J. Robbins

Kinetic and pharmacological properties of the M-current in rodent neuroblastoma x glioma hybrid cells.

1. The M‐like current IK(M,ng) in differentiated NG108‐15 mouse neuroblastoma x rat glioma hybrid cells has been studied using tight‐seal, whole‐cell patch‐clamp recording. 2. When calculated from steady‐state current‐voltage curves, the conductance underlying IK(M,ng) showed a Boltzmann dependence on voltage with half‐activation voltage Vo = ‐44 mV (in 3 mM [K+]) and slope factor (a) = 8.1 mV/e‐fold increase in conductance. In 12 mM [K+] Vo = ‐38 mV and a = 6.9 mV. The deactivation reciprocal time constant accelerated with hyperpolarization with slope factor 17 mV/e‐fold voltage change. 3. The reversal potential for deactivation tail currents varied with external [K+] as if PNa/PK were 0.005. 4. Steady‐state current was increased on removing external Ca2+. In the presence of external Ca2+, reactivation of IK(M, ng) after a hyperpolarizing step was delayed. This delay was preceded by an inward Ca2+ current, and coincided with an increase in intracellular [Ca2+] as measured with Indo‐1 fluorescence. Elevation of intracellular [Ca2+] with caffeine also reduced IK(M, ng). 5. IK(M, ng) was inhibited by external divalent cations in decreasing order of potency (mM IC50 in parentheses): Zn2+ (0.011) greater than Cu2+ (0.018) greater than Cd2+ (0.070) greater than Ni2+ (0.44) greater than Ba2+ (0.47) greater than Fe2+ (0.69) greater than Mn2+ (0.86) greater than Co2+ (0.92) greater than Ca2+ (5.6) greater than Mg2+ (16) greater than Sr2+ (33). This was not secondary to inhibition of ICa since: (i) inhibition persisted in Ca(2+)‐free solution; (ii) La3+ did not inhibit IK(M, ng) at concentrations which inhibited ICa; and (iii) organic Ca2+ channel blockers were ineffective. Inhibition comprised both depression of the maximum conductance and a positive shift of the activation curve. Addition of Ca2+ (10 microM free [Ca2+]) or Ba2+ (1 mM total [Ba2+]) to the pipette solution did not significantly change IK(M, ng). 6. IK(M, ng) was reduced by 9‐amino‐1,2,3,4‐tetrahydroacridine (IC50 8 microM) and quinine (30 microM) but was insensitive to tetraethylammonium (IC50 greater than 30 mM), 4‐aminopyridine (greater than 10 mM), apamin (greater than 3 microM) or dendrotoxin (greater than 100 nM). 7. IK(M, ng) was inhibited by bradykinin (1‐10 microM) or angiotensin II (1‐10 microM), but not by the following other receptor agonists: acetylcholine (10 mM), muscarine (10 microM), noradrenaline (100 microM), adrenaline (100 microM), dopamine (100 microM), histamine (100 microM), 5‐hydroxytryptamine (10 microM), Met‐enkephalin (1 microM), glycine (100 microM), gamma‐aminobutyric acid (100 microM) or baclofen (500 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

On the mechanism of M‐current inhibition by muscarinic m1 receptors in DNA‐transfected rodent neuroblastoma x glioma cells.

