Cholesterol stimulates the cellular uptake of L-carnitine by the carnitine/organic cation transporter novel 2 (OCTN2)

The carnitine/organic cation transporter novel 2 (OCTN2) is responsible for the cellular uptake of carnitine in most tissues. Being a transmembrane protein OCTN2 must interact with surrounding lipid microenvironment to function. Among main species that constitute eukaryotic cells, cholesterol has highly dynamic levels under number physiopathological conditions. This work describes how plasma membrane modulates transport L-carnitine human embryonic kidney 293 cells overexpressing (OCTN2-HEK293) and proteoliposomes harboring OCTN2. We manipulated content intact assessed by thin layer chromatography, through short exposures empty and/or cholesterol-saturated methyl-?-cyclodextrin (m?cd), whereas free was used enrich reconstituted proteoliposomes. measured using [3H]L-carnitine, expression localization surface biotinylation Western blotting. A 20-min preincubation m?cd reduced inhibited influx 50% comparison controls. Analogously, insertion OCTN2-proteoliposomes stimulated dose-dependent manner. Carnitine incubated preserve comparable controls, suggesting effect on dependent. Cholesterol markedly increasing affinity significantly enhancing Na+ and, turn, maximal capacity. Because antilipogenic antioxidant features L-carnitine, stimulatory might represent protective against lipid-induced toxicity oxidative stress. vitamin-like compound cardinal translocation mid- long-chain fatty acids from cytosol into mitochondrial matrix, where acid ?-oxidation takes place (1Longo N. Frigeni M. Pasquali oxidation.Biochim. Biophys. Acta. 2016; 1863: 2422-2435Crossref PubMed Scopus (250) Google Scholar, 2Vaz F.M. Wanders R.J. biosynthesis mammals.Biochem. J. 2002; 361: 417-429Crossref (491) Scholar). An important experimental model comprehension role cell metabolism been still juvenile visceral steatosis (jvs) mouse. 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Surface labeling coupled blotting employed assess upon m?cd. 2A, m?cd-treated heterogeneous, concentrating cholesterol-rich sphingomyelin-rich (membrane rafts). Several studies have spatial reorganization because shattering cholesterol-enriched (39Zidovetzki Levitan Use cyclodextrins manipulate content: evidence, misconceptions strategies.Biochim. 1768: 1311-1324Crossref (712) addition, rat astrocytes demonstrated localizes primarily it caveolin-1, enriched rafts (40Czeredys Samluk Michalec Tulodziecka Skowronek Nalecz K.A. Caveolin-1--a partner (Octn2): C this astrocytes.PLoS 8e82105Crossref (16) order examine whether localized what extent affected m?cd, raft (insoluble) nonraft (soluble) fractions 2B shows that, depletion, there significant disruption fraction, redistribution CAVEOLIN-1 FLOTILLIN-1 insoluble (rafts) soluble fraction (nonrafts). 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Measurement intracellular slices microelectrodes.Biochim. 903: 56-67Crossref (14) monitoring ions membrane. 3B equilibrium, ouabain, inhibits causing partial depolarization (p 0.018). Conversely, slightly, albeit significantly, hyperpolarized reflected lower ion 0.049). dependent, “cholesterol-matched” experiments performed. varying ratios m?cd:cholesterol-saturated (RAMEB) mixtures showed 4A). 3:1 m?cd:RAMEB ratio after (3:1) (0.009 NS), dependent 4B). study limited overloading 4A) deleterious more extensive likely jeopardize viability. Hence, better characterize dependence content, egg yolk phospholipid-liposomes. 5A, illustrates Na+, demonstrates functional. obtained incorporation exogenous such appeared dose 5B). Finally, potassium diffusion valinomycin 5C). Taken together, data secondary pump. understand cholesterol-dependent stimulation transport, kinetics mM. Km (19.9 7.79 76.9 26.5 ?M, slight, significant, capacity (0.63 0.13 0.92 0.12 protein/min, 0.04) 6A). Although kinetic values Hanes–Woolf transformation 6B) those calculated hyperbola derived Michaelis–Menten equation, noteworthy latter, overestimated saturation reaction could reached. Kinetic parameters summarized Table 1. augmentation concentrations Na+. 6C regression lines nearly intersected x-axis, (3.60 0.26 5.13 0.70 mM, NS). analysis Vmax manner no changes toward L-carnitine. 25% (25 ?g cholesterol/mg lipids) higher than (6.15 1.34 2.47 0.3 0.0003) 7, A–B, 1). change titrating buffer. cholesterol-loaded ?4 times stronger 0.03), loading 7C).Table 1Kinetic OCTN2AnalysisTreatmentKmp-valueVmaxp-valueIntact Michaelis–MentenControl19.9 7.790.020.63 0.130.042.5 m?cd76.9 26.50.92 Hanes–WoolfControl17.3 4.240.0030.62 0.14NS2.5 m?cd53.3 8.310.78 0.23Proteoliposomes Michaelis–MentenControl115 38.3NS2.47 0.300.000325% Cholesterol108 48.36.15 1