2D model of human NPC1L1. The membrane topology of human NPC1L1 was predicted with HMMTOP and TMHMM servers available through http://expasy.org/tools/#ptm and manually refined. Predicted loops are labeled throughout the protein sequence (A–N). The pentahelical SSD is highlighted in purple. Key residues for [³H]AS binding (Phe/Tyr-532 and Met/Ile-543) and sequence variants found exclusively in low (red circle) or high (light blue circle) absorbers of cholesterol as identified in the Dallas Heart study (17) are shown. The hotspot of hypoabsorption polymorphisms adjacent to key residues determining [³H]AS binding are highlighted (red triangle).

Binding of [³H]AS to TsA-201 cells expressing dog or mouse NPC1L1. (A) Saturation studies. TsA-201 cells were transiently transfected with dog NPC1L1 and incubated with increasing concentrations of [³H]AS for 4 h at 37°C as indicated in Experimental Procedures. Nonspecific binding is determined in the presence of 100 μM EZE-gluc. Specific binding, defined as the difference between total and nonspecific binding, is presented. Specific binding for dog NPC1L1 (red circle) is a saturable function of [³H]AS concentration and displays a K_d of 1.37 nM (Dog NPC1L1 B_max = 11455 cpm). (Inset) TsA-201 cells were transiently transfected with mouse NPC1L1 and [³H]AS binding was determined as described in A. Total (black circle), specific (red circle), and nonspecific (blue circle) binding are presented. (B) Specific binding data from A are presented relative to the maximum receptor occupancy. (C) Pharmacology of [³H]AS binding to mouse NPC1L1. Cells were incubated with 150 nM [³H]AS in the presence or absence of increasing concentrations of PS (●) for 4 h at 37°C. Inhibition of binding was assessed relative to an untreated control. Specific binding was fit to a single-site inhibition model, yielding a K_i value of (●) 51 nM (PS).

Two residues in loop C are critical for high-affinity [³H]AS binding to NPC1L1. (A) Sequence alignment of a 61-aa subdivision across multiple species of NPC1L1. The amino acid sequence of NPC1L1 (residues 510–571) corresponding to high-affinity (dog, rat, and human) or low-affinity (mouse) [³H]AS binding is shown. Nonconserved positions for which there is a correlation between ligand binding affinity and amino acid identity are highlighted in red, whereas those nonconserved positions where there is no correlation between ligand-binding affinity and amino acid identity are highlighted in blue. (B) Gain of [³H]AS-binding NPC1L1 mutants. TsA-201 cells were transfected with dog NPC1L1 (red circle) or mouse NPC1L1 with the mutations Tyr-532→Phe (green square), Ile-543→Met (yellow inverted triangle), or Tyr-532→Phe/Ile-543→Met (magenta triangle) and incubated with increasing concentrations of [³H]AS, as indicated in Experimental Procedures. Specific binding data were fit to a single class of [³H]AS-binding sites and are presented relative to the maximum receptor occupancy; dog NPC1L1 [(red circle) K_d 0.81 nM, B_max 3,878 cpm], Tyr-532→Phe [(green square) K_d 6.6 nM, B_max 7,873 cpm], Ile-543→Met [(yellow inverted triangle) K_d 5.6 nM, B_max 11,313 cpm], and Tyr-532→Phe/Ile-543→Met [(magenta triangle) K_d 2.29 nM, B_max 12,701 cpm]. (C) Loss of [³H]AS-binding NPC1L1 mutants. TsA-201 cells were seeded and transfected with dog NPC1L1 (red circle) or dog NPC1L1 with the mutations Phe-532→Tyr (dark blue square), Met-543→Ile (cyan inverted triangle), or Phe-532→Tyr/Met-543→Ile (black triangle) and incubated with increasing concentrations of [³H]AS, as indicated in Experimental Procedures. Specific binding data were fit to a single class of [³H]AS-binding sites and are presented relative to the maximum receptor occupancy; dog NPC1L1 [(red circle) K_d 1.23 nM, B_max 11,445 cpm], Phe-532→Tyr [(dark blue square) K_d 2.69 nM, B_max 8,101 cpm], Met-543→Ile [(cyan inverted triangle) K_d 1.38 nM, B_max 8,258 cpm] and Phe-532→Tyr/Met-543→Ile [(black triangle) K_d 12.4 nM, B_max 7,940 cpm].

