Dual functions of NPC1L1 for cholesterol transport: Cholesterol capture and regulation of inner-leaflet cholesterol accessibility

Niemann-Pick C1-like 1 (NPC1L1) is essential for intestinal cholesterol absorption, but the detailed mechanisms underlying its non-vesicular cholesterol transport remain unclear. Using fluorescent cholesterol probes, we found that NPC1L1 decreased accessible cholesterol in the outer leaflet while increasing it in the inner leaflet in an ezetimibe-sensitive manner. This activity was independent of outer-leaflet sphingomyelin levels and was unaffected by a dysfunctional mutation in the N-terminal domain (NTD), which mediates extracellular cholesterol binding and insertion into the outer leaflet. In contrast, mutations in the sterol-sensing domain (SSD) abolished NPC1L1-dependent redistribution of accessible cholesterol between plasma membrane leaflets. These findings support a two-step model in which the NTD captures and inserts cholesterol into the outer leaflet, whereas the SSD promotes its redistribution toward the inner leaflet. Our study identifies a previously unrecognized step in NPC1L1-mediated non-vesicular cholesterol transport and provides mechanistic insight into SSD function.