1. Acetylcholine (ACh) produces two membrane current changes when applied to NG108‐15 mouse neuroblastoma x rat glioma hybrid cells transformed (by DNA transfection) to express m1 muscarinic receptors: it activates a Ca(2+)‐dependent K+ conductance, producing an outward current, and it inhibits a voltage‐dependent K+ conductance (the M conductance), thus diminishing the M‐type voltage‐dependent K+ current (IK(M)) and producing an inward current. The present experiments were undertaken to find out how far inhibition of IK(M) might be secondary to stimulation of phospholipase C, by recording membrane currents and intracellular Ca2+ changes with indo‐1 using whole‐cell patch‐clamp methods. 2. Bath application of 100 microM ACh reversibly inhibited IK(M) by 47.3 +/‐ 3.2% (n = 23). Following pressure‐application of 1 mM ACh, the mean latency to inhibition was 420 ms at 35 degrees C and 1.79 s at 23 degrees C. Latencies to inhibition by Ba2+ ions were 148 ms at 35 degrees C and 92 ms at 23 degrees C. 3. The involvement of a G‐protein was tested by adding 0.5 mM GTP‐gamma‐S or 10 mM potassium fluoride to the pipette solution. These slowly reduced IK(M), with half‐times of about 30 and 20 min respectively, and rendered the effect of superimposed ACh irreversible. Effects of ACh were not significantly changed after pretreatment for 24 h with 500 ng ml‐1 pertussis toxin or on adding up to 10 mM GDP‐beta‐S to the pipette solution. 4. The role of phospholipase C and its products was tested using neomycin (to inhibit phospholipase C), inositol 1,4,5‐trisphosphate (InsP3) and inositol 1,3,4,5‐tetrakisphosphate (InsP4), heparin, and phorbol dibutyrate (PDBu) and staurosporin (to activate and inhibit protein kinase C respectively). Both neomycin (1 mM external) and InsP3 (100 microM intrapipette) inhibited the ACh‐induced outward current and/or intracellular Ca2+ transient but did not block ACh‐induced inhibition of IK(M). Intrapipette heparin (1 mM) blocked activation of IK(Ca) and reduced Ach‐induced inhibitions of IK(M), but also reduced inhibition of ICa via endogeneous m4 receptors. PDBu (with or without intrapipette ATP) and staurosporin had no significant effects.(ABSTRACT TRUNCATED AT 400 WORDS)

A transient outward current in NG108-15 neuroblastoma × glioma hybrid cells

Outward currents were recorded from voltage-clamped NG108-15 mouse neuroblastoma × rat glioma hybrid cells, differentiated with prostaglandin E1. Depolarising voltage steps from −70 mV, evoked a transient outward current from a threshold of −30 mV. The outward current showed complete inactivation at potentials positive to -10 mV. Inactivation was removed by hyperpolarisation with half-inactivation at −53 mV. The time course of the inactivation could be best fitted by two exponentials with mean time constants of 280 ms and 1.6 s at + 80 mV. Tail current measurements showed a shift in the reversal potential with changes in external K+ concentration, consistent with K+ as the current-carrying ion. The outward current amplitude was reversibly reduced by 4-aminopyridine, and the time course of inactivation modified. In the presence of other K+ channel blockers (tetraethylammonium, barium and tetrahydroaminoacridine) the amplitude of the outward current was also reversibly reduced, but with a negliglible effect on its time course. The current was unaffected by dendrotoxin, d-tubocurarine, apamin, Cd2+ and Ni2+, and by replacing external Ca2+ with Co2+ or Mg2+. In current clamp, action potential duration was greatly increased by 4-aminopyridine. The findings show that the NG108-15 cell line displays a transient outward current that resembles IK(A) but with a higher than usual threshold and relatively slow inactivation, and that this current is likely to be important for action potential repolarisation.

Neurotransmitters inhibit the omega-conotoxin-sensitive component of Ca current in neuroblastoma × glioma hybrid (NG108-15) cells, not the nifedipine-sensitive component

Voltage-dependent calcium currents (ICa) in NG 108-15 cells consisted of three pharmacologically distinct components: a transient low-voltage-activated (LVA) current, sensitive to Ni2+; a high-voltage-activated (HVA) current sensitive to the dihydropyridine antagonist, nifedipine and a HVA current sensitive to omega-conotoxin GVIA (CgTx). The voltage sensitivities and decay kinetics of the two HVA currents were indistinguishable. The neurotransmitters acetylcholine (ACh) and noradrenaline inhibited ICa. This inhibition was not occluded by Ni2+ or nifedipine, but was abolished by CgTx. It is therefore concluded that the neurotransmitter-sensitive component of ICa is restricted to that component of HVA current inhibitable by omega-conotoxin.

