Effect of Cholesterol on the Organic Cation Transporter OCTN1

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International Journal of Molecular Sciences

Article Eﬀect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

Lorena Pochini, Gilda Pappacoda, Michele Galluccio , Francesco Pastore, Mariafrancesca Scalise and Cesare Indiveri *

Department of Biology, Ecology and Earth Sciences (DiBEST), Unit of Biochemistry and Molecular Biotechnology, University of Calabria, via P. Bucci 4c, 87036 Arcavacata di Rende, Italy; [email protected] (L.P.); [email protected] (G.P.); [email protected] (M.G.); [email protected] (F.P.); [email protected] (M.S.) * Correspondence: [email protected]; Tel.: +39-0984-492939

Received: 18 December 2019; Accepted: 3 February 2020; Published: 6 February 2020

Abstract: The eﬀect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MβCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MβCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct inﬂuence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.

Keywords: acetylcholine; cholesterol; membrane transport; OCTN1; proteoliposomes

1. Introduction OCTN1 belongs to a small sub-family of organic cation transporters that includes only three members, OCTN1, 2 and 3 [1,2]. However, the gene coding for OCTN3 was lost in humans [3,4], that possess only OCTN1 and OCTN2 [5]. Differently from OCTN2, which is a well-acknowledged carnitine transporter, human OCTN1 presents still uncertainties concerning the substrate specificity and, hence, the physiological role. It is expressed in several districts [6] including peritoneum, where the transporter should be involved in control of inflammatory processes that are critical in this district [7]. Transport function of OCTN1 was firstly characterized in intact cells transfected with OCTN1 cDNA [1,8–10], in OCTN1 cRNA injected Xenopus oocytes [9] or in membrane vesicles obtained by OCTN1 expressing cells [11]. It was defined as an organic cation transporter on the basis of specificity towards the prototype cation TEA, with a low affinity for carnitine. Then, studies performed using a metabolomic approach, highlighted transport competence for ergothioneine, a fungi

Int. J. Mol. Sci. 2020, 21, 1091; doi:10.3390/ijms21031091 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 1091 2 of 17 metabolite [12]. Even though the high affinity towards this molecule was documented, the physiological significance of this function still remains uncertain [13–15]. To further investigate the function of this transporter on the single protein, the human OCTN1 was over-expressed in E. coli and its transport activity was assayed in proteoliposomes harboring the recombinant transporter. The specificity for TEA was confirmed and other substrates were identified such as acetylcholine and choline for which a bi-directional transport mode was demonstrated [16]. The transport sensitivity to physiological ions and effectors, suggested that the acetylcholine transport could occur in vivo mainly as efflux from cells. This also correlated to the stimulation of efflux by internal ATP and K+ [8,17,18]. This function was acknowledged to be possibly involved in the non-neuronal cholinergic system whose fundamental role in cells emerged in the last decades [19,20]. In this frame, the described association of the OCTN1503F variant with the Crohn’s disease could be explained by the impaired efflux of acetylcholine exhibited by this variant [16]. Studies performed in primary mesothelial [7] and HeLa cell lines [21] supported the capacity of OCTN1 in mediating acetylcholine release in vivo, as well. This finding could contribute to explain some results obtained in knockout mouse, in wild type cells or in silenced cells that could not be explained by the sole impairment of ergothioneine transport [22–24]. Very recently, spermine was also suggested as a physiological substrate of OCTN1 [25]. The role of OCTN1 in drug disposition and drug interaction has been well assessed [26–34]. Recently, it has been shown that cholesterol regulates a number of SLC transporters [35–40]. Therefore, we have investigated whether OCTN1 activity could be influenced by the presence of cholesterol in the phospholipid bilayer. To this aim, we have used two approaches, one based on cholesterol sequestration from the native membranes the other based on cholesterol addition to the artificial phospholipid bilayer of proteoliposomes.

2. Results

2.1. Effect of Cholesterol Removal on the Native or Recombinant OCTN1 To evaluate the possible effect of cholesterol on the native human OCTN1, the protein extracted from HeLa cells was exploited. Indeed, it was previously assessed by us [21] and then also by another research group [41] that this cell line expresses a functional OCTN1 protein. OCTN1 was extracted by the non-ionic detergent Triton X-100 and reconstituted in proteoliposomes, as previously described [21]. To evaluate the possible effect of cholesterol on the transport function, the cell extract was incubated prior to reconstitution with MβCD which is widely used to sequester cholesterol from native membranes [35]. Then, MβCD-incubated or untreated HeLa extract, was reconstituted for transport assay in proteoliposomes as [14C]-TEA uptake. The same reconstitution procedure and transport assay was performed on cell extracts obtained from HeLa cells incubated with or without MβCD prior to solubilization (Figure1b). To evaluate the OCTN1 specific transport activity the concentration of the protein in the cell extract was determined by a method based on western blot normalization using as a standard the recombinant OCTN1 protein [21]. Figure1a shows that the MβCD treatment caused a decrease of the native OCTN1 activity of 35% with respect to the control, suggesting a role of cholesterol in the transport function. Similar results, with a decrease of 23% with respect to the control, were obtained by incubating cells with MβCD prior to solubilization (Figure1b). Each time course experiment was performed with a unique proteoliposome preparation to take the protein amount and cholesterol concentration constant within a time data series. Int. J. Mol. Sci. 2020, 21, 1091 3 of 17 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 17

FigureFigure 1. Effect 1. Effect of cholesterol of cholesterol removal removal on the on native the native OCTN1. OCTN1. (a) HeLa (a) HeLa cell protein cell protein extract extract or (b) or HeLa (b) HeLa cell samplecell sample was incubated was incubated in the in absence the absence ( ) or (○ in) theor in presence the presence of 10 mMof 10 M mMβCD M (βCD) and (●) then and thethen the • extractsextracts obtained obtained in the in two the di twofferent different conditions #conditions (a or b ()a and or b corresponding) and corresponding respectively respectively to 2.1 to0.04 2.1 ± 0.04 ± (a) and(a 2.2) and0.07 2.2 ±µ 0.07g protein µg protein (b) were (b) reconstitutedwere reconstituted in liposomes in liposomes as described as described in Section in Section 4.4. Transport 4.4. Transport ± was startedwas started adding adding 0.1 mM 0.1 [14 mMC]-TEA [14C]-TEA at time at zerotime to zero proteoliposomes to proteoliposomes and stopped and stopped at the at indicated the indicated timestimes as described as described in Section in Section 4.7. Time 4.7. Time course course data weredata were interpolated interpolated in a first in a order first order rate equation. rate equation. The valuesThe values are means are meansSD from± SD threefrom dithreefferent different experiments. experiments. (c) Immunodetected (c) Immunodetected OCTN1 OCTN1 from HeLafrom HeLa ± cells incubatedcells incubated in the in absence the absence (lane 1)(lane or in 1) the or in presence the presence (lane 2)(lane of M 2)β ofCD. M ActinβCD. Actin is shown is shown as a control as a control of totalof lysate total lysate loading. loading. OCTN1 OCTN1 or Actin or Actin was immunodetected was immunodetected by anti-OCTN1 by anti-OCTN1 or anti-Actin or anti-Actin antibody, antibody, respectively,respectively, as described as described in Section in Section 4.9. 4.9.

