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
L. Bettendorff et P. Wins, 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, The Journal of biological chemistry, 269(20), 1994, pp. 14379-14385
Nerve cells are particularly sensitive to thiamine deficiency. We stud
ied thiamine transport in mouse neuroblastoma (Neuro 2a) cells. At low
external concentration, [C-14]thiamine was taken up through a saturab
le high affinity mechanism (K-m = 35 nM). This was blocked by low conc
entrations of the Na+ channel activators veratridine (IC50 = 7 +/- 4 m
u M) and batrachotoxin (IC50 = 0.9 mu M). These effects were not antag
onized by tetrodotoxin and were also observed in cell lines devoid of
Na+ channels, suggesting that these channels are not involved in the m
echanism of inhibition. At high extracellular concentrations, thiamine
uptake proceeds essentially via a low affinity carrier (K-m = 0.8 mM)
, insensitive to veratridine but blocked by divalent cations. In both
cases, the uptake was independent on external sodium, partially inhibi
ted (10-35%) by depolarization and sensitive to metabolic inhibitors.
A linear relationship between the rate of thiamine transport and intra
cellular 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 fr
ee thiamine was barely significant. These data suggest a secondary act
ive transport of thiamine, the main driving force being thiamine phosp
horylation rather than the sodium gradient.