Diversity of nicotinic receptors mediating Cl- current in Lymnaea neurons distinguished with specific agonists and antagonist.

Diversity of nicotinic acetylcholine receptors (nAChRs) mediating Cl- current in voltage-clamped identifiable Lymnaea stagnalis neurons was studied using acetylcholine (ACh), three agonists and alpha-conotoxin ImI (ImI). Cytisine, nicotine, and choline, full agonists at alpha7 subunit-containing nAChRs of vertebrates, were found to evoke at saturating concentration 84-92% of the maximal current elicited by ACh. ImI, known to block selectively alpha7 and alpha9 nAChRs, markedly diminished the responses to ACh. The average maximal ImI-induced block was 80%, leaving a residual current which had very slow kinetics. The choline-, cytisine-, and nicotine-induced currents were blocked by ImI almost completely, suggesting that they activate only ImI-sensitive receptors. Two groups of cells which differ in desensitization kinetics and in sensitivity to ImI were revealed. IC50 values for ImI against ACh were 10.3 and 288 nM, respectively, with the rapidly desensitizing current being the more sensitive to ImI. The data obtained suggest the existence of at least three pharmacologically distinct subtypes of nicotinic receptors in Lymnaea neurons. Two of the subtypes are similar to alpha7 nAChRs of vertebrates, but differ from each other in their affinity for ImI and in their desensitization kinetics. The third subtype is quite distinct, in that it is resistant to ImI, is not activated by nicotine, cytisine or choline, and mediates a very slowly developing current.

Nicotinic receptors in Lymnaea stagnalis neurons are blocked by alpha-neurotoxins from cobra venoms.

The influence of cobra neurotoxins on the Cl-dependent responses to acetylcholine (ACh) of Lymnaea neurons was studied by the voltage-clamp technique. It was found that a short chain neurotoxin II (NT II), a long chain cobratoxin (CTX) and weak neurotoxin (WTX) diminished the ACh-induced currents, the block being concentration-dependent and competitive. The IC(50) values of 130 nM for CTX, 11 microM for NT II, and 67 microM for WTX were determined. The block induced by NT II was quickly reversible upon toxin washout, whereas the action of CTX and WTX was only partially reversible even after an hour of intensive washing. The data obtained suggest that acetylcholine receptors (AChRs) in Lymnaea neurons have common features with cation-selective alpha 7 AChRs of vertebrates and one type of Aplysia Cl-conducting AChRs.

Intracellular Ca2+ modulates Cl- current evoked by acetylcholine in Lymnaea neurons.

The influence of voltage-gated Ca2+ current (ICa) on Cl current (ICl) initiated by nicotinic receptors (AChRs) in dialysed voltage-clamped Lymnaea neurons was studied. Depolarising steps applied before or during ACh application decreased ICl transiently and slowed down both the rising phase and decay of ICl. The effect of ICa depended on the interval between ICa and ICl; it was prevented by intracellular buffering with BAPTA or Ca2+ channel blocking with Ni2+. ISr had a similar action but the recovery was slower than after ICa; IBa was ineffective. The data suggest that inactivation of AChR channels is mediated by Ca2+ binding to a site in the AChR or the regulatory protein.

Intracellular ATP modulates desensitization of acetylcholine receptors controlling chloride current in Lymnaea neurons.

Chloride current activated by nicotinic acetylcholine receptors (AChR) was examined in dialysed voltage-clamp neurons of Lymnaea stagnalis. Fast superfusion of acetylcholine (ACh) evoked an inward current rapidly rising to a peak followed by a decline due to desensitization. When adenosine triphosphate with Mg2+ (MgATP, 2-10 mM) was added intracellularly the peak of the ACh-induced current was increased and its decay was slowed down. ATP without Mg2+ did not affect desensitization. Mg2+ alone accelerated desensitization. Intracellular treatment with an inhibitor of ATP synthesis, sodium arsenate, increased the desensitization rate and decreased the peak current. MgATP after arsenate wash-out restored the initial characteristics of the response; a mixture of glycolytic substrates had a similar effect. A non-hydrolysable analogue of ATP, adenosine [gamma-thio]triphosphate mimicked ATP action after arsenate removal but was weaker; another non-hydrolysable analogue, adenylyl imidodiphosphate, did not affect desensitization at all. Intracellular treatment of the neurons with alkaline phosphatase accelerated current decay. The data suggest that a change in intracellular ATP concentration modulates AChR desensitization via an enzymatic process that might be phosphorylation of AChR or some associated protein(s). Involvement of Ca2+ homeostasis cannot be excluded. The results are compared with the data obtained on vertebrate tissues under conditions promoting phosphorylation.