To obtainTo obtain more more reliable reliable information information on the on eff theect effe of cholesterolct of cholesterol on the on hOCTN1, the hOCTN1, the recombinant the recombinant proteinprotein was exploited,was exploited, whose whose transport transport features features overlap overla thosep those of of the the nativenative protein [21]. [21]. Before Before testing testingthe the effect effect of of addition ofof cholesterolcholesterol to to the the cholesterol-free cholesterol-free recombinant recombinant protein, protein, the cholesterolthe cholesterol concentrationconcentration in the phospholipidin the phospholipid preparations preparations used for reconstitution used for reconstitution was measured was by anmeasured enzymatic by an assayenzymatic (see Material assay and (see methods). Material and 10 µ methods).g/ mg total 10 lipidsµg/ mg corresponding total lipids corresponding to 1.0% cholesterol to 1.0% incholesterol the phospholipidin the phospholipid mixture [7, 21mixture] used [7,21] for reconstitution used for reconsti wastution calculated. was calculated. The effect The of incubation effect of incubation of the of lipidsthe with lipids Mβ withCD was MβCD firstly was assessed, firstly assessed, by adding by adding MβCD M toβ theCD proteoliposometo the proteoliposome preparation. preparation. After After this treatment,this treatment, the concentration the concentration of cholesterol of cholesterol was loweredwas lowered to 6.5 toµ g6.5/mg µg/mg total lipidstotal lipids corresponding corresponding to 0.65%to 0.65% cholesterol. cholesterol. After After removal removal of M ofβ CD,MβCD, transport transport was was measured measured as TEAas TEA uptake. uptake. As As shown shown by by FigureFigure2a the2a the M βMCDβCD treatment treatment caused caused a decrease a decrease of transport, of transp withort, with respect respect to the to control. the control. This This correlatedcorrelated well withwell with the presence the presence of cholesterol of cholesterol in the in phospholipid the phospholipid mixture mixture and withand with the data the ofdata of FigureFigure1 obtained 1 obtained with the with native the native protein. protein. To gain To information gain information on the on e fftheect effect of cholesterol of cholesterol removal removal on on the propertiesthe properties of the of transporter, the transporter, the kinetics the kinetics of transport of transport catalyzed catalyzed by the by recombinant the recombinant protein protein were were analyzedanalyzed studying studying the dependence the dependence of the of transport the transport rate on rate the on substrate the substrate concentration. concentration. Data plottedData plotted accordingaccording to Lineweaver–Burk to Lineweaver–Burk (Figure2 b)(Figure showed 2b) a transport showed impairment a transport at lowerimpairment TEA concentration, at lower TEA whileconcentration, at higher concentration while at higher the transport concentration rate increased. the transport This rate anomalous increased. behavior This anomalous resulted in behavior an increaseresulted of both in thean increase Vmax and of both the Km.the Vmax The catalytic and the eKm.fficiency The catalytic (Vmax/Km) effici decreasedency (Vmax/Km) from 25.3 decreased to 1 1 19.8 (ml min mg ) after the−1 treatment−1 with MβCD. from· 25.3− to· 19.8− (ml ⋅ min ⋅ mg ) after the treatment with MβCD.

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Figure 2. Effect of cholesterol removal from proteoliposomes on the recombinant OCTN1. The recombinant purified OCTN1 was reconstituted in liposomes as described in Section 4.5. Proteoliposomes were incubated in the absence ( ) or in the presence of 10 mM MβCD ( ). • (a) TransportFigure was2. Effect started of adding cholesterol 0.1 mM removal [14C]-TEA from at # timeproteoliposomes zero to proteoliposomes on the recombinant and stopped OCTN1. at the The indicatedrecombinant times as describedpurified inOCTN1 Section was4.7. Timereconstituted course data in wereliposomes interpolated as described in a first orderin Section rate 4.5. 14 equation.Proteoliposomes (b) The transport were rate incubated was measured in the absence adding [(○C]-TEA) or in the at the presence indicated of concentrations10 mM MβCD to(●). (a) proteoliposomes.Transport was The started transport adding reaction 0.1 wasmM stopped[14C]-TEA at 10at min,time i.e.,zero within to proteoliposomes the initial linear and range stopped of the at the time course.indicated Data times were as plotted described according in Section to Lineweaver–Burk 4.7. Time course as data reciprocal were transportinterpolated rate in vs a reciprocalfirst order rate TEA concentration. The values in both (a) and (b) are means SD14 from three different experiments. equation. (b) The transport rate was measured adding± [ C]-TEA at the indicated concentrations to proteoliposomes. The transport reaction was stopped at 10 min, i.e., within the initial linear range of 2.2. Dependence of CHS Addition on the Recombinant OCTN1 the time course. Data were plotted according to Lineweaver–Burk as reciprocal transport rate vs The dependencereciprocal TEA of theconcentratio transportn. The activity values on in cholesterol both (a) and concentration (b) are means was ± SD then from studied three different on the recombinantexperiments. OCTN1. To this aim, CHS, a widely used cholesterol analogue which is more suitable than free cholesterol for incorporating into membranes [35], was added to the phospholipid mixture. In these2.2. experiments, Dependence of besides CHS Additi TEA,on transport on the Recombinant of acetylcholine OCTN1. was also measured, that is a physiological substrate ofThe OCTN1 dependence [5,15,20 of,42 the]. Figure transport3a,b activity show the on time cholesterol courses ofconcentration TEA and acetylcholine was then studied uptake, on the respectively,recombinant in the OCTN1. presence To of this increasing aim, CHS, CHS a widely concentrations. used cholesterol As in Figureanalogue1, eachwhich time is more course suitable experimentthan free was cholesterol performed for with incorporating a unique proteoliposomeinto membranes preparation[35], was added to avoid to the variations phospholipid of both mixture. proteinIn andthese CHS experiments, amount within besides a data TEA, time series.transport The of transport acetylcholine of both was substrates also measured, increased upthat to is a µ aboutphysiological double the control substrate (without of OCTN1 added [5,15,20,42]. cholesterol) Figure at a CHS 3a,b concentration show the time of 8.3% courses (83 gof CHSTEA/ and mg totalacetylcholine lipids). This uptake, value respectively, does not include in the the presence 1% cholesterol of increasing initially CHS present concentrat in theions. phospholipid As in Figure 1, preparations.each time Initial course transport experiment rates was for TEAperformed and acetylcholine, with a unique calculated proteoliposome as the product preparation of k (theto avoid first ordervariations rate constant) of both protein and the and transport CHS amount at equilibrium within a data from time Figure series.3a,b, The are transport reported inof Figureboth substrates3c. The rateincreased of transport up tonearly about doubleddouble the at thecontrol highest (without cholesterol added concentration cholesterol) withat a CHS respect concentration to the control of 8.3% for either(83 µg the CHS/ substrates. mg total Inclusion lipids). This of more value cholesterol does not include into proteoliposomes the 1% cholesterol caused initially a decrease present of in the transportphospholipid (Figure3a, preparations. dotted line). Initial transport rates for TEA and acetylcholine, calculated as the product of k (the first order rate constant) and the transport at equilibrium from Figure 3a,b, are reported in Figure 3c. The rate of transport nearly doubled at the highest cholesterol concentration with respect to the control for either the substrates. Inclusion of more cholesterol into proteoliposomes caused a decrease of transport (Figure 3a, dotted line).