POSTSYNAPTIC ACTIONS OF ACETYLCHOLINE - THE COUPLING OF MUSCARINIC RECEPTOR SUBTYPES TO NEURONAL ION CHANNELS

Publisher Summary In recent years, the application of molecular biological techniques has established that there are at least five subtypes of muscarinic receptor, each with a unique pattern of distribution. Radio-ligand binding displacement studies of antagonist pharmacology of the individual receptors expressed in appropriate host cells have shown that each receptor does have a discrete antagonist profile, but that there are no single antagonists with sufficient selectivity to be clearly diagnostic for any particular receptor type. This has hampered the elucidation of the roles of muscarinic receptor subtypes, particularly, in cells which may simultaneously express more than one subtype, as is probably the case for neurones. The use of cell lines transfected with a gene encoding a single muscarinic receptor subtype has extended the characterization of muscarinic receptor subtypes beyond the pharmacology of the recognition site and has generated an additional classification on the basis of the downstream cellular mechanisms to which each subtype preferentially couples. This approach can clearly provide indications of likely coupling mechanisms of subtypes in, for example, the CNS neurones. Initial biochemical experiments with cell lines, such as the neuroblastoma x glioma hybrid NG108-15 have established two major groupings of muscarinic receptor subtypes, with ml, m3, and m5 receptors coupling preferentially to stimulation of phospholipase C (PLC) through a Pertussis toxin-insensitive G-protein, with subsequent elevation of IP3, while m2 and m4 receptors couple through a Pertussis toxin-sensitive G-protein to preferentially inhibit adenylyl cyclase.This chapter describes the electrophysiological consequences of muscarinic receptor activation using chemically differentiated NG108-15 cells transfected with ml-m4 receptor genes. These cells are especially useful for electrophysiological studies, because they express a range of K + and Ca 2+ channels similar to those found in many neuronal cell types. However, NGl08-15 cells do natively express the m4 muscarinic receptor.

Intracellular Mg2+ inhibits the IP3-activated IK(Ca) in NG108-15 cells. [Why intracellular citrate can be useful for recording IK(Ca)]

Receptor-mediated formation of inositol 1,4,5-trisphosphate (IP3) can induce an outward Ca2+-activated K+ current [IK(Ca)] some neural cells. We have investigated IK(Ca) activated by intracellular injections of IP3 in whole-cell patch-clamped neuroblastoma×glioma hybrid cells. The current could only be recorded reliably using citrate as the anion in the pipette, but not using acetate, aspartate, chloride, fluoride, gluconate or methylsulphate. This could be attributed to buffering of intracellular Mg2+ by citrate. Theoretical calculations suggested free [Mg2+] of 1.0 and 0.07 mM respectively in the acetate- and citrate-based recording solutions. Further, IP3-activated IK(Ca) could be recorded when the free Mg2+ level in the acetate, chloride or methylsulphate solutions was lowered to the range (0.05 mM) calculated for the citrate solution. Thus, raised [Mg2+] blocks IK(Ca). This appeared to be due to inhibition of the response to released Ca2+, since high [Mg2+] also blocked the response to intracellular injections of Ca2+ ions. Mean Mg2+ levels in intact neuroblastoma×glioma hybrid cells measured by Mag-Indo-1/AM fluorescence were estimated to be less than 0.14 mM. We therefore conclude that IP3-induced IK(Ca) is expressed under normal conditions, but may be subject to regulation by intracellular Mg2+.

