Mechanism of thiamine transport in neuroblastoma cells. Inhibition of a high affinity carrier by sodium channel activators and dependence of thiamine uptake on membrane potential and intracellular ATP.
Nerve cells are particularly sensitive to thiamine deficiency. We studied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low external concentration, [14C]thiamine was taken up through a saturable high affinity mechanism (Km = 35 nM). This was blocked by low concentrations of the Na+ channel activators veratridine (IC50 = 7 +/- 4 microM) and batrachotoxin (IC50 = 0.9 microM). These effects were not antagonized by tetrodotoxin and were also observed in cell lines devoid of Na+ channels, suggesting that these channels are not involved in the mechanism of inhibition. At high extracellular concentrations, thiamine uptake proceeds essentially via a low affinity carrier (Km = 0.8 mM), insensitive to veratridine but blocked by divalent cations. In both cases, the uptake was independent on external sodium, partially inhibited (10-35%) by depolarization and sensitive to metabolic inhibitors. A linear relationship between the rate of thiamine transport and intracellular ATP concentration was found. When cells grown in a medium of low thiamine concentration (6 nM) were exposed to 100 nM extracellular thiamine, a 3-fold increase in intracellular thiamine diphosphate was observed after 2 h while the concomitant increase in intracellular free thiamine was barely significant. These data suggest a secondary active transport of thiamine, the main driving force being thiamine phosphorylation rather than the sodium gradient.
Bettendorff
L
L
Laboratory of General and Comparative Biochemistry, University of Liège, Belgium.
Wins
P
P
D000157:Aconitine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001496:Batrachotoxins / Q000494:pharmacology*; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology; D013831:Thiamine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014701:Veratridine / Q000494:pharmacology*
D001496:Batrachotoxins; D015222:Sodium Channels; D013779:Tetrodotoxin; D014701:Veratridine; D000584:Amiloride; D000255:Adenosine Triphosphate; D013831:Thiamine; D000157:Aconitine