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FigureFigure 3. 3.EffectEffect of increasing of increasing cholesterol cholesterol (CHS) (CHS) concentr concentrationsations on OCTN1 on OCTN1 mediated mediated transport transport of TEA of and acetylcholine.TEA and acetylcholine. The recombinant The recombinantOCTN1 was reconstituted OCTN1 was in reconstituted liposomes as in described liposomes in as Section described 4.5. except in thatSection (a and 4.5 b.) except proteoliposomes that (a and bbatches) proteoliposomes used for each batches time used course for experiment each time course were experimentreconstituted were in the presencereconstituted of 0 (○ in), the2.1 presence(●), 4.2 (□ of), 08.3 ( (),■) 2.1 % (CHS), 4.2 (corresponding (), 8.3 () % CHS to 0, (corresponding 21, 42 and 83 µg to 0,CHS/mg 21, 42 and total 83 lipids,µg • 14 respectively).CHS/mg total (a) lipids, Transport respectively). was started# (a) adding Transport 0.1 wasmM started[14C]-TEA adding (b) or 0.1 0.1 mM mM [ [C]-TEA3H]-acetylcholine, (b) or 0.1 mMat time 3 zero[ H]-acetylcholine, to proteoliposomes at time and zero stopped to proteoliposomes at the indicated and times stopped as described at the indicated in Section times 4.7. as Time described course in data wereSection interpolated 4.7. Time in course a first order data wererate equation. interpolated The invalues a first are order means rate ± SD equation. from three The different values are experiments. means 14 3 SD from three different14 experiments. (c3) Transport rates of [ C]-TEA ( ) or [ H]-acetylcholine ( ) (c)± Transport rates of [ C]-TEA (○) or [ H]-acetylcholine (●) uptake, calculated as K x transport• at equilibriumuptake, calculated from (a) asand K x(b transport), reported at as equilibrium function of from the % (a ,CHS.b), reported as function# of the % CHS.

TheThe initial initial transport transport rate rate gives gives information information on on the the specific specific activity activity of theof the transporter, transporter, while while the the transporttransport at equilibriumat equilibrium can can be correlatedbe correlated to theto the number number or toor theto the size size of proteoliposomes,of proteoliposomes, i.e., i.e., the the fractionfraction of vesiclesof vesicles harboring harboring the the active active transporter transporte incorporatedr incorporated into into the the membrane membrane with with respect respect to to thethe total total liposome liposome vesicles. vesicles. To discriminateTo discriminate between between the the two two possibilities, possibilities, the the intraliposomal intraliposomal volume volume andand the the protein protein incorporated incorporated into into the the proteoliposomes proteoliposomes were were measured. measured. The The internal internal volumes volumes ranged ranged µ fromfrom 3.2 and3.2 and 3.4 3.4L/ mgµL/mg phospholipids phospholipids in the in variousthe variou sampless samples with with no significant no significant variations variations (Figure (Figure4, upper4, upper panel). panel). Therefore, Therefore, CHS CHS did notdid causenot cause variations variations in the in proteoliposomethe proteoliposome volume. volume. Surprisingly Surprisingly no changesno changes in incorporatedin incorporated purified purified OCTN1 OCTN1 protein protein detected detected by aby specific a specific antibody antibody were were found, found, too too (Figure(Figure4, lower 4, lower panel). panel).

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Figure 4. Effect of CHS on the internal volume of the proteoliposomes. The recombinant OCTN1 was Figure 4. Effect of CHS on the internal volume of the proteoliposomes. The recombinant OCTN1 was reconstitutedFigure 4. Effect in liposomesof CHS on asthe described internal volume in Section of th 4.5.e proteoliposomes. except that in proteoliposomes The recombinant batches OCTN1 used was reconstituted in liposomes as described in Section 4.5. except that in proteoliposomes batches used forreconstituted each time course in liposomes experiment as described were reconstituted in Section in4.5. the except presence that ofin 0proteoliposomes (○), 2.1 (●), 4.2 ( □batches), 8.3 (■ used) % for each time course experiment were reconstituted in the presence of 0 ( ), 2.1 ( ), 4.2 (), 8.3 ()% CHSfor each (corresponding time course toexperiment 0, 21, 42, wereand 83 reconstituted µg CHS/mg in total the presencelipids, respectively). of 0 (○•), 2.1 (Internal●), 4.2 ( □volume), 8.3 (■ )of % CHS (corresponding to 0, 21, 42, and 83 µg CHS/mg total lipids, respectively).# Internal volume of proteoliposomesCHS (corresponding was measuredto 0, 21, 42, as anddescribed 83 µg in CHS/mg Section total4.5. (upper lipids, panel).respectively). Immunodetected Internal volume OCTN1 of proteoliposomes was measured as described in Section 4.5. (upper panel). Immunodetected OCTN1 bandsproteoliposomes (anti-His antibody, was measured as described as described in Section in 4.Section9) from 4.5. the (upper different panel). proteoliposomes Immunodetected preparations OCTN1 bands (anti-His antibody, as described in Section 4.9) from the different proteoliposomes preparations (lowerbands panel).(anti-His The antibody, relative concentrationas described in of Section the immuno-stained 4.9) from the different proteins, proteoliposomes normalized to the preparations 0 % CHS (lower panel). The relative concentration of the immuno-stained proteins, normalized to the 0 % CHS (considered(lower panel). as 100%)The relative was 109 concentration ± 14% (2.1% of CHS); the immuno-stained 104 ± 5.8% (4.2% proteins, CHS); 106normalized ± 9.1% (8.3% to the CHS), 0 % CHS as (considered as 100%) was 109 14% (2.1% CHS); 104 5.8% (4.2% CHS); 106 9.1% (8.3% CHS), derived(considered from asthree 100%) differe was±nt 109 experiments ± 14% (2.1% (± S.D.).CHS); ± 104 ± 5.8% (4.2% CHS);± 106 ± 9.1% (8.3% CHS), as as derived from three different experiments ( S.D.). derived from three different experiments± (± S.D.). Thus, the increase in transport at equilibrium shown in Figure 3a,b could only be ascribed to an Thus, the increase in transport at equilibrium shown in Figure3a,b could only be ascribed to an increased fraction of active (appropriately folded) protein induced by the presence of CHS. This also increased fractionThus, the of activeincrease (appropriately in transport at folded) equilibrium protein show inducedn in Figure by the presence3a,b could of only CHS. be This ascribed also to an correlates with the increased transport rate associated with the addition of CHS to proteoliposomes. correlatesincreased with the fraction increased of active transport (appropriately rate associated folded) with protein the addition induced of by CHS the topresence proteoliposomes. of CHS. This also As a further proof of the relationships between cholesterol concentrations and protein activity, Ascorrelates a further with proof the of increased the relationships transport between rate associated cholesterol with concentrations the addition of and CHS protein to proteoliposomes. activity, proteoliposomes prepared with the optimal CHS concentration were then incubated with MβCD to proteoliposomesAs a further prepared proof with of the relationships optimal CHS between concentration cholesterol were concentrations then incubated and with protein MβCD activity, remove the CHS previously incorporated. A decrease in OCTN1 activity was detected in to removeproteoliposomes the CHS previously prepared with incorporated. the optimal A CHS decrease concentration in OCTN1 were activity then incubated was detected with M inβCD to proteoliposomes after MβCD treatment. Also, in this case, the effect was observed both on the initial proteoliposomesremove the after CHS M βpreviouslyCD treatment. incorporated. Also, in thisA case,decrease the einffect OCTN1 was observed activity bothwas ondetected the in transport rate and on the transport at equilibrium (Figure 5). initial transportproteoliposomes rate and after on theMβ transportCD treatment. at equilibrium Also, in this (Figure case,5 ).the effect was observed both on the initial transport rate and on the transport at equilibrium (Figure 5).