Intracellular ATP modifies the voltage dependence of the fast transient outward K+ current in Lymnaea stagnalis neurones.

1. The action of intracellular ATP on the fast transient outward K+ current (A-current) was studied in dialysed voltage-clamped Lymnaea stagnalis neurones. 2. When introduced intracellularly in millimolar concentrations ATP caused a shift of the steady-state inactivation curve along the voltage axis in the direction of positive potentials and decreased A-current at all test voltages. 3. Intracellular treatment with an inhibitor of ATP synthesis, sodium arsenate, led to the opposite changes. The action of arsenate was not reversed upon its removal. After wash-out of arsenate ATP restored the initial voltage dependence. 4. Addition of Mg2+ to the solution weakened the action of ATP in proportion to the Mg2+: ATP concentration ratio. On the other hand, in neurones pretreated with arsenate, Mg2+ did not affect the ATP action. 5. When a mixture of glycolytic substrates was applied after arsenate wash-out the activation and inactivation curves shifted towards positive voltages. A substrate of oxidative phosphorylation was ineffective in the same conditions. 6. Non-hydrolysable analogues of ATP, adenosine-5'-O-gamma-thiotriphosphate and adenylyl imidodiphosphate, did not mimic the ATP action. This means that the ATP effect is mediated by some enzymatic process(es). 7. Elevation of total cytosolic Ca2+ concentration as well as intracellular application of agents increasing intracellular free Ca2+ reduced A-current amplitude but failed to alter its voltage dependence. Therefore, ATP action cannot be related to activation of Ca2+ transport. 8. Treatment of the neurones with alkaline phosphatase evoked a shift of the inactivation voltage dependence towards hyperpolarizing potentials and increased the A-current amplitudes at all test voltages. 9. The data indicate that a change in intracellular ATP concentration modulates the A-current voltage dependence. The effect of ATP is probably the result of phosphorylation of a channel protein or some associated proteins, but lowering of free Mg2+ concentration cannot be excluded. The possible physiological significance of the phenomenon is discussed.

Modulation of nicotinic acetylcholine receptors by intracellular calcium and cyclic AMP in Lymnaea stagnalis neurones.

Acetylcholine (ACh)-receptor ion channels were investigated under the modulatory action of calcium and cyclic AMP in completely isolated Lymnaea stagnalis neurones using the noise analysis technique. Elevation of the intracellular Ca2+ concentration in dialyzed neurones produced a reduction in the amplitude of ACh induced current accompanied by slight decrease in the mean channel open time and a simultaneous 1.5-fold increase in mean channel conductance. Direct introduction of cyclic AMP into neurones or elevation of intracellular cyclic AMP level by application of serotonin or forskolin produced 20-40% reduction in ACh-induced conductance without significant effect on the measured parameters of the ion channels. The inhibitory effects of calcium and cyclic AMP appear to be independent. Our findings indicate that reduction in ACh induced conductance under calcium and cyclic AMP modulation results from an alteration in the channel gating mechanism. Since the efficiency of ion transfer is independent of cyclic AMP, and it even rises with the elevation of calcium concentration, the inhibition of ACh responses may be accounted for by a decrease in the rate constant for channel opening, so that channels activated by acetylcholine remain in a closed state over longer intervals.

Intracellular Mg2+ modulates the A-current and its blockage by catechol in isolated Lymnaea neurons.

