The human uptake transporter NaCT is important for human brain development, brain function and energy metabolism and mediates the uptake of citrate and other intermediates of the tricarboxylic acid cycle from blood into neurons and hepatocytes. Mutations in the SLC13A5 gene encoding NaCT are associated with epileptic encephalopathy. To gain more insights into the transport mechanisms we analyzed the functional consequences of mutations in the SLC13A5 gene on NaCT-mediated transport function. Using HEK293 cells expressing wild-type and eight mutated NaCT proteins, we investigated the mRNA and protein amount as well as the protein localization of all NaCT variants. Furthermore, the impact on NaCT-mediated citrate uptake was measured. In addition, a structural model of the transport pore was generated to rationalize the consequences of the mutations on a structural basis. We demonstrated that all proteins were synthesized with an identical molecular weight as the wild-type transporter but several mutations (NaCTp.G219R, −p.G219E, −p.T227M, −p.L420P and −p.L488P) lead to a complete loss of NaCT-mediated citrate transport. This loss of transport activity can be explained on the basis of the developed structural model. This model may help in the further elucidation of the transport mechanism of this important uptake transporter.

Low plasma concentration of L-homoarginine is an independent predictor of cardiovascular events and total mortality. Experimental data indicate that supplementation of L-homoarginine may have protective effects. We aimed to elucidate the mechanisms involved in the cellular uptake of L-homoarginine, which are little understood, so far. Using human embryonic kidney (HEK293) cell lines stably overexpressing the human cationic amino acid transporters CAT1 [solute carrier family 7 (SLC7A1)], CAT2A (SLC7A2A) or CAT2B (SLC7A2B) we assessed the transport kinetics of L-homoarginine and interactions with the CAT substrates L-arginine and asymmetric dimethylarginine (ADMA). Significant uptake of L-homoarginine was observed for all three CATs with apparent KM-values of 175 ± 7 µM for CAT1 and 523 ± 35 µM for CAT2B. Saturation of CAT2A-mediated L-homoarginine uptake could not be reached. Uptake of L-homoarginine by any of the three CATs could be inhibited by L-arginine and ADMA. Significant inhibition of CAT1-mediated uptake of L-homoarginine by L-arginine already occurred in the physiological concentration range. Taken together these data demonstrate that L-homoarginine is a substrate of CAT1, CAT2A and CAT2B and that CAT1 is a key site with regard to physiological relevance and interactions with related substrates such as L-arginine.

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Current guidelines recommend as treatment option in patients on oral anticoagulation (OAC) undergoing percutaneous coronary intervention (PCI) an antiplatelet monotherapy with clopidogrel if there is an increased risk for bleeding. However, retrospective data suggested a potential interaction of clopidogrel and the vitamin K antagonist (VKA) phenprocoumon leading to a diminished antiplatelet effect. This would increase the ischemic risk of patients treated with this combination. Thus, this prospective study sought to evaluate the pharmacodynamic effect of clopidogrel monotherapy in patients on phenprocoumon undergoing PCI and assessed clinical outcomes. This study enrolled 100 patients on aspirin plus clopidogrel (DAPT-cohort, without indication for VKA) and 100 patients on clopidogrel monotherapy plus phenprocoumon (OAC-cohort) undergoing elective PCI. Platelet reactivity was assessed by impedance aggregometry on day 1 following PCI. Ischemic (death, stroke, or myocardial infarction) and bleeding (BARC 2-5) events within 12 months were compared in a propensity score adjusted model. Platelet reactivity was not different in the OAC- and DAPT-cohort (187 [127-242] vs. 167 [126-218] AU×min; p = 0.23). Overall, 17 ischemic and 34 bleeding events were recorded during follow-up. The OAC-cohort showed a nonsignificant trend to an 80% higher incidence for ischemic and bleeding events in unadjusted analyses, which disappeared following adjustment (ischemic events HR 1.07, 95%-CI 0.32-3.59, p = 0.91; bleeding events HR 1.25, 95%-CI 0.46-3.40, p = 0.67). Following PCI, the pharmacodynamic effect of a clopidogrel monotherapy together with phenprocoumon is similar as compared to DAPT without a VKA, and not associated with an increased risk for ischemic events beyond the higher underlying baseline risk.