FigureFigure 5. Eff 5.ect Effect of removal of removal of CHS of CHS after after addition addition to proteoliposomes.to proteoliposomes The. The recombinant recombinant OCTN1 OCTN1 was was reconstitutedreconstitutedFigure 5. Effect in in liposomes ofliposomes removal as asof described CHSdescribed after in Sectionadditionin Section 4.5 to . 4.5.proteoliposomes except except that that in the in. The presencethe recombinant presence of 8.3% of OCTN1 CHS8.3% CHS was µ correspondingcorrespondingreconstituted to 83 toing CHS83liposomes µg/mg CHS/mg total as lipids. describedtotal lipids. Proteoliposomes in Proteoliposomes Section 4.5. were except incubated were that incubated in the absenceinpresence the absence ( of) or 8.3% in(○) orCHS in the presencethe presence of 10 mMof 10 M mMβCD M (β).CD Transport (●). Transport was started was started adding adding 0.1 mM 0.1 [14 mMC]-TEA [14C]-TEA at time at# zerotime to zero to corresponding to 83 µg CHS/mg• total lipids. Proteoliposomes were incubated in the absence (○) or in proteoliposomesproteoliposomesthe presence and of stopped 10and mM stopped at M theβCD indicatedat the(●). indicated Transport times astimes was described asstarted described in adding Section in Section 0.14.7 .mM Time 4.7. [14 courseTimeC]-TEA course data at time were data zero were to interpolated in a first order rate equation. The values are means SD from three different experiments. interpolatedproteoliposomes in a first and order stopped rate atequation. the indicated The values times areas described means± ± SD in fromSection three 4.7. different Time course experiments. data were To confirminterpolated the CHS in a removal first order after rate Mequation.βCD treatment, The values CHSare means concentration ± SD from three was different measured experiments. (see the To confirm the CHS removal after MβCD treatment, CHS concentration was measured (see the method in Section 4.6) and compared to that of the untreated sample. The initial concentration of CHS methodTo inconfirm Section the 4.6) CHS and removal compared after to M thatβCD of treatment, the untreated CHS sample.concentration The initial was measuredconcentration (see theof of 8.3% (83 µg CHS/mg total lipids) was reduced to 5% (50 µg CHS/mg total lipids) corresponding to a CHSmethod of 8.3% in Section (83 µg CHS/mg 4.6) and total compared lipids) was to thatreduced of the to 5%untreated (50 µg CHS/mg sample. total The lipids) initial corresponding concentration to of CHS of 8.3% (83 µg CHS/mg total lipids) was reduced to 5% (50 µg CHS/mg total lipids) corresponding to

Int.Int. J. Mol. J. Mol. Sci. Sci. 20192020, 20, 21, x, FOR 1091 PEER REVIEW 7 of7 of 17 17 a reduction of 40% of the initial CHS concentration. The decrease of CHS by MβCD was comparable toreduction the 31% decrease of 40% of in the transport initial CHS activity concentration. observed in The Figure decrease 5. of CHS by MβCD was comparable to the 31% decrease in transport activity observed in Figure5. 2.3. Effects of CHS on Regulation of OCTN1 by Physiological or Non-Physiological Effectors. 2.3. Effects of CHS on Regulation of OCTN1 by Physiological or Non-Physiological Effectors It was previously reported that OCTN1 is regulated by intraliposomal (intracellular) ATP [8,17]. Thus, Itthe was effect previously of ATP reportedwas tested that on OCTN1 the protei is regulatedn treated bywith intraliposomal the optimal (intracellular)CHS concentration. ATP [8 As,17 ]. shownThus, theby eFigureffect of 6a, ATP ATP was stimulated tested on the the protein transport treated when with measured the optimal either CHS as concentration. TEA or acetylcholine As shown uptake.by Figure The6 a,possible ATP stimulated influence of the cholesterol transport whenwas also measured tested on either the hOCTN1 as TEA or regulation acetylcholine by external uptake. + NaThe+ which possible inhibits influence the transport. of cholesterol The was dose-response also tested onof thethe hOCTN1external Na regulation+ effect on by [ external14C]-TEA Na or [which3H]- inhibits the transport. The dose-response of the external Na+ effect on [14C]-TEA or [3H]-acetylcholine acetylcholine uptake was studied (Figure 6b) and an IC50 value of 1.7 ± 0.5 or 2.2 ± 0.2 mM, respectivelyuptake was for studied TEA or (Figure acetylcholine6b) and anwas IC derived.50 value Thus, of 1.7 the0.5 OCTN1 or 2.2 added0.2 mM, with respectively CHS is regulated for TEA ± ± + byor ATP acetylcholine and Na+ in was a similar derived. fashion Thus, as the the OCTN1 transporter added reconstituted with CHS isunder regulated basic byconditions ATP and (no Na CHSin a added)similar [16,17]. fashion as the transporter reconstituted under basic conditions (no CHS added) [16,17].

