Immuno-detection of OCTN1 (SLC22A4) in HeLa cells and characterization of transport function

Pochini, Lorena; Scalise, Mariafrancesca; Indiveri, Cesare

doi:10.1016/j.intimp.2015.04.040

Cited by 19 publications

(32 citation statements)

References 26 publications

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“…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][23][24].…”

Section: Introductionmentioning

confidence: 86%

“…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].…”

Section: Effect Of Cholesterol Removal On the Native Or Recombinant Omentioning

confidence: 96%

“…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].…”

Section: Effect Of Cholesterol Removal On the Native Or Recombinant Omentioning

confidence: 96%

“…The same reconstitution procedure and transport assay was performed on cell extracts obtained from HeLa cells incubated with or without MβCD prior to solubilization (Figure 1b). 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]. Figure 1a 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.…”

Section: Effect Of Cholesterol Removal On the Native Or Recombinant Omentioning

confidence: 99%

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

Pochini

Pappacoda

Galluccio

et al. 2020

IJMS

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The effect of cholesterol was investigated on the OCTN1 transport activity measured as [ 14 C]-tetraethylamonium or [ 3 H]-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 [ 14 C]-tetraethylamonium or [ 3 H]-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.

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Section: Introductionmentioning

confidence: 86%

Section: Effect Of Cholesterol Removal On the Native Or Recombinant Omentioning

confidence: 96%

Section: Effect Of Cholesterol Removal On the Native Or Recombinant Omentioning

confidence: 96%

Section: Effect Of Cholesterol Removal On the Native Or Recombinant Omentioning

confidence: 99%

See 2 more Smart Citations

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

Pochini

Pappacoda

Galluccio

et al. 2020

IJMS

Self Cite

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“…Interestingly, OCTN1 has been immune-detected in HeLa cells (23), and the relatively high uptake of cytarabine in CHO cells was previously connected with the number of NBMPR-binding sites (24). Reconstructed OCTN1 and ENT1 cDNAs (Origene) with a Flag-tag were subcloned into the pMIG II vector, engineered from a MSCV-IRES-GFP vector.…”

Section: Methodsmentioning

confidence: 99%

OCTN1 Is a High-Affinity Carrier of Nucleoside Analogues

et al. 2017

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Resistance to xenobiotic nucleosides used to treat acute myeloid leukemia (AML) and other cancers remains a major obstacle to clinical management. One process suggested to participate in resistance is reduced uptake into tumor cells via nucleoside transporters, although precise mechanisms are not understood. Through transcriptomic profiling, we determined that low expression of the ergothioneine transporter OCTN1 (SLC22A4; ETT) strongly predicts poor event-free survival and overall survival in multiple cohorts of AML patients receiving treatment with the cytidine nucleoside analog cytarabine. Cell biological studies confirmed OCTN1-mediated transport of cytarabine and various structurally-related cytidine analogs, such as 2′deoxycytidine and gemcitabine, occurs through a saturable process that is highly sensitive to inhibition by the classic nucleoside transporter inhibitors dipyridamole and nitrobenzylmercaptopurine ribonucleoside (NBMPR). Our findings have immediate clinical implications given the potential of the identified transport system to help refine strategies that could improve patient survival across multiple cancer types where nucleoside analogs are used in cancer treatment.

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The ergothioneine transporter (ETT): substrates and locations, an inventory

2022

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In all vertebrates including mammals, the ergothioneine transporter ETT (obsolete name OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). ETT is not expressed ubiquitously and only cells with high ETT cell‐surface levels can accumulate ET to high concentration. Without ETT, there is no uptake because the plasma membrane is essentially impermeable to this hydrophilic zwitterion. Here, we review the substrate specificity and localization of ETT, which is prominently expressed in neutrophils, monocytes/macrophages, and developing erythrocytes. Most sites of strong expression are conserved across species, but there are also major differences. In particular, we critically analyze the evidence for the expression of ETT in the brain as well as recent data suggesting that the transporter SLC22A15 may also transport ET. We conclude that, to date, ETT remains the only well‐defined biomarker for intracellular ET activity. In humans, the ability to take up, distribute, and retain ET depends principally on this transporter.

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Immuno-detection of OCTN1 (SLC22A4) in HeLa cells and characterization of transport function

Cited by 19 publications

References 26 publications

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

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

OCTN1 Is a High-Affinity Carrier of Nucleoside Analogues

The ergothioneine transporter (ETT): substrates and locations, an inventory

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