Elsevier

Vitamins & Hormones

Volume 66, 2003, Pages 403-456
Vitamins & Hormones

Membrane Transport of Folates

https://doi.org/10.1016/S0083-6729(03)01012-4Get rights and content

Abstract

The chapter reviews the current understanding of the transport mechanisms for folates in mammalian cells—their molecular identities and organization, tissue expression, regulation, structures, and their kinetic and thermodynamic properties. This encompasses a variety of diverse processes. Best characterized is the reduced folate carrier, a member of the SLC19 family of facilitative carriers. But other facilitative organic anion carriers (SLC21), largely expressed in epithelial tissues, transport folates as well. In addition to these bi-directional carrier systems are the membrane-localized folate receptors alpha and beta, that mediate folate uptake unidirectionally into cells via an endocytotic process. There are also several transporters, typified by the family of multidrug resistance-associated proteins, that unidirectionally export folates from cells. There are transport activities for folates, that function optimally at low pH, related in part to the reduced folate carrier, with at least one activity that is independent of this carrier. The reduced folate carrier-associated low-pH route mediates intestinal folate transport. This review considers how these different transport processes contribute to the generation of transmembrane folate gradients and to vectorial flows of folates across epithelia. The role of folate transporters in mouse development, as assessed by homologous deletion of folate receptors and the reduced folate carrier, is described. Much of the focus is on antifolate cancer chemotherapeutic agents that are often model surrogates for natural folates in transport studies. In particular, antifolate transport mediated by the reduced folate carrier is a major determinant of the activity of, and resistance to, these agents. Finally, many of the key in vitro findings on the properties of antifolate transporters are now beginning to be extended to patient specimens, thus setting the stage for understanding response to these drugs in the clinical setting at the molecular level.© 2003, Elsevier Science (USA).

Introduction

The membrane transport of folate compounds in mammalian cells is mediated by a variety of diverse processes. Best studied is the reduced folate carrier (RFC), a member of the major facilitator superfamily (Saier et al., 1999). Recently, other facilitative carriers largely expressed in epithelial tissues have been shown to be folate transporters. Complementing transport fluxes mediated by these bidirectional systems are those involving the membrane-localized folate receptors (FRs), including FRα and FRβ, that mediate folate uptake into cells via an endocytotic process, and assorted transporters typified by the family of multidrug resistance-associated proteins (MRPs) (Borst and Elferink, 2002) that are unidirectional exporters. Further, there are transport activities for folates that function optimally at low pH and play a major role in intestinal transport. Although this activity is related, in part, to RFC, there are folate transport processes optimal at low pH that are RFC independent. This chapter summarizes the current understanding of these various transport routes, their molecular identities, their regulation, and their kinetic and thermodynamic properties.

Section snippets

Reduced Folate Carrier (RFC), a Member of the SLC19 Family of Transporters

This SLC superfamily of transporters (Saier et al., 1999) is composed of more than 36 families of facilitative carriers in mammalian cells that are distinct from the ATP-binding cassette (ABC) unidirectional exporters (Dean et al., 2001). Folates are transported by a member of the SLC19 family, termed RFC or SLC19A1, first cloned in 1994 (Dixon et al., 1994). More recently, SLC19A2 (Diaz 1999, Dutta 1999, Fleming 1999, Labay 1999) and SLC19A3 (Rajgopal et al., 2001a) were cloned. These

Transport of Folates by SLC21 Organic Anion Carriers

In the early 1990s, members of a new family of facilitative carriers (SLC21) began to be cloned that transport organic anions in epithelial tissues. Many of these carriers transport folates (Russel et al., 2002). The rat OAT- K1, expressed in liver and kidney, transports MTX with a higher affinity than for a variety of other organic anions including other folates, bromosulfopthalein (BSP), taurocholate, and probenecid. The MTX influx Kt for this Na+-independent system is ∼1 μM (Saito et al.,

Folate Transporters that Operate Optimally at Low pH: The Mechanism of Folate Transport in Intestinal Cells

Although RFC-mediated transport in murine leukemia cells has a pH optimum of ∼7.5 (Sierra 1997, Sirotnak 1968), this is not the case for folate transport in many other tissues that express RFC. The basis for this discrepancy is not clear. This issue is of special relevance to the mechanism of absorption of folates in the small intestine. RFC is highly expressed in intestine (Chiao et al., 1997) and is localized to the apical brush border of large and small intestinal cells of the mouse (Wang et

Structures and Specificities of FRsα,β, andγ

FR designates a family of high-affinity folate-binding proteins encoded by three distinct genes designated α, β, and γ (Brigle 1992, Elwood 1989, Lacey 1989, Ratnam 1989, Sadasivan 1989, Shen 1994), localized to chromosome 11q13.3–q13.5 (Ragoussis et al., 1992). The cDNA for FRα was originally isolated from human KB cells, placenta, and CaCo-2 cells (Elwood 1989, Lacey 1989, Sadasivan 1989), whereas those for FRβ and γ were obtained from human placenta (Ratnam et al., 1989) and malignant

Multidrug Resistance-Associated Proteins (MRPs) and their Impact on the Transport of Folates

At the same time that the characteristics of RFC-mediated transport began to emerge, data were also appearing indicating that there were energy-dependent processes that limit MTX accumulation in cells. This was based upon the observation that energy inhibitors augmented net uptake of MTX and aminopterin, an effect due almost entirely to inhibition of MTX efflux (Goldman 1969, Hakala 1965). It soon became apparent that these observations were related to inhibition of exporters distinct from RFC

Transport of Folates by Other ABC Exporters

There is now evidence emerging that there are other exporters of the ABC gene family that transport MTX and, likely, folates, as well. Of potential importance is the ABC half-transporter, breast cancer resistance protein (BCRP) (Rocchi et al., 2000). Over expression of this gene has been identified in tumor cell lines selected for resistance to the antracycline, mitoxantrone, that are also cross-resistant to MTX due to decreased net accumulation of MTX (Volk 2002, Volk 2000). Arg-482 was

Factors that Influence Concentrative Folate Transport in Cells

Concentrative transport of folates is determined by the net effect of complementary and opposing transporters that determine the free monoglutamyl folate level in cells (Fig. 4). Several studies have evaluated the impact of alterations in expression of RFC on concentrative transport. When RFC was transfected into murine leukemia cells that express both RFC and energy-dependent exporter activity, influx of MTX was markedly enhanced, export was also increased but only by a factor approximately

The Localization of Folate Transporters in Cells and Their Roles in Vectorial Transport in Epithelia

The relative localization of transporters in apical versus basolateral membranes is an important determinant of the vectorial flows of substrates across epithelia (Cui et al., 2001). At least one of the transporters involved in this process must provide the driving force for this flow. Some information is emerging on the localization of folate transporters in epithelial cells, and this can now be integrated with information on the localization of other transporters in these tissues.

FRα is

The Role of Folate Transporters in Mouse Development

Both FRα and RFC are required for mouse development. For the former, homozygous deletion of the gene was embryonic lethal at the stage of neural tube formation (Piedrahita et al., 1999). Deletion of FRβ or the deletion of one FRα allele resulted in normal development. Likewise, although deletion of one RFC allele produced no phenotype, targeting of both alleles was embryonic lethal at a very early stage of development (Zhao et al., 2001). Some of the latter animals could, nonetheless, be

Acknowledgements

This work was supported by Grants CA53535, CA76641, and CA82621 from the National Cancer Institute, National Institutes of Health.

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