Figure 6. Effect of CHS on regulation of OCTN1 by ATP or NaCl. The recombinant OCTN1 was Figure 6. Effect of CHS on regulation of OCTN1 by ATP or NaCl. The recombinant OCTN1 was reconstituted in liposomes as described in Section 4.5. except that in the presence of 8.3% CHS reconstituted in liposomes as described in Section 4.5. except that in the presence of 8.3% CHS corresponding to 83 µg CHS/mg total lipids and (a) in the presence of the indicated ATP concentrations. corresponding to 83 µg CHS/mg total lipids and (a) in the presence of the indicated ATP Transport was started adding 0.1 mM [14C]-TEA (white column, ) or [3H]-acetylcholine (grey column, concentrations. Transport was started adding 0.1 mM [14C]-TEA (white column, ○) or [3H]- ) at time zero to proteoliposomes. (b) Increasing NaCl extraliposomal# concentration were added acetylcholine (grey column, □) at time zero to proteoliposomes. (b) Increasing NaCl extraliposomal together to the radioactive substrates. The transport reaction was stopped at 20 min. Percent residual concentration were added together to the radioactive substrates. The transport reaction was stopped activity with respect to the control data were interpolated in an IC50 equation (Dose-response curves). atThe 20 valuesmin. Percent are means residualSD activity from three with experiments. respect to Significantlythe control data different were for interpolated * p < 0.01 as in calculated an IC50 ± equationfrom Student’s (Dose-response t test analysis. curves). The values are means ± SD from three experiments. Significantly different for * p < 0.01 as calculated from Student’s t test analysis.

Inhibition of OCTN1 by HgCl2 due to interaction of the metal with Cys residues was previously describedInhibition [43 ].of TheOCTN1 addition by HgCl of CHS2 due did to interaction not change of the the mercurial metal with inhibitory Cys residues eﬀect. was The previously measured describedIC value [43]. was The 0.27 addition0.05 mM of CHS or 0.2 did not0.03 change mM, respectively the mercurial in theinhibitory absence effect. or in theThe presence measured of IC CHS50 50 ± ± value(Supplementary was 0.27 ± 0.05 Figure mM S1), or indicating0.2 ± 0.03 thatmM, cholesterol respectively does in the not absence interfere or with in the accessibilitypresence of CHS of the (SupplementaryCys residues to Figure the Hg ++1),. indicating that cholesterol does not interfere with the accessibility of the Cys residuesThe eﬀ ectto the of theHg acetylcholine++. analogue hemicholinium or the TEA analogue TEBA was studied on theThe recombinant effect of the hOCTN1acetylcholine in the analogue absence hemich or in theolinium presence or the of CHS. TEA Toanalogue this aim, TEBA [3H]-acetylcholine was studied onor the [14 recombinantC]-TEA uptake, hOCTN1 respectively, in the absence was measured or in the presence in the presence of CHS. ofTo increasingthis aim, [3H]-acetylcholine concentrations of or [14C]-TEA uptake, respectively, was measured in the presence of increasing concentrations of the the analogues (Figure7a,b). The dose-response analysis revealed an IC 50 for hemicholinium of analogues0.53 0.046 (Figure mM or7a,b). 0.30 The0.052 dose-response mM, and an analysis IC for revealed TEBA of an 1.6 IC500.3 for mM hemicholinium or 1.9 0.7 mM, of 0.53 respectively ± 0.046 ± ± 50 ± ± in the absence or in the presence of CHS.

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 of 17 mMInt. or J. Mol. 0.30 Sci. ± 20200.052, 21 mM,, 1091 and an IC50 for TEBA of 1.6 ± 0.3 mM or 1.9 ± 0.7 mM, respectively in 8the of 17 absence or in the presence of CHS.

Figure 7. Eﬀect of hemicholinium and TEBA on the OCTN1 activity measured in the presence or Figureabsence 7. Effect of CHS. of Thehemicholinium recombinant and OCTN1 TEBA was on reconstitutedthe OCTN1 activity in liposomes measured as described in the presence in Section or 4.5 . absenceexcept of that CHS in.the The absence recombinant ( ) or OCTN1 in the presence was reconstituted ( ) of 8.3% CHSin liposomes corresponding as described to 83 µ ing CHSSection/mg 4.5. total •3 exceptlipids. that (a )in Transport the absence was (○ started) #or in the adding presence 0.1 mM (●) [ofH]-acetylcholine 8.3% CHS corresponding at time zero to 83 to proteoliposomesµg CHS/mg total in lipids.the presence(a) Transport of increasing was started external adding hemicholinium 0.1 mM [3H]-acetylcholine concentrations at time (0-0.1-0.3-0.5-0.8-1-1.5-2.6 zero to proteoliposomes mM)in 14 theor presence (b) adding of increasing 0.1 mM extern [ C]-TEAal hemicholinium in the presence concentrations of increasing (0-0.1-0.3-0.5-0.8-1-1.5-2.6 external TEBA concentrations mM) or (b(0-0.5-1-2.5-5-10-20) adding 0.1 mM [14 mM).C]-TEA The in transportthe presence reaction of increasing was stopped extern atal 20 TEBA min. Percentconcentrations residual (0-0.5-1-2.5- activity with 5-10-20respect mM). to the The control transport data reaction were interpolated was stopped in anat 20 IC 50min.equation Percent (Dose-response residual activity curves). with respect The values to are means SD from three experiments. the control data± were interpolated in an IC50 equation (Dose-response curves). The values are means 2.4.± ESDﬀects from of three CHS experiments. on the Kinetics of OCTN1 Mediated Transport

2.4. EffectsKinetic of CHS analysis on the Kinetics was performed of OCTN1 varying Mediated TEA Transport. or acetylcholine concentration (Figure8a,b), in the absence or in the presence of CHS. Double reciprocal (Lineweaver–Burk) plot analysis Kinetic analysis was performed varying TEA or acetylcholine concentration (Figure 8a,b), in the showed that the presence of CHS doubled the Vmax of transport both in the case of TEA and absence or in the presence of CHS. Double reciprocal (Lineweaver–Burk) plot analysis showed that acetylcholine. The Vmax of TEA transport increased from 36.1 8.6, in absence of CHS, to or the presence of CHS doubled1 the Vmax1 of transport both in the case ±of TEA and acetylcholine. The 66.6 16.4 nmol min− mg protein− , in the presence of CHS. The Vmax of acetylcholine transport −1 Vmax ±of TEA transport· increased· from 36.1 ± 8.6, in absence of CHS, to1 or 66.6 ± 16.41 nmol ⋅ min ⋅ increased from 49.3 12.4, in absence of CHS to 119 19.8 nmol min− mg protein− , in the presence mg protein−1, in the presence± of CHS. The Vmax of acetylcholine± transport· · increased from 49.3 ± 12.4, of CHS. While, no signiﬁcant diﬀerences of Km were found. The Km for TEA was 0.9 0.29 or in absence of CHS to 119 ± 19.8 nmol ⋅ min−1 ⋅ mg protein−1, in the presence of CHS. While,± no 0.8 0.28 mM in the absence or presence of CHS, respectively. The Km for acetylcholine was 1.1 0.37 significant± differences of Km were found. The Km for TEA was 0.9 ± 0.29 or 0.8 ± 0.28 mM in± the or 1.25 0.27 mM in the absence or in the presence of CHS, respectively [35]. absence or± presence of CHS, respectively. The Km for acetylcholine was 1.1 ± 0.37 or 1.25 ± 0.27 mM in the absence or in the presence of CHS, respectively [35].

