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ABCC6 is a basolateral plasma membrane protein.

ABSTRACT: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein.In a recent article in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment.We performed immunofluorescent labeling of frozen mouse and human liver sections, as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and is not associated with the mitochondria, mitochondria-associated membrane, or the endoplasmic reticulum.Our findings support the model that ABCC6 is in the basolateral membrane, mediating the sinusoidal efflux of a metabolite from the hepatocytes to systemic circulation.

SUBMITTER: Pomozi V

PROVIDER: S-EPMC3727999 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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ABCC6 is a basolateral plasma membrane protein.

Pomozi Viola V Le Saux Olivier O Brampton Christopher C Apana Ailea A Iliás Attila A Szeri Flóra F Martin Ludovic L Monostory Katalin K Paku Sándor S Sarkadi Balázs B Szakács Gergely G Váradi András A

Circulation research 20130426 11

<h4>Rationale</h4>ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein.<h4>Objective</h4>In a recent article in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the su ...[more]

PMID: 23625951

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Project description:BackgroundPseudoxanthoma elasticum (PXE), a monogenic disorder resulting in calcification affecting the skin, eyes and peripheral arteries, is caused by mutations in the ABCC6 gene, and is associated with low plasma inorganic pyrophosphate (PPi). It is unknown how ABCC6 genotype affects plasma PPi.MethodsWe studied the association of ABCC6 genotype (192 patients with biallelic pathogenic ABCC6 mutations) and PPi levels, and its association with the severity of arterial and ophthalmological phenotypes. ABCC6 variants were classified as truncating or non-truncating, and three groups of the 192 patients were formed: those with truncating mutations on both chromosomes (n = 121), those with two non-truncating mutations (n = 10), and a group who had one truncating and one non-truncating ABCC6 mutation (n = 61). The hypothesis formulated before this study was that there was a negative association between PPi level and disease severity.ResultsOur findings confirm low PPi in PXE compared with healthy controls (0.53 ± 0.15 vs. 1.13 ± 0.29 µM, p < 0.01). The PPi of patients correlated with increasing age (β: 0.05 µM, 95% CI: 0.03-0.06 per 10 years) and was higher in females (0.55 ± 0.17 vs. 0.51 ± 0.13 µM in males, p = 0.03). However, no association between PPi and PXE phenotypes was found. When adjusted for age and sex, no association between PPi and ABCC6 genotype was found.ConclusionsOur data suggest that the relationship between ABCC6 mutations and reduced plasma PPi may not be as direct as previously thought. PPi levels varied widely, even in patients with the same ABCC6 mutations, further suggesting a lack of direct correlation between them, even though the ABCC6 protein-mediated pathway is responsible for ~60% of this metabolite in the circulation. We discuss potential factors that may perturb the expected associations between ABCC6 genotype and PPi and between PPi and disease severity. Our findings support the argument that predictions of pathogenicity made on the basis of mutations (or on the structure of the mutated protein) could be misleading.

Project description:Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

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ABCC6 is a basolateral plasma membrane protein.

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ABCC6 is a basolateral plasma membrane protein.

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