Fig. 8.

Illustration of the transporters that impact upon the proton gradient and PCFT-mediated net folate/antifolate levels in cells. In R1-11-PCFT-h cells, RFC is absent whereas PCFT is overexpressed. At the steady state, influx of MTX is mediated solely by PCFT; efflux is mediated largely by PCFT owing to its extremely high level of expression. While multidrug-resistant proteins pump folates out of cells, their activity should be decreased relative to PCFT under these experimental conditions. In HEPES-based buffer, an inward proton gradient is the basis for the MTX or pemetrexed gradient mediated by PCFT at pH 7.4. Na⁺/K⁺-ATPase provides the sodium gradient that drives the export of H⁺ in exchange for Na⁺ via Na⁺/H⁺ antiporter(s), resulting in the intracellular alkalization. Either inhibition of Na⁺/K⁺-ATPase by ouabain or suppression of Na⁺/H⁺ antiporters by EIPA diminishes H⁺ export, decreasing the inward proton gradient at neutral pH. In the presence of monensin, which functions as Na⁺/H⁺ exchanger, the activity of the Na⁺/H⁺ antiporters is obviated. FCCP is a proton ionophore that eliminates the proton gradient.