Binding of [³H]AS to dog/mouse NPC1L1 chimeras. (A) Schematic of dog/mouse chimeras. Dog (orange) and mouse (blue) NPC1L1 sequences are shown, with their predicted TMDs indicated in dark color. SSD, between amino acids 629 and 806 is indicated by an arrow. (B) [³H]AS binding to dog/mouse chimeras 1–3. Saturation studies. TsA-201 cells transfected with dog NPC1L1 (●) and chimeras 1 (▼), 2 (♦), or 3 (■) were incubated with increasing concentrations of [³H]AS, as indicated in Experimental Procedures. Specific binding data were fit to a single class of [³H]AS-binding sites and are presented relative to the maximum receptor occupancy; dog NPC1L1 [(●) K_d 0.91 nM, B_max 9,873 cpm], 1 [(▼) K_d 102 nM, B_max 464 cpm], 2 [(♦) K_d 1.66 nM, B_max 15250 cpm] and chimera 3 [(■) K_d 3.54 nM, B_max 8,277 cpm]. (C) [³H]AS binding to dog/mouse chimeras 4–6. Saturation studies. TsA-201 cells transfected with dog NPC1L1 (●) and chimeras 4 (■), 5 (▼) or 6 (●) were incubated with increasing concentrations of [³H]AS, as indicated in Experimental Procedures. Specific binding was fit to a single class of [³H]AS-binding sites and is presented relative to the maximum receptor occupancy; dog NPC1L1 [(●) K_d 0.91 nM, B_max 9,873 cpm], 4 [(■) K_d 1.91 nM, B_max 9,802 cpm], 5 [(▼) K_d 0.81 nM, B_max 13,014 cpm] and 6 [(●) K_d 1.01 nM, B_max 15,443 cpm]. (D) [³H]AS binding to dog/mouse loop C exchange chimeras 7–8. Saturation studies. TsA-201 cells were transfected with dog NPC1L1 (●) and loop C exchange chimeras 7 (▲) or 8 (■) and incubated with increasing concentrations of [³H]AS, as indicated in Experimental Procedures. Specific binding data were fit to a single class of [³H]AS-binding sites and are presented relative to the maximum receptor occupancy; dog NPC1L1 [(●) K_d 1.23 nM, B_max 8,597 cpm], 7 [(▲) K_d 3.38 nM, B_max 43,683 cpm] and 8 [(■) K_d 63 nM, B_max 2081 cpm]. (E) [³H]AS binding to dog/mouse loop C exchange chimeras 9–11. Saturation studies. TsA-201 cells were transfected with dog NPC1L1 (●) and chimeras 9 (▼), 10 (■), or 11 (▲) and incubated with increasing concentrations of [³H]AS, as indicated in Experimental Procedures. Specific binding data were fit to a single class of [³H]AS-binding sites and are presented relative to the maximum receptor occupancy; dog NPC1L1 [(●) K_d 0.43 nM, B_max 6,976 cpm], 9 [(▼) K_d 46.8 nM, B_max 2,321 cpm], 10 [(■) K_d 71 nM, B_max 5,759 cpm] and 11 [(▲) K_d 1.38 nM, B_max 11,297 cpm].

