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

The mechanism of mammalian polyamine transport is poorly understood. We have investigated the role of plasma-membrane potential (ΔΨ_pm) 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 ΔΨ_pm, as determined by the accumulation of [³H]tetraphenylphosphonium bromide (TPP). Inward transport was unaffected by a selective decrease in mitochondrial potential (ΔΨ_mit) induced by valinomycin at low [K⁺]_o, but was reduced by ≈ 60% by the rheogenic protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which rapidly (≤ 15 min) collapsed both ΔΨ_pm and ΔΨ_mit. 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 μM) 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 [K_i 4.0±0.9 (S.D.) μM], 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 ΔΨ_pm, but not ΔΨ_mit. 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.