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 (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.
Poulin
R
R
Laboratory of Molecular Endocrinology, Laval University Medical Research Centre, 2705 Laurier Blvd., Ste.Foy, Quebec, Canada G1V4G2.
Zhao
C
C
Verma
S
S
Charest-Gaudreault
R
R
Audette
M
M
D000584:Amiloride / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D001943:Breast Neoplasms; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D005260:Female; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008322:Mammals; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D009861:Onium Compounds / Q000493:pharmacokinetics; D009943:Organophosphorus Compounds / Q000493:pharmacokinetics; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D011700:Putrescine / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000378:metabolism*; D013095:Spermidine / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014634:Valinomycin / Q000494:pharmacology
D007202:Indicators and Reagents; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D017923:Sodium-Hydrogen Exchangers; C013407:benzamil; D014634:Valinomycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000584:Amiloride; D011188:Potassium; D013095:Spermidine; C013289:tetraphenylphosphonium; D011700:Putrescine; C039614:ethylisopropylamiloride