Regulation of the high‐affinity choline transporter activity and trafficking by its association with cholesterol‐rich lipid rafts

Abstract

The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-b-cyclodextrin (MbC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MbC. Kinetic analysis of binding of [ 3 H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MbC decreased both the apparent binding affinity (K D ) and maximum number of binding sites (B max ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.

Disruption of Lipid Raft Function Increases Expression and Secretion of Monocyte Chemoattractant Protein-1 in 3T3-L1 Adipocytes

“…As expected, the 4 h MβCD treatment reduced membrane cholesterol to 50% of the Ctrl cells (Fig 6B). In contrast, filipin treatment did not affect membrane cholesterol (Fig 6B), which is consistent with its function to sequester membrane cholesterol without depleting it [36, 37]. Thus, MβCD treatment, which depletes cellular and membrane cholesterol, may increase MCP-1 expression and secretion and activate ERK and NF-κB by disrupting lipid raft integrity or function.…”

“…Depletion of membrane cholesterol may lead to the disruption of lipid rafts, which are the microdomains of plasma membrane enriched in cholesterol and sphingolipids, as well as caveolae, a subset of lipid raft domains consisting of structure protein caveolin [11, 35]. To determine if another cholesterol-binding reagent would also induced MCP-1 similarly to MβCD, we treated 3T3-L1 adipocytes with the polyene antibiotic filipin, which can also bind and sequester membrane cholesterol [36]. We performed a dose response experiment of filipin treatment.…”

Inhibitors of Acetylcholinesterase Derived from 7-Methoxytacrine and Their Effects on the Choline Transporter CHT1

“…Previous experiments on synaptosomes or cell cultures revealed that the disruption of lipid rafts using methyl-β-cyclodextrine treatment attenuated the HACU levels through a reduction in membrane-bound CHT1 and also through altered conformation. In addition, CHT1 from cholesterol-depleted synaptosomes appeared to be more vulnerable to Aβ actions [23,61]. Since perturbation of the lipid raft integrity are involved in AD pathogenesis [62], we suppose that the attenuated effects of some AChE inhibitors on CHT1 from cholesterol-depleted synaptic membranes (indicating, e.g., their stronger requirement to CHT1 conformation) should be advantageous with regard to AD therapy.…”

“…CHT1 is specifically localized on presynaptic cholinergic nerve terminals and is directly involved in the high-affinity choline uptake (HACU) transport, a key regulatory step in the synthesis of ACh. CHT1 transporters actively transport choline from the synapsis back to the presynaptic terminals, where it is used for the de novo synthesis of ACh [18,19,20,21,22,23,24]. In contrast to AChE, however, the 3D structure of the transmembrane CHT1 is not known in details yet.…”

“…Recent research indicates that CHT1 is localized predominantly within lipid rafts in neuronal membranes, where it colocalizes with the amyloid precursor protein (APP). It appears that APP regulates the cell surface levels of CHT1 via increased endocytosis and thus directly attenuates HACU activity [23,24]. The disruption of lipid rafts, e.g., by removing membrane cholesterol by methyl-β-cyclodextrine, thus enables in vitro investigations of CHT1 interactions under pathological conditions.…”