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.

Modulation of calcium current in snail neurones by dopamine: the role of intracellular free calcium.

The inhibition of voltage-gated Ca-current in Limnaea stagnalis (L.) neurones by dopamine cannot be prevented by intracellular administration of the Ca-chelating agent, EGTA. At the same time, substitution of external Ba2+ for Ca2+ substantially weakens the dopamine-induced inhibition. The Ca-ionophore, A23187, also produces an inhibition, but the inhibitory effect of A23187 and of dopamine on Ca-current are non-additive. These findings allow one to suggest that the entry and increase of free Ca concentration presumably in the narrow submembrane space, but not in the bulk of cytoplasm, is important for development of inhibition of neuronal Ca-current by dopamine.

Neurotransmitter-induced modulation of neuronal Ca current is not mediated by intracellular Ca2+ or cAMP.

Dopamine (1 microM) inhibits the Ca component of action potential and corresponding electroexcitable Ca current (ICa) in isolated snail neurons. Adrenaline and serotonin also reduce ICa. The inhibition is not related to changes in intracellular Ca2+ or cAMP concentrations: internal application of 10 mM ethyleneglycoltetraacetic acid or 10 microM cAMP in the mixture with 1 mM Mg-ATP and 2 mM theophylline does not influence the action of neurotransmitters on ICa.

Effect of "calcium antagonist" D-600 on the postsynaptic membrane.

Using the technique of dialysis of the cell interior (Kostyuk et al., 1978), the effect of "calcium antagonist" D-600 on voltage clamped isolated neurones of mollusc Limnaea stagnalis was studied. Identified giant neurones responding to acetylcholine (ACh) application with changes in conductance for Cl- ions only were used. 1. D-600, 10-100 microM, blocks electroexcitable inward Ca2+-Na+ current in the neurone soma membrane. In the same concentrations, D-600 reduces the amplitude of ACh-induced current and also the time constant of desensitization onset. 2. Voltage jump relaxation experiments show D-600 to decrease the average lifetime, tau, of the Cl- channels opened by ACh, although the effect of d-tubocurarine (d-TC), 10-20 microM, on tau is insignificant. 3. The lifetime of open Cl- channels increases with membrane hyperpolarization; but while the voltage dependence of tau is not altered by d-TC, it is reduced by D-600. 4. The results obtained allow us to suggest that D-600 blocks open Cl- channels. The calculated rate constant of association of D-600 with open channels is of the order of 10(5) M-1 s-1.

Voltage clamp analysis of acetylcholine receptor desensitization in isolated mollusc neurones.

1. Desensitization produced by acetylcholine (ACh) in completely isolated Limnaea stagnalis neurones with chloride-selective membrane channels was studied using a voltage-clamp technique. 2. A difference in the time course of the neurone responses to ACh, depending on whether the measured parameter was voltage or current, was observed and explained on the basis of an equivalent electric scheme of the neurone soma membrane. 3. Desensitization onset was shown not to depend on membrane potential in the range of -30 to -120 mV. 4. Variation of external Ca2+, Na+ and Cl- concentrations over a wide range had little influence on the onset of desensitization and recovery from it. 5. An obvious difference is shown to exist between features of desensitization in mollusc neurone and frog muscle end-plate ACh receptors.

Chemical nature of functional cholinoreceptor groups of Lymnaea stagnalis neurons.

1. The effect of pH and the influence of some alkylating agents on the properties of the cholinoreceptive membrane of the mollusc Lymnaea stagnalis neurons have been studied using the microelectrode voltage clamp technique. 2. Lowering below 7.5 of the pH of the bathing solution had to decrease the neuronal responses to ACh. A twofold decrease in cholinoreceptive membrane conductivity was found at the pH 6.7 +/- 0.1 (n=10). Raising the pH to 10.6 did not influence the response to ACh. 3. The pH effect is not associated with the influence on the properties of ionic channels but appears to be due to reduction of a functional group at the ChR active site by proton. 4. No highly reactive SH-groups were found at the ChR active site, but some functionally important carboxyl groups have been discovered. 5. The effect of pH is probably connected with reduction of --COO-- or imidazol group with a pKa of about 6.7.