Palytoxin-induced single-channel currents from the sodium pump synthesized by in vitro expression

Palytoxin, the most potent animal toxin, is proposed to convert Na+/K(+)-ATPase into a cation-selective ion channel. Because of the ubiquity of pumps and channels in the living tissues used to study its mechanism of action, it is difficult to rule out that another site may be involved. In order to show that palytoxin selectively acts on Na+/K(+)-ATPase, two entirely in vitro methods were employed: (1) a cell-free expression system to synthesize the rat alpha 3 and beta 1 subunit proteins, and (2) single-channel recording of the synthetic Na+/K(+)-ATPase reconstituted in a planar lipid bilayer. Upon addition of palytoxin, single-channel currents were induced which had a conductance of 10 pS, in agreement with previous studies. In control experiments, when the cDNAs for Na+/K(+)-ATPase subunits were omitted, no single-channel currents were induced with palytoxin. Thus, the results show unambiguously that the Na+/ K(+)-ATPase is the site of action for palytoxin. Because palytoxin turns the Na+/K(+)-ATPase into a channel which can be detected by the exquisitely sensitive single-channel recording technique, the present results are the first to demonstrate the activity of in vitro synthesized Na+/K(+)-ATPase.