Characterization of a large conductance, cation-selective channel from sea urchin eggs that is sensitive to sulfhydryl reducing agents

Abstract

Vesicles containing large conductance cation selective channels were isolated from sea urchin (Strongylocentrotus purpuratus) eggs. Addition of the vesicles to one side of lipid bilayer led to the rapid appearance of 200 or more identical channels. These channels would then inactivate within 2 to 10 min. The inactivation could be prevented by the addition of sulfhydryl reducing agents (e.g., dithiothreitol or glutathione) to the cis side of the membrane. Only one channel type is present. The channel is cation selective, with a conductance of 572 ps in symmetrical 0.5 M KCl. The relative cation selectivity is K (1.0) > Cs (0.53) ~ Na (0.52) > Li (0.2). The permeability ratio (P(X)/P(K) is 1.37 (Li) > 1.27 (Na) > 0.57 (Cs). Most organic cations (choline, tetraethylamine, tetrabutylamine, gallamine, lysine, histidine, arginine, etc.) and multivalent cations (La+3, alkali earth family, Zn+2, Eu+3, etc.) produced a significant channel block. The highest observed affinity was for La+3 which produced a 50% decrease in conductance in 500 mM KCl at a concentration of 8 μM. The biophysical properties of this channel are similar to those of a nonselective channel found in ascidian egg plasma membrane (Dale and DeFelice, 1984). A soluble extract of the egg supernatant can also prevent the inactivation of the channels. Using deactivated channels reconstituted into a planar lipid bilayer as an assay, this factor was partially purified. It is heat and acetone stable with a molecular weight of between 10 and 20 K. One of the major bands remaining in the purest fraction cross reacted with antibodies raised against E. coli thioredoxin.