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Properties of single sodium channels translated by Xenopus oocytes after injection with messenger ribonucleic acid.
Sigel E.
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1. The properties of fast transient Na channels induced in the Xenopus laevis oocyte plasma membrane after injection of the oocyte with foreign messenger ribonucleic acid (mRNA) were investigated with the whole-cell voltage clamp and with the patch-clamp technique. 2. The time course of expression and the effect of the metabolic inhibitors actinomycin D and tunicamycin were studied. The rate of channel insertion reached a maximum only about 3 days after injection with mRNA and corresponded to the incorporation of 20 active channels/s, into a single oocyte. When applied intracellularly tunicamycin blocked the appearance of active channels nearly completely while actinomycin D added to the medium had no effect. 3. The whole-cell currents showed activation and inactivation properties reminiscent of skeletal muscle Na+ currents. The maximal peak current amplitude was 6 microA. Tetrodotoxin blocked the observed transient inward current. 50% inhibition was observed at 10 nM concentration. Veratridine depressed inactivation of the current and led to prolonged tail currents. 4. After removal of the surrounding layers of the oocyte tight seals were obtained with a patch-clamp electrode pushed on the surface membrane. Single-channel currents endogenous to the oocyte and Na+-channel currents induced by injected mRNA could be recorded. The single-channel slope conductance of the latter was 12-15 pS. Two different types of kinetic behaviour were evident from an analysis of single-channel currents and ensemble average currents. One type showed fast inactivation (tau less than 1 ms) and brief channel openings (less than 1 ms) whereas the second type was characterized by slower inactivation and a bursting behaviour. 5. When veratridine (75 microM) was present in the pipette solution the single-channel behaviour was modified in a complex manner. In addition to the channel openings with normal conductance a second open state was observed with a slope conductance of 3.5 pS. This second type of channel opening could still be recorded after return to the holding potential. Its final closure followed an exponential time course with a constant time constant of 0.5 s at -100 mV. These events probably underlie the tail currents in the whole-cell configuration. 6. The Xenopus oocyte represents a useful system for the study of the expression of channels induced by foreign mRNA, for the characterization of their single-channel behaviour and for the investigation of the action of pharmacologically active substances on these channels. This system may prove useful for the study of channels that are not accessible to patch-clamp experiments 'in situ'.
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