Int. J. Mol. Sci. 2020, 21, 1091 9 of 17 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 9 of 17

Figure 8. Effect of CHS on OCTN1 transport kinetics. The recombinant OCTN1 was reconstituted Figure 8. Effect of CHS on OCTN1 transport kinetics. The recombinant OCTN1 was reconstituted in in liposomes as described in Section 4.5. except that in the absence ( ) or in the presence ( ) of liposomes as described in Section 4.5. except that in the absence (○) or in the presence (●) of 8.3% CHS• 8.3% CHS corresponding to 83 µg CHS/mg total lipids. The transport# rate was measured adding corresponding to 83 µg CHS/mg total lipids. The transport rate was measured adding [14C]-TEA (a) [14C]-TEA (a) or [3H]-acetylcholine (b) at the indicated increasing concentrations to proteoliposomes. or [3H]-acetylcholine (b) at the indicated increasing concentrations to proteoliposomes. The transport The transport reaction was stopped at 10 min. Data were plotted according to Lineweaver–Burk as reaction was stopped at 10 min. Data were plotted according to Lineweaver–Burk as reciprocal reciprocal transport rate vs reciprocal TEA (a) or acetylcholine (b) concentration. The values are means transport rate vs reciprocal TEA (a) or acetylcholine (b) concentration. The values are means ± SD SD from three experiments. from± three experiments. 2.5. In Silico Analysis of the Interaction of OCTN1 with Cholesterol 2.5. In Silico Analysis of the Interaction of OCTN1 with Cholesterol To verify if cholesterol could interact with OCTN1, CARC and CRAC motifs were searched inTo the verify sequence. if cholesterol These sequencescould interact are with acknowledged OCTN1, CARC as cholesterol-binding and CRAC motifs motifswere searched in membrane in theproteins sequence. [44 ].These As shownsequences in Figure are 9acknowledgeda, several of these as cholesterol-binding motifs are present inmotifs the OCTN1 in membrane sequence proteinsand most [44]. ofAs these shown motifs in Figure overlap 9a, several to the predictedof these motifs transmembrane are present segments,in the OCTN1 highlighted sequence by and solid mostrectangles. of these Amotifs prediction overlap of the to cholesterol the predicte interactiond transmembrane sites based segments, on docking highlighted analysis was by performed solid rectangles.using the A OCTN1prediction homology of the cholesterol model previously interaction obtained sites based [5]. Many on docking cholesterol analysis poses was were performed predicted usingby the OCTN1 docking, homology with a wide model range previously of interaction obtained energies. [5]. Many However, cholesterol it is poses well knownwere predicted that in the by presencethe docking, of strong with a hydrophobic wide range interactions,of interaction as energies. in the case However, of cholesterol, it is well docking known procedures that in the are presencenot always of strong straightforward hydrophobic [ 45interactions,]. Therefore, as onlyin the those case posesof cholesterol, which fulfill docking both procedures higher scores are (lowernot alwaysbinding straightforward energy) and the[45]. vicinity Therefore, to some only CARC-CRAC those poses motifs which were fulfill shown both in higher the figures scores (Figure (lower9b,c). bindingThe cholesterol energy) and molecules the vicinity are to located some CARC-CRA at the interfaceC motifs between were someshown hydrophobic in the figures moieties (Figures of9b the andprotein c). The and cholesterol the membrane. molecules As are expected, located this at th lipide interface may mediate between the some interactions hydrophobic between moieties the protein of theand protein themembrane and the membrane. in agreement As withexpected, the stimulation this lipid ofmay the mediate transport the function. interactions between the protein and the membrane in agreement with the stimulation of the transport function.

Int.Int. J. Mol. J. Mol. Sci. Sci.2020 2019, 21,, 20 1091, x FOR PEER REVIEW 10 10of 17 of 17

Figure 9. Bioinformatic analysis. (a) Primary structure of the human OCTN1 transporter. Figure 9. Bioinformatic analysis. (a) Primary structure of the human OCTN1 transporter. The The transmembrane α-helices (TM1-12) are depicted as light gray rectangles. The region containing the transmembrane α-helices (TM1-12) are depicted as light gray rectangles. The region containing the amino acids from 50 to 133 (big extracellular loop, absent in the homology model) is depicted as dotted amino acids from 50 to 133 (big extracellular loop, absent in the homology model) is depicted as line. Hypothetical cholesterol-binding motifs CRAC and CARC are indicated as blue and orange boxes, dotted line. Hypothetical cholesterol-binding motifs CRAC and CARC are indicated as blue and respectively. (b,c) side and top view, respectively, of the homology structural model of the OCTN1 orange boxes, respectively. (b) and (c) side and top view, respectively, of the homology structural docked with three molecules of cholesterol (green). In this ribbon representation, only the best three model of the OCTN1 docked with three molecules of cholesterol (green). In this ribbon representation, (lowest binding energy) out of the twenty-two predicted by Achilles blind docking server, are reported. only the best three (lowest binding energy) out of the twenty-two predicted by Achilles blind docking 3. Discussionserver, are reported.

