A COMPARATIVE STUDY OF ANAESTHETIC AGENTS ON HIGH VOLTAGE ACTIVATED CALCIUM CHANNEL CURRENTS IN IDENTIFIED MOLLUSCAN NEURONS

SUMMARYUsing the two electrode voltage clamp configuration, a high voltage activated whole-cell Ca2+ channel current (IBa) was recorded from a cluster of neurosecretory ‘Light Yellow’ Cells (LYC) in the right parietal ganglion of the pond snail Lymnaea stagnalis.Recordings of IBa from LYCs show a reversible concentration-dependent depression of current amplitude in the presence of the volatile anaesthetics halothane, isoflurane and sevoflurane, or the non-volatile anaesthetic pentobarbitone at clinical concentrations.In the presence of the anaesthetics investigated, IBa measured at the end of the depolarizing test pulse showed proportionally greater depression than that at measured peak amplitude, as well as significant decrease in the rate of activation or increase in inactivation or both.Within the range of concentrations used, the concentration-response plots for all the anaesthetics investigated correlate strongly to straight line functions, with linear regression R2 values > 0.99 in all instances.For volatile anaesthetics, the dose-response regression slopes for IBa increase in magnitude, in order of gradient: sevoflurane, isoflurane and halothane, a sequence which reflects their order of clinical potency in terms of MAC value.

Characterisation of δ-Conotoxin TxVIA as a Mammalian T-Type Calcium Channel Modulator

The 27-amino acid (aa)-long δ-conotoxin TxVIA, originally isolated from the mollusc-hunting cone snail Conus textile, slows voltage-gated sodium (NaV) channel inactivation in molluscan neurons, but its mammalian ion channel targets remain undetermined. In this study, we confirmed that TxVIA was inactive on mammalian NaV1.2 and NaV1.7 even at high concentrations (10 µM). Given the fact that invertebrate NaV channel and T-type calcium channels (CaV3.x) are evolutionarily related, we examined the possibility that TxVIA may act on CaV3.x. Electrophysiological characterisation of the native TxVIA on CaV3.1, 3.2 and 3.3 revealed that TxVIA preferentially inhibits CaV3.2 current (IC50 = 0.24 μM) and enhances CaV3.1 current at higher concentrations. In fish bioassays TxVIA showed little effect on zebrafish behaviours when injected intramuscular at 250 ng/100 mg fish. The binding sites for TxVIA at NaV1.7 and CaV3.1 revealed that their channel binding sites contained a common epitope.

Retinoic acid affects calcium signaling in adult molluscan neurons

Retinoic acid, the active metabolite of vitamin A, is important for nervous system development, regeneration, as well as cognitive functions of the adult central nervous system. These central nervous system functions are all highly dependent on neuronal activity. Retinoic acid has previously been shown to induce changes in the firing properties and action potential waveforms of adult molluscan neurons in a dose- and isomer-dependent manner. In this study, we aimed to determine the cellular pathways by which retinoic acid might exert such effects, by testing the involvement of pathways previously shown to be affected by retinoic acid. We demonstrated that the ability of all- trans retinoic acid (atRA) to induce electrophysiological changes in cultured molluscan neurons was not prevented by inhibitors of protein synthesis, protein kinase A or phospholipase C. However, we showed that atRA was capable of rapidly reducing intracellular calcium levels in the same dose- and isomer-dependent manner as shown previously for changes in neuronal firing. Moreover, we also demonstrated that the transmembrane ion flux through voltage-gated calcium channels was rapidly modulated by retinoic acid. In particular, the peak current density was reduced and the inactivation rate was increased in the presence of atRA, over a similar time course as the changes in cell firing and reductions in intracellular calcium. These studies provide further evidence for the ability of atRA to induce rapid effects in mature neurons.