The effects of intracellular Mg2+ (2-8 mM) upon the transient outward current (the A-current) under normal conditions and under catechol-induced blockage were studied in molluscan neurons by using the voltage-clamp and intracellular dialysis techniques. Identified giant Lymnaea stagnalis L. neurons were investigated at room temperature (20-22 degrees C). When applied intracellularly, Mg2+ caused both time- and dose-dependent shifts of the voltage dependence of the steady-state activation and inactivation of the A-current to more negative membrane potentials. Upon external application, catechol suppressed (5-6 mM) or eliminated (9-10 mM) the A-currents, slowed down the current decay and shifted the activation and inactivation curves to more positive membrane voltages. Intracellular Mg2+ decreased the blocking ability of extracellularly applied catechol, whereas catechol antagonized the Mg2(+)-induced negative shift of the steady-state activation and inactivation curves of the A-currents.

Depression of neuron responses to acetylcholine by combined application of norepinephrine and substrates of the tricarboxylic acid cycle.

The possible relationship between the function of nicotinic acetylcholine receptors in Lymnaea stagnalis neurons and energy metabolism was studied. Oxidative phosphorylation was activated by treatment of neurons with substrates of the tricarboxylic acid cycle and norepinephrine. Transmembrane currents induced by acetylcholine in isolated neurons were measured by voltage clamp. Succinate dehydrogenase activity was determined histochemically in the same neurons. Cyclic adenosine monophosphate concentration in ganglia were assayed by the protein saturation method of Gilman (1970). When used alone, succinate depressed the responses of about 50% of neurons to acetylcholine. Norepinephrine did not affect the acetylcholine-induced currents but almost doubled the inhibitory action of succinate. The mixture of norepinephrine and isocitrate also diminished the responses to acetylcholine but to a lesser extent than norepinephrine with succinate. A short-term exposure of the ganglia to succinate with norepinephrine led to the activation of succinate dehydrogenase in neurons and a threefold increase in cyclic adenosine monophosphate concentrations in ganglia. When used alone, norepinephrine doubled the cyclic adenosine monophosphate concentration. The results obtained suggest energy-dependent regulation of acetylcholine receptors.

Two-component desensitization of nicotinic receptors induced by acetylcholine agonists in Lymnaea stagnalis neurones.

The kinetics of desensitization induced by different agonists of acetylcholine (ACh) as well as the kinetics of recovery from densensitization, have been studied using the voltage-clamp technique in isolated, identified Lymnaea stagnalis neurones. Desensitization follows the sum of two exponentials: one fast and one slow. The time constant of the fast desensitization component (tau Ids) under ACh application is in the range of seconds at room temperature (18-23 degrees C). It increases upon cooling (Q10 = 2.8 +/- 0.9), decreases with increasing ACh concentration and is independent of membrane voltage. The time constant of the slow component of densensitization (tau Ids) is in the range of tens of seconds. It decreases with increasing drug concentration and is weakly dependent upon temperature (Q10 = 1.3 +/- 0.4). The relative amplitude of the fast component, estimated by back extrapolation to the position of the peak current, increases with agonist concentration and decreases upon cooling. Recovery from desensitization follows the sum of two exponentials with time constants (tau Ir and tau IIr) of the order of seconds and minutes, respectively. Cooling prolongs the slow component (Q10 of tau IIr is approx. 3) and reduces its contribution during recovery. A comparison of the desensitization induced by various agonists indicates that for the small monoquaternary agonists the onset and recovery of desensitization resemble the onset and recovery observed with ACh. For more bulky agonists, like ethoxysebacylcholine, sebacylcholine and suberylcholine, the decay of the response during prolonged application of the agonist may involve an additional blocking process.

Interaction between acetylcholine and other agonists at cholinoreceptive membrane of mollusc neurons.

The effect of pretreatment with agonists of various chemical structure on acetylcholine action at isolated Limnaea stagnalis neurones was studied. Threshold concentrations of monoquaternary agonists or dicholinic ester of adipinic acid increase and inhibit the responses to low and high acetylcholine concentrations respectively. In contrast, suberyldicholine and compound IEM-1054 (containing two charged ammonium groups in a molecule with an internitrogen distance of 2.0-2.2 nm) decrease the action of any acetylcholine concentration. The results are discussed in terms of the cooperativity model of acetylcholine receptors (AChR) and the hypothesis about regular arrangement of AChRs in the membrane, the so called 'C-16 structure'.