3. ToDiscussion describe the role of cholesterol on the function of hOCTN1 we used the proteoliposome experimental tool that is the most suitable for easily changing the lipid composition of the membrane. However,To cholesteroldescribe the is role diffi cultof cholesterol to insert into on the phospholipidfunction of hOCTN1 bilayer ofwe proteoliposomes. used the proteoliposome Thus, theexperimental widely used tool soluble that analogueis the most CHS suitable [35] for has easily been changing included the in the lipid proteoliposome composition of preparations the membrane. harboringHowever, the cholesterol cholesterol-free is difficult recombinant to insert hOCTN1 into the expressed phospholipid in E. colibilayer. Together of proteoliposomes. with this strategy, Thus, the the nativewidely transporter used soluble extracted analogue from HeLa CHS cells [35] was has exploited. been included Starting fromin the the proteoliposome hypothesis that thepreparations protein extractedharboring from the cells cholesterol-free harbors a native recombinant cholesterol hOCTN1 shell, the expressed cholesterol in E. sequestering coli. Together agent with M thisβCD strategy, was usedthe to native remove, transporter at least partially, extracted the from native HeLa membrane cells was cholesterol exploited. as Starting previously from described the hypothesis [35,36,38 that,40]. the Interestingly,protein extracted the removal from/reduction cells harbor of thes a cholesterolnative cholesterol shell from shell, the the native cholesterol protein or sequestering the addition agent of cholesterolMβCD was (CHS) used to theto cholesterol-freeremove, at least protein partially, gave oppositethe native eff ects,membrane i.e., impairment cholesterol or stimulationas previously of transport,described respectively.[35,36,38,40]. EvenInterestingly, though we the cannot removal/reduction exclude the presence of the cholesterol of some di shellfferences from between the native CHSprotein and cholesterol or the addition effects, of thesecholesterol findings (CHS) concur to the in demonstratingcholesterol-free that protein cholesterol gave opposite is required effects, for ai.e., properimpairment functioning or stimulation of the OCTN1 of transport, transporter respective and thatly. the Even lipid though has a we physiological cannot exclude role, asthe found presence for of othersome transporters differences [35 between,36,38,40 CHS,46–48 and]. Interestingly, cholesterol effect it cames, these evident findings that a smallconcur fraction in demonstrating of cholesterol, that cholesterol is required for a proper functioning of the OCTN1 transporter and that the lipid has a physiological role, as found for other transporters [35,36,38,40,46–48]. Interestingly, it came evident

Int. J. Mol. Sci. 2020, 21, 1091 11 of 17 measured by an enzymatic assay, is present in the lipid mixture used for proteoliposome assembly. This explains why a functionally active OCTN1 was found in proteoliposomes reconstituted with the recombinant (cholesterol-free) protein without adding CHS, as well (Figure2). This finding correlates with previous works describing the OCTN1 function in proteoliposomes [16,17]. However, the functional protein in absence of CHS accounts for less than 50% of the full activity, even though the Km and the main regulatory properties such as the ATP stimulation or the Na+ inhibition are virtually identical. Addition of further cholesterol, as CHS, increases the transport activity of OCTN1, about doubling the amount of functional protein under optimal condition of CHS/phospholipid mixture. Altogether the collected experimental results indicate that the protein is in its functional state in the presence of cholesterol. Thus, addition of CHS increases the number of active proteins. This correlates well with the kinetic analysis that shows an increase of the Vmax of transport while the Km is not influenced. It might be that cholesterol participates to the folding of the protein also mediating the correct positioning of some hydrophobic transmembrane segments of OCTN1 in the membrane, according to the acknowledged role of this lipid on membrane proteins [44]. Indeed, the computational analysis predicts the cholesterol poses in some locations that are at the boundary between the hydrophobic stretches of the protein and the membrane (Figure9b). The e ffect of cholesterol on OCTN1 correlates with previous findings on SLC38A9, SLC7A5 (LAT1), SLC1A5 (ASCT2) and SLC22A2 (OCT2) transporters [35,36,38,40]. Similarities as well as differences emerge from this work in the mechanism of action of cholesterol with respect to the other transporters. The increase of Vmax seems to be a common finding for the transporters characterized so far [35,38–40,49,50]. While, mostly invariant Km was found for OCTN1 as well as, ASCT2 and SLC38A9. Clearly different effects of cholesterol on the substrate affinity were observed in the case of OCT2 [40]. Indeed, OCT2 showed a strong variation of substrate affinity and changes in the allosteric binding of 1-methyl-4-phenylpyridinium (MPP+). While, OCTN1 shows no or very small changes in Km as well as in regulation by several effectors, including TEBA and hemicholinium that have been tested on OCTN1 for the first time in this work. The differences between OCTN1 and OCT2 may be unexpected since the two proteins belong to the same SLC22 family. However, such differences correlate with the relatively low identity of 32.3% existing between the sequences of the two proteins, (see Supplementary Figure S2). This also correlates with different locations of some CRAC-CARC motifs (see Supplementary Figure S2 in comparison with Figure9a), which should be the sites of interaction of cholesterol. Interestingly, a more extensive removal of cholesterol from proteoliposomes harboring the recombinant OCTN1, leads to a small variation of the Km (Figure2b) that can be observed also in the case of LAT1, even though in an opposite fashion: a Km increase (reduced affinity) for OCTN1, a Km decrease (increased affinity) for LAT1. However, in both cases the variations have a scarce significance, thus, this finding indicates a limited action of cholesterol on the conformation of the substrate binding sites of these transporters, differently from OCT2. Thus, except than for OCT2, it appears that cholesterol essentially increases the rate of the conformational changes underlying the transport path, in line with an action on improving the folding of the proteins. This might be in agreement with the predicted interaction of more than one cholesterol molecule with each protein and with the location of cholesterol molecules at the interface between transmembrane α-helices and the membrane, as suggested by the computational analysis for both LAT1 and OCTN1. The described results open new perspectives in the study of the role of cholesterol in cancer cells in which the OCTN1 transporter is expressed and may play important roles in inflammatory and/or immunomodulatory processes.

4. Materials and Methods

4.1. Materials Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcoline 60%), CHS, Triton-X100, Sephadex G-75, TEA and acetylcholine were purchased from Merck Life Science s.r.l., 20149 Milano, Italy; tetraethylammonium bromide [ethyl-1-14C] was from ARC (American Radiolabeled Chemicals, Int. J. Mol. Sci. 2020, 21, 1091 12 of 17

St. Louis, MO 63146 USA); acetylcholine iodide [acetyl-3H] was from Perkin-Elmer Italia S.p.A. 20126 Milano, MI, Italy. All the other reagents were of analytical grade.

4.2. CHS Solubilization CHS was solubilized in 20 mM Tris/HCl (pH 8.8) and 5% Triton X-100 by two sonication cycles (2 min, no pulse, 40 W) with a Vibracell VCX-130 soniﬁer (Sonics, 53 Church Hill Road, Newtown, CT 06470, USA). Solution was centrifuged for 5 min at 10.000 g. The supernatant was then used for the reconstitution procedure. The pH was adjusted to pH 7.5 before reconstitution.

4.3. Cell culture HeLa cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS, GIBCO, ThermoFisher Scientific, Milan, Italy), 1 mM glutamine, 2 1 mM sodium pyruvate and Pen/strep as antibiotics. Cells were grown on 10 cm plates at 37 ◦C in a humidified incubator and a 5% CO2 atmosphere.

4.4. Reconstitution of the OCTN1 Transporter from HeLa Cell Extract into Proteoliposomes HeLa cells pellet was treated with 150 µL TX-100 10%. After vortexing cells have been incubated 30 min on ice and then centrifuged 10 min 12.000 g at 4 C. The cell membrane extract (supernatant) × ◦ was used for the reconstitution [21]. 30 µL supernatant were included to the reconstitution mixture containing 120 µL 10% egg yolk phospholipids in the form of sonicated liposomes prepared as previously described [51], 80 µL 10% Triton X-100 solution, 16 mM ATP, 7 µL of 500 mM Tris/HCl (pH 7.5) in a ﬁnal volume of 700 µL.

