelerated the kinetics of IQ at -100 mV with an average 24% reduction in its activation time constant. The second phase reflected an additional inward current that was Cs(+)-resistant, displayed little apparent voltage sensitivity and had a mean extrapolated reversal potential, determined in the presence of external Cs+ (< or = 5 mM), of approximately -20 mV. In a small proportion of cells the second phase of inward current was followed (or overlapped) by an outward current, also associated with a conductance increase, which reversed at approximately -70 mV. These carbachol actions were prevented by extracellular 300 microM Cd2+ and 2 mM Mn2+, by high levels (> 5 mM) of extracellular Mg2+ or Ca2+, and by omission of Ca2+ or reduction of extracellular Na+ to 25 mM by substitution of NaCl with Tris or N-methyl-D-glucamine. Carbachol action was not mimicked by oxotremorine (< or = 60 microM), but was irreversibly blocked by this drug. Likewise, atropine (100 nM) irreversibly and gallamine (10 microM) reversibly antagonized carbachol's action. The action of carbachol was blocked shortly after prior exposure of slices to 2-5 mM caffeine. Chronic or acute incubation of slices with 2 mM Li+ potentiated (between 1- and 2-fold) carbachol responses. The data indicate that muscarinic activation increases cationic flux by a calcium-dependent potentiation of IQ and activation of a non-selective conductance. The probability that inositol phospholipid metabolism is involved in triggering these events is discussed.

Platelet activating factor-induced increase in cytosolic calcium and transmembrane current in human macrophages.

Platelet-activating factor (PAF) is synthesized and secreted by macrophages in responding to inflammatory stimuli. When exogenously applied to human monocyte derived macrophages (HMDMs), PAF induces a rapid rise in cytosolic free calcium (Cai) believed to be an early triggering event in macrophage activation. We investigated PAF-induced Ca2+ signaling in HMDMs using the calcium indicator Fura-2, combining single cell ratio fluorimetry and digital video imaging with whole-cell recording techniques. Application of PAF (20 ng/ml) to adherent macrophages induced transient increases in Cai that were biphasic, consisting of an initial phase that could be observed in Ca(2+)-free solutions and a second phase that was critically dependent upon Ca2+ entry. When Mn2+ was applied to cells in the presence and absence of Ca2+, PAF increased the rate of Mn2+ entry rate only when Ca2+ was absent. PAF increased the rate of Ba2+ entry even when measured in the presence of external Ca2+. Ca2+ entry was reversibly inhibited in the presence of external La3+ (1 mM). Data obtained from simultaneous voltage-clamp/microfluorimetry experiments demonstrated the activation of a nonselective cation current which closely paralleled the rising phase of the Cai transient. We investigated whether the non-selective cation conductance provided for the bulk of the agonist-induced Ca2+ influx. Changes in Cai following removal of extracellular Ca2+ (Cao) during the agonist-induced Cai response were not associated with changes in whole-cell current. The inability to detect whole-cell current changes correlated with a decrease in Cao suggests that the bulk of the Ca2+ influx was not through the nonselective conductance and either does not occur through a conductance pathway or occurs via a parallel pathway consisting of channels which are both low conductance and highly Ca2+ selective.

Extracellular ATP induces a large nonselective conductance in macrophage plasma membranes.

Extracellular ATP in its tetra-anionic form (ATP4-) induces ion fluxes and membrane depolarization in the mouse macrophage-like cell line J774.2 and in resident mouse macrophages. We analyzed the effects of extracellular ATP4- by both patch-clamp and intracellular microelectrode techniques. Whole-cell patch-configuration membrane potential measurements on J774.2 cells revealed that ATP4- -induced depolarization occurred within 40 ms of pulsed application of ATP and was completely reversible. The depolarizations were accompanied by a dramatic increase in membrane conductance and showed no sign of adaptation to ATP over a period of 30 min. At 5 mM total ATP (ATPt) the whole-cell conductance was approximately 10 nS, and an upper limit of 20 pS for a single-channel conductance has been established. The reversal potential associated with the ATP-induced depolarization at asymmetric K+, Na+, Ca2+, and Cl- concentrations across the membrane was 0 mV. In patch-clamped cells depolarization was complete at 20 microM ATP4-, and repolarization from full depolarization occurred in approximately 5 s. In contrast, in intact cells measured by microelectrode impalement, complete depolarization occurred at approximately 2 mM ATP4- and repolarization was much slower (approximately 100 min). These findings indicate that the changes in intracellular ionic composition that occur after ATP treatment affect the rate of cell repolarization. At lower concentrations of ATP, potassium conductances modulated the depolarizing effect of ATP. ATP also depolarized mouse peritoneal macrophages, but a variant cell line (ATPR B2), derived from J774.2 cells by prolonged exposure to ATP, was insensitive to ATP. Our results provide a membrane electrophysiological description and analysis of a large nonselective plasma membrane conductance of macrophages induced by extracellular ATP.

The effects of various "nicotine-like" agents in the cat superior cervical ganglion in situ.

The effects of nicotine, lobeline, anabasine, cytisine, coniine, sparteine, piperidine, acetylcholine, tetramethylammonium (TMA) and dexamphet-amine, given as i.a. bolus injections, were studied in the cat superior cervical ganglion (SCG) in situ and compared with those of (DMPP) 1,1-dimethyl-4-phenylpiperazinium described in a preceding paper. Two main events are thought to determine the ganglionic response to these agents. A non-selective conductance increase of the ganglion cells by stimulation of nicotinic receptors is responsible for depolarization, firing, facilitation of ganglionic transmission and depolarization block. The accumulation of Na+ resulting from the altered conductance activates an electrogenic Na +-pump which tends to increase the membrane potential and causes a delayed unspecific depression of ganglionic excitability. After agents with a brief agonistic action (acetylcholine, DMPP, TMA), the two mechanisms lead to a distinct biphasic effect on ganglionic polarity (initial depolarization, later hyperpolarization) and transmission (early and later block); with the nicotinic agonists of long duration of action the effect of the electrogenic Na+-pump was obscured by the long-lasting activation of nicotinic receptors and was usually revealed only by special pharmacological procedures. An additional preganglionic depression of transmitter release is very likely. A desensitization of nicotinic receptors occurred after high single or repeated doses of nicotine, resulting in a selective unsurmountable block. A competitive block of nicotinic receptors occurred after coniine. Local anaesthetic properties of lobeline and dexamphetamine interfered with the two main events when high doses were given. The only effect of sparteine was to produce a short-lasting hexamethonium-like block of transmission.