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  • br Materials and methods br Results In this study

    2021-09-29


    Materials and methods
    Results In this study we re-examined the kinetics of ET transport and tested the ability of various suggested substrates of OCNT1 to inhibit ET uptake. Using a native ET transporter expressed in the human HeLa cell line ET was found to be taken up with a Km of 51 μM and Vmax of 118 pmol/min per million cells (Fig. 1B). HeLa cells were found to accumulate ET faster than HEK293 cells in line with the fact that HEK293 cells express little OCNT1 [20] (Fig. 1C). Even low concentrations (300 nM) led to considerable intracellular content of ET (Fig. 1A). Using a HeLa volume estimate of 1540μm3 an intracellular concentration of 23 μM can be calculated. This is much greater than the exterior. As the rate of efflux was only 67% of the uptake rate despite the internal concentration being much higher than the extracellular concentration, it suggests that the transport of ET is highly selective in the inward direction over that of efflux. We next examined the ability of compounds claimed by various groups to be substrates for OCTN1 to inhibit ET uptake (Fig. 2A,B,C). Strong inhibition of ET transport was only seen with pyrilamine, quinidine and verapamil. Carnitine demonstrated an inhibition coefficient of 33, the only other compound shown to compete at a less than 100-fold lower effectiveness. No other compounds tested demonstrated any significant inhibition of ET transport out to 10x concentration over ET.
    Discussion Following the discovery that OCTN1 transports ET [7] numerous substrates and alternate physiological roles have been suggested for OCTN1. Earlier studies have examined this [13,26] however this work was performed using overexpression systems, which may not always recapitulate all the aspects required for successful assay of substrates that compete with ET, e.g. transporters could be expressed in the wrong conformation or sometimes inserted in the wrong orientation in the plasma membrane or certain intracellular regulatory factors could be missing. In general our data support the conclusions of Grundemann et al. [7], Grigat et al. [21] and Tschirka et al. [13] in suggesting that OCNT1 is primarily an ET transporter.
    Summary In recent years numerous groups have claimed that OCTN1 functions to transport a wide array of compounds. Our study shows that it seems unlikely that OCTN1 plays any role in the non-neuronal ha tag peptide system, neither is it likely that OCTN1 plays a role in the transport of metformin or any significant role in the transport of carnitine or acetylcarnitine. In agreement with Urban et al. [15] gabapentin was found to enhance ET uptake, possibly in an electroneutral exchange process and could conceivably enhance ET retention in the body. ET transport is not notably interfered with compounds of very similar chemical structure, (S-methyl ergothioneine and hercynine) suggesting high selectivity for ET. The only substrates found to inhibit the transport of ET were 3 highly promiscuous transport inhibitors whose actions have been previously demonstrated by Yabuuchi et al. [6] and Grigat et al. [21].
    Conflicts of interest
    Acknowledgments We wish to acknowledge Tetrehedron, (Paris, France) for kindly supplying ET, S-methyl ergothioneine, hercynine and deuterated standards. We are grateful to the National Medical Research Council Singapore (Individual Research Grant NMRC/1264/2010/082/12) and Tan Chin Tuan Foundation for research support.
    Introduction Carbohydrates (or sugars) are widespread in ha tag peptide nature and are utilized by all domains of life (Toukach and Egorova, 2015). It has multiple roles such as osmoprotection, intracellular signaling and source of carbon and energy in different organisms (Iturriaga et al., 2009). In order to perform these various metabolic activities, cells uptake sugars from extracellular environment with the aid of three major classes of transporters viz. (1) primary active transporters, (2) secondary transporters and (3) group translocators, exclusive in bacteria (Saier, 2000a). The primary active transporters include the largest superfamily of transporters known as the ATP-binding cassette (ABC) transporters (Transporter Classification ID or TCID: 3.A.1), which utilizes the energy produced by ATP hydrolysis as the driving force for nutrient transport (Saurin et al., 1999; Saier, 2000b). ABC transporters are classified into exporters and importers, depending upon the direction of the substrate transport. Architecturally, ABC exporters comprise of a transmembrane domain (TMD) for substrate translocation and a nucleotide-binding domain (NBD) for the ATP hydrolysis process, whereas ABC importers contain an additional substrate (or solute)-binding protein (SBP) for substrate recognition. Among these subunits, SBPs confer substrate affinity, specificity and directionality of ABC transporters (Maqbool et al., 2015). The primary function of SBPs is to sequester substrates from an extracellular environment and subsequently deliver them to TMDs for their transport (Davidson et al., 2008). ABC transporters employ 45 families for the uptake of different nutrients, of which, carbohydrate uptake transporters 1 and 2 (referred to as CUT1 and CUT2) are known to be specific to carbohydrate transport (Saier, 2000a). Members of the CUT1 family are specific to oligosaccharides, polyols, glycerol and glycerol-phosphate transport while those of the CUT2 family transport only monosaccharides (Schneider, 2001).