Dependence of mammalian putrescine and spermidine transport on plasma-membrane potential: identification of an amiloride binding site on the putrescine carrier

Abstract

The mechanism of mammalian polyamine transport is poorly understood. We have investigated the role of plasma-membrane potential (DeltaPsipm) in putrescine and spermidine uptake in ZR-75-1 human breast cancer cells. The rate of [3H]putrescine and [3H]spermidine uptake was inversely correlated to extracellular [K+] ([K+]o) and to DeltaPsipm, as determined by the accumulation of [3H]tetraphenylphosphonium bromide (TPP). Inward transport was unaffected by a selective decrease in mitochondrial potential (DeltaPsimit) induced by valinomycin at low [K+]o, but was reduced by approximately 60% by the rheogenic protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which rapidly (<=15 min) collapsed both DeltaPsipm and DeltaPsimit. Plasma-membrane depolarization by high [K+]o or CCCP did not enhance putrescine efflux in cells pre-loaded with [3H]putrescine, suggesting that decreased uptake caused by these agents did not result from a higher excretion rate. On the other hand, the electroneutral K+/H+ exchanger nigericin (10 microM) co-operatively depressed -3H-TPP, [3H]putrescine and [3H]spermidine uptake in the presence of ouabain. Suppression of putrescine uptake by nigericin+ouabain was Na+-dependent, suggesting that plasma-membrane repolarization by the electrogenic Na+ pump was required upon acidification induced by nigericin, due to the activation of the Na+/H+ antiporter. The sole addition of 5-N, N-hexamethylene amiloride, a potent inhibitor of the Na+/H+ antiporter, strongly inhibited putrescine uptake in a competitive fashion -Ki 4.0+/-0.9 (S.D.) microM-, while being a weaker antagonist of spermidine uptake. The potency of a series of amiloride analogues to inhibit putrescine uptake was clearly different from that of the Na+/H+ antiporter, and resembled that noted for Na+ co-transport proteins. These data demonstrate that putrescine and spermidine influx is mainly unidirectional and strictly depends on DeltaPsipm, but not DeltaPsimit. This report also provides first evidence for a high-affinity amiloride-binding site on the putrescine carrier, which provides new insight into the biochemical properties of this transporter.