Interaction between anions and the reduced folate/methotrexate transport system in L1210 cell plasma membrane vesicles: Directional symmetry and anion specificity for differential mobility of loaded and unloaded carrier

Abstract

The effect of various anions on the mediated influx and efflux of [3H]methotrexate by L1210 cell plasma membrane vesicles in a HEPES buffer system was studied. Our results show that flux is stimulated to the same extent in either direction when SO4, Pi, or folate compounds (1,L5-CHO-folate-H4, methotrexate), but not Cl- was present in the opposite compartment. This implies the property of directional symmetry, a condition in which differential mobility of loaded and unloaded carriers occurs in both directions. We also observed a similarity in the specificity of the interaction between various anions and carrier in each orientation of the membrane, in the order, Cl- much less than Pi approximately equal to SO2-4 much less than methotrexate less than 1,L5-CHO-folate-H4. Also, the absolute differential in mobility of loaded and unloaded carrier (assumed from the extent of transstimulation obtained) varied substantially among the anions examined. No stimulation was obtained with Cl-, and stimulation was twofold with Pi, SO2-4 and methotrexate and fourfold with 1,L5-CHO-folate-H4. Transstimulation of flux from either external or internal compartment only occurred when a positive gradient of total anions was maintained in the opposite compartment. Also, no stimulation occurred when the same equivalence of two different anions are present in opposing compartments. The concentration of anions required to transstimulate [3H]methotrexate influx was increased four- to 10-fold when vesicles were equilibrated in 145 mM NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the Proton-Coupled Folate Transporter (PCFT-SLC46A1) by Bicarbonate and Other Anions

“…The inhibitory effects of nitrate and nitrite were even smaller (∼15%) but still significant (P , 0.01). These results are consistent with prior observations on the inhibitory effect of anions on RFC-mediated transport (Henderson and Zevely, 1983;Yang et al, 1984). However, no significant difference was observed between bisulfite and sulfate or between nitrate and nitrite, in contrast to what was observed for PCFT-mediated 5-CHO-THF influx.…”

“…This is quite opposite to the high K i for folic acid and very low K i for PT523 for RFC-mediated transport (Rosowsky et al, 1998;Zhao et al, 2011a). Unlike PCFT, which is a proton-coupled process that operates optimally at low pH (Qiu et al, 2006;Nakai et al, 2007;Umapathy et al, 2007;Inoue et al, 2008), RFC is an anion exchanger that operates most efficiently at pH 7.4 and is inhibited by inorganic and organic anions (Henderson and Zevely, 1983;Yang et al, 1984;Zhao et al, 2001). Although some organic anions are weak inhibitors of PCFTmediated transport, with inhibitor constants in the submillimolar to millimolar range (Inoue et al, 2008), little is known about the impact of inorganic anions on PCFTmediated transport, particularly anions that have physiologic relevance.…”

Resistance to antifolates

“…This carrier system appears to generate small transmembrane chemical gradients through an exchange with organic phosphates, in particular the adenine nucleotides that are synthesized and accumulate to high levels within cells. This ability of structurally unrelated anions to exchange with folates and augment concentrative folate and antifolate transport was confirmed by studies with membrane vesicles (Yang et al, 1984). Recently, thiamine mono-and pyrophosphate were shown to utilize RFC in murine leukemia cells (Zhao et al, 2000d(Zhao et al, , 2002.…”

Neural Tube and Craniofacial Defects With Special Emphasis On Folate Pathway Genes

“…These gradients exist to drive the uphill transport of folate into tissues under physiological conditions which would otherwise be unfavorable for folate internalization (Schron et al, 1985). The reduced folate carrier allows folylmonoglutamates to move in both directions across the membrane (Kamen et al, 1991).The RFC shows a sensitivity to structurally unrelated inorganic and organic anions in the sense that transport inhibition can be induced by the addition of anions at concentrations varying from the typical physiological concentration (Goldman, 1971;Henderson and Zevely, 1984;Yang et al, 1984). The RFC has a Km for transport of 5-10 i M for natural and reduced folates (Sirotnak, 1985; Westerhof et al, 1995b), and a Vmax of 1-30 pmol/min per 106 cells.…”

“…The RFC shows a sensitivity to structurally unrelated inorganic and organic anions in the sense that transport inhibition can be induced by the addition of anions at concentrations varying from the typical physiological concentration (Goldman, 1971;Henderson and Zevely, 1984;Yang et al, 1984). The RFC has a Km for transport of 5-10 i M for natural and reduced folates (Sirotnak, 1985; Westerhof et al, 1995b), and a Vmax of 1-30 pmol/min per 106 cells.…”