The transport mechanism and specificities of ionophore ETH-29 have been investigated in a highly defined phospholipid vesicle system, with the goal of facilitating the application of this compound to biological problems. ETH-129 transports Ca2- via an electrogenic mechanism, in contrast to A23187 and ionomycin, which function in a charge neutral manner. The rate of transport is a function of membrane potential, increasing by 3.9-fold per 59 mV over a broad range of that parameter. Rate is independent of the transmembrane pH gradient and strongly stimulated by the uncoupler carbonyl cyanide m-chlorophenylhydrazone when no external potential has been applied. The effect of uncoupler reflects the collapse of an opposing potential arising during Ca2+ transport, but also reflects the formation of a mixed complex between the uncoupler, ETH-129, and Ca2+ that readily permeates the vesicle membrane. Oleate does not substitute for the uncoupler in either regard. ETH-129 transports polyvalent cations according to the selectivity sequence La3+ > Ca2- > Zn2+ ≈ Sr2+ > Co2+ ≈ Ni2+ ≈ Mn2+, with the magnitude of the selectivity coefficients reflecting the cation concentration range considered. There is little or no activity for the transport of Na+, K+, and Mg2+. These properties suggest that ETH-129 will be useful for investigating the consequences of a mitochondrial Ca2+ overload in mammalian cells, which is difficult to pursue through the application of electroneutral ionophores.
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