[³H]AS binds directly to dog NPC1L1-K_v1.1. (A) 2D model of dog NPC1L1-K_v1.1. The membrane topology of dog NPC1L1 was predicted with HMMTOP and TMHMM servers available through http://expasy.org/tools/#ptm and manually refined. The pentahelical SSD is highlighted in purple. (B and C) Dog NPC1L1-K_v1.1 is functional. Dog NPC1L1-K_v1.1/MDCKII-Flp (B) or dog NPC1L1/MDCKII-Flp (C) cells were seeded on 96-well plates and incubated with increasing concentrations of [³H]AS for 4 h at 37°C. Specific binding was fit to a single-site saturation model yielding K_d/B_max values of 1.15 nM/3370 cpm for Dog NPC1L1-K_v1.1/MDCKII-Flp cells (●) and 1.28 nM/6436 cpm for dog NPC1L1/MDCKII-Flp cells (▲). (Inset) [³H]cholesterol flux into dog NPC1L1-K_v1.1/MDCKII-Flp cells (●) and dog NPC1L1/MDCKII-Flp cells (▲) was performed as described in the Experimental Procedures in the presence of increasing concentrations of PS. [³H]Ch flux was fit to a single-site inhibition model, yielding IC₅₀ values of 0.21 (B) and 0.24 nM (C) for dog NPC1L1-K_v1.1/MDCKII-Flp cells (●) and dog NPC1L1/MDCKII-Flp cells (▲), respectively. (D) Strategy for affinity purification of dog NPC1L1-K_v1.1. (E and F) Immunoprecipitation of dog NPC1L1-K_v1.1 from membranes and cells. Membranes from dog NPC1L1-K_v1.1/MDCKII-Flp (E) or dog NPC1L1/MDCKII-Flp (F) cells were incubated with 20 nM [³H]AS overnight and solubilized with 1% digitonin/0.03% sodium taurocholate for 30 min at 4°C, as described in Experimental Procedures. Solubilization of 35% or 26% of membrane bound [³H]AS activity from either dog NPC1L1-K_v1.1 or dog NPC1L1 membranes, respectively, was obtained. Solubilized [³H]AS activity (S) was incubated with protein A Sepharose beads coated with an anti-K_v1.1 antibody for 3 h at 4°C. Unbound [³H]AS activity (U) was collected, and the beads were washed three times before determination of [³H]AS bound (P). [³H]AS in U and P has been corrected to account for the dissociation of bound [³H]AS (t_1/2 ≈6 h) during the time of immunoprecipitation. [³H]AS recovered in P of dog NPC1L1/MDCKII-Flp is identical to that obtained in the absence of anti-K_v1.1 antibody. Dog NPC1L1-K_v1.1/MDCKII-Flp (E Inset) or dog NPC1L1/MDCKII-Flp (F Inset) cells were incubated with 20 nM [³H]AS overnight. Free [³H]AS was removed from cells by aspiration as described in Experimental Procedures, and the [³H]AS activity bound to cells was solubilized with 1% digitonin/0.03% sodium taurocholate for 30 min at 4°C. The solubilized [³H]AS activity (S) was immunoprecipitated as indicated in D. Data in E and F are representative of 10 and 8 independent experiments from membranes and cells, respectively. (G) Characterization of affinity-purified dog NPC1L1-K_v1.1. The solubilized (S), unbound (U), and purified (P) material of the immunoprecipitation from dog NPC1L1-K_v1.1/MDCKII-Flp and dog NPC1L1/MDCKII-Flp membranes was resolved by SDS/PAGE, transferred onto a PVDF membrane, and analyzed with an anti-K_v1.1 antibody. Two proteins of M_r 125 and 165 kDa are specifically recognized by the anti-K_v1.1 antibody in the three NPC1L1-K_v1.1 but not NPC1L1 samples. (H) Purified material (P) from G was resolved by SDS/PAGE and visualized by silver staining. Two bands at 125 and 165 kDa (indicated by red arrows) are present in the material purified from dog NPC1L1 K_v1.1 but not dog NPC1L1. (I) Quantification histogram displaying the relative specificities scored by relative peptide queries (rPQ) of affinity-purified proteins identified by LC-MS/MS sequencing of the gel lanes in H. * indicates proteins for which MS/MS spectra are assigned from dog NPC1L1 K_v1.1 but not dog NPC1L1-material. Values were 12 queries for NPC1L1 (gi 148223061) and 2 queries for Trx-related protein (gi 73963782).