Characterization and Subcellular Localization of l ‐[3H]Carnitine Binding Sites in Rat Brain

In the present study, we investigated the existence of a binding site for l‐carnitine in the rat brain. In crude synaptic membranes, l‐[3H]carnitine bound with relatively high affinity (KD = 281 nM) and in a saturable manner to a finite number (apparent Bmax value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a Koff of 0.018 min−1 and a Kon of 0.187 × 10−3 min−1 nM−1. Binding was highest in spinal cord, followed by medulla oblongata‐pons ≥ corpus striatum ≥ cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. l‐[3H]Carnitine binding was stereoselective for the l‐isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of l‐[3H]carnitine binding was l‐carnitine followed by propionyl‐l‐carnitine. Acetyl‐l‐carnitine and isobutyryl‐l‐carnitine showed an affinity ∼500‐fold lower than that obtained for l‐carnitine. The precursor γ‐butyrobetaine had negligible activity at 0.1 mM. l‐Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrine, 5‐hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mMl‐carnitine. Finally, a subcellular fractionation study showed that synaptic vesicles contained the highest density of l‐carnitine membrane binding sites whereas l‐carnitine palmitoyltransferase activity was undetectable, thus excluding the possibility of the presence of an active site for carnitine palmitoyltransferase. This finding indicated that the localization of the l‐[3H]carnitine binding site should be essentially presynaptic.