The naphthalenesulphonamide calmodulin antagonist W7 and its 5-iodo-1-C8 analogue inhibit potassium and calcium currents in NG108-15 neuroblastoma × glioma cells in a manner possibly unrelated to their antagonism of calmodulin

Patch clamp techniques were used to record voltage-sensitive calcium and potassium currents from NG108-15 cells. N-(6-aminohexyl)-5-chloro-1-naphthalene- sulphonamide (W7), a calmodulin (CaM) antagonist and its more potent (10 times) 5-iodo-1-C8 analogue (J8) inhibited these currents in a dose-dependent manner. The inhibition was not dependent on internal or external Ca2+. W7 was about four times more potent as an inhibitor of the transient potassium current (IC50 = 8 microM) than of the M-current or of the calcium current. J8 was also selective for the potassium currents (IC50 values: transient current 4 microM, M-current 11 microM) compared to the calcium current (IC50 36 microM). It is suggested that the inhibition does not result from an anti-CaM action of the compounds.

Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS, of protein kinase C.

SummaryDifferentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (IK) and the M-current (IM). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E1 (PGE1) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: Ik, a small current through a “T-like” Ca2+ channel, and no M-current.Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22±7%. In contrast to the stimulation of Ik observed with cardiac cells, PKC activation produced only a small inhibition of Ik, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of Ik in neuroblastoma cells.Supported in part by research grants from the National Institutes of Health (DK-40145 and EY-08343) and from the U.K. Medical Research Council.

Agonist-induced inhibition of inositol-trisphosphate-activated IK(ca) in NG108-15 neuroblastoma hybrid cells

IK(ca) activated by intracellular ionophoresis of inositol trisphosphate (IP3) or pressure-applied acetylcholine was inhibited by bradykinin and acetylcholine in NG108-15 cells transfected with m1 receptors. The inhibition of the IP3-evoked current was complete at 10 μM acetylcholine. This inhibition was not seen if the current was evoked by intracellular ionophoresis of calcium ions. Only receptors the activate the phosphoinositide system in these cells produced this inhibition, i.e. transfected muscarinic m1 and m3 and bradykinin receptors, but not muscarinic m2, m4 or adrenergic α2 receptors. This inhibition was not sensitive to pertussis toxin or staurosporine. The concentrations of acetylcholine needed to inhibit the evoked current were identical to those needed to raise intracellular calcium but tenfold less than those needed for the agonist to activate IK(Ca). In a normal calcium-containing superfusate, recovery from inhibition required around 8 min (half-time 4 min) after removal of acetylcholine. When the experiment was performed in calcium-free medium no recovery was seen after 8 min washing in drug-free solution, but complete recovery was seen within 3 min (half-time 1.5 min) after adding calcium. Responses to repeated pressure applications of acetylcholine could be reversibly inhibited by acetylcholine and bradykinin. It seems, then, that there is no direct action of acetylcholine or bradykinin on the IK(Ca) channels themselves but that concentrations below those needed to activate IK(Ca) can empty and inhibit the IP3-sensitive calcium store. This may provide a mechanism for heterologous desensitization for phospholipase-C-linked receptor-mediated responses.

NEUROTRANSMITTER MODULATION OF CALCIUM CHANNELS IS DEPENDENT ON THE CHARGE CARRIER USED IN THE RECORDING OF CURRENTS

Currents through calcium channels were recorded using calcium, barium and strontium as charge carriers in NG108-15 cells. The mean normalised peak current amplitude at 0 mV was not significantly different between the charge carriers; however, the sustained component (measured at the end of the 500 ms command step) was ca. 3 times larger in barium and strontium. Further, the inhibition by acetylcholine or noradrenaline, although the same at the peak of the current envelope, was significantly greater on the sustained portion of the current for barium and strontium. Increasing internal calcium-buffering (to reduce calcium-dependent inactivation with calcium as the charge carrier) did not increase the amount of inhibition of the sustained portion of current. These results suggest a cautious approach to analysis of neurotransmitter modulation of calcium currents using other charge carriers than calcium.