4.5. Reconstitution of the Recombinant OCTN1 Transporter into Liposomes OCTN1 over-expressed in E. coli and purified as previously described [17] was used for reconstitution in liposomes. The composition of the initial mixture used for reconstitution (except when differently indicated) was 200 µL of the purified protein (5 µg protein in 0.05% DDM), CHS at the indicated concentrations solubilized in 240 µL of 20 mM Tris/HCl (pH 7.5) and 5% Triton X-100 or buffer and Triton X-100 alone in the case of samples without CHS, 120 µL of 10% egg yolk phospholipids in the form of sonicated liposomes prepared as previously described [51], 16 mM ATP and (where indicated) in a final volume of 700 µL. The purified OCTN1 inserted in liposomal membrane by removing the detergent from mixed micelles containing detergent, protein, and phospholipids with Amberlite XAD-4 in a batch-wise procedure. Where indicated, proteoliposome internal volume was measured using a colorimetric phosphate method [52]. In detail, 30 mM dipotassium phosphate (K2HPO4) was added to the reconstitution mixture. After elution from Sephadex G75 pre-equilibrated with 5 mM Tris/HCl (pH 7.5), 100 µL of sample were used for the colorimetric reaction. A solution containing 150 µL of 10% SDS was used to solubilize liposomal membranes. Then, 700 µL of a solution prepared with 10 mM hexammonium heptamolybdate 4-hydrate, 0.3 mM H2SO4 and 0.1 mM FeSO4 was added starting an incubation of 30 min in the dark. At the end, absorbance from each sample was measured at 578 nm. Internal volume in µL was derived from nmol of phosphate incorporated in liposomes.

4.6. MβCD Treatment

Each HeLa cell sample or HeLa cell extract was incubated with 10 mM MβCD for 90 min at 37 ◦C under stirring. Then, the respective extracts were reconstituted in proteoliposomes. Proteoliposomes reconstituted with the recombinant OCTN1 and added or not with CHS were incubated with MβCD as above described and then, MβCD was removed by chromatography on Sephadex G-75 column as described in Section 4.7. Int. J. Mol. Sci. 2020, 21, 1091 13 of 17

4.7. Transport Measurements Proteoliposomes (550 µL) were passed through a Sephadex G-75 column (0.7 cm diameter x 15 cm height) pre-equilibrated with 5 mM Tris/HCl (pH 7.5). The first 550 µL turbid eluate was collected and divided in aliquots (samples) of 100 µL each. Transport was started by adding 0.1 mM [14C]-TEA or 3 [ H]-acetylcholine to the proteoliposome samples and stopped by adding 0.5 mM HgCl2 at the desired time interval. In control samples, the inhibitor was added at time zero according to the inhibitor stop method [53]. Finally, each sample of proteoliposomes (100 µL) was passed through a Sephadex G-75 column (0.6 cm diameter 8 cm height) in order to separate the external from the internal radioactivity. × Liposomes were eluted with 1 mL of 50 mM NaCl and collected in 4ml of scintillation mixture, vortexed, and counted. The experimental values were corrected by subtracting the respective controls [16,17]. The uptake time course data were interpolated using a first order rate equation from which rate constants were derived as the product of k (the first order rate constant) and the transport at the equilibrium. Data from dose-response experiments were fitted into an IC50 equation. For kinetic experiments the initial rate of transport expressed as nmol min 1 mg protein 1, was measured by stopping the · − · − reaction after 10 min, i.e., within the initial linear range of [14C]-TEA or [3H]-acetylcholine uptake into the proteoliposomes. Kinetic constants were estimated from interpolation of the experimental data into a Lineweaver–Burk equation. The Grafit (version 5.0.3) software was used for calculations.

4.8. Quantitation of Cholesterol Quantitation was performed using the Cholesterol Quantitation Kit MAK043 by Merck Life Science s.r.l., 20149 Milan, Italy,). Assay was performed in the presence of cholesterol esterase with absorbance measured on a microplate reader at 570 nm.

4.9. Electrophoretic and Western Blotting Analysis Protein amount was measured by densitometry of SDS-PAGE stained by Coomassie Blue, using the ChemiDoc imaging system equipped with Quantity One software (Bio-Rad Laboratories) [54]. Immunoblotting analysis was performed using anti-His antibody 1:1000 Merck Life Science s.r.l., 20149 Milan, Italy,), Rabbit anti-OCTN1 antibody [21] or Mouse Monoclonal β actin Antibody 1: 2000 (Sigma) incubated 1h in 3% BSA under shaking at room temperature and then revealed by chemiluminescence assay (AmershamTM ECLTM Prime Western Blotting Detection Reagent, GE Healthcare s.r.l., Milan, Italy) in the case of anti-His or after incubation with the secondary antibody, anti-rabbit or anti-mouse IgG, 1:2000 respectively (Cell Signalling, EuroClone, Pero (Milan), Italy).

4.10. Docking The homology structural model of the hOCTN1 was built by Iterative Threading ASSEmbly Refinement, (I-TASSER) available at https://zhanglab.ccmb.med.umich.edu/I-TASSER/. The large hydrophilic loop between the transmembrane domain I and II (amino acids 42-141) was removed from the structure by Chimera 1.13.1 software [41]. The 3D structure of cholesterol was downloaded from PubChem Database (https://pubchem.ncbi.nlm.nih.gov/#) as .sdf file and saved as .pdb by Chimera. The ligand and the transporter protein were prepared using ADT [55]. Gasteiger charge was assigned and the files were saved in PDBQT format. Achilles blind docking server available at https://bio-hpc.ucam.edu/achilles/entry was used to predict possible cholesterol-binding sites. AutoDock Vina was used [56].

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/3/1091/ s1. Author Contributions: L.P., G.P. and F.P. were involved in protein purification, reconstitution procedure and transport assay. M.G. was involved in cloning, expression and modeling. M.S. was involved in human cell growth and sample preparation. L.P. and C.I. conceived, designated the experiments, contributed to interpretation of Data and wrote the manuscript; L.P., M.S. were involved in data curation, formal analysis and methodology Int. J. Mol. Sci. 2020, 21, 1091 14 of 17 investigation. C.I. coordinated and supervised the work and the manuscript writing. All authors have read and agreed to the published version of the manuscript. Funding: This work was in supported by PON (Programma Operativo Nazionale) Project No. 01_00937 granted by MIUR (Ministry of Education, University and Research) Italy. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

OCTN Novel Organic Cation Transporters SLC SoLute Carrier CHS Cholesteryl HemiSuccinate MβCD Methyl-beta-CycloDextrin DDM n-Dodecyl-β-D-maltoside TEA Tetraethylammonium TEBA Benzyltriethylammonium

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