Preliminary observations of mitochondrial dysfunction in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a complex multisystem disorder because of errors in genomic imprinting with severe hypotonia, decreased muscle mass, poor suckling, feeding problems and failure to thrive during infancy, growth and other hormone deficiency, childhood-onset hyperphagia, and subsequent obesity. Decreased energy expenditure in PWS is thought to contribute to reduced muscle mass and physical activity but may also relate to cellular metabolism and disturbances in mitochondrial function. We established fibroblast cell lines from six children and adults with PWS and six healthy controls for mitochondrial assays. We used Agilent Seahorse XF extracellular flux technology to determine real-time measurements of several metabolic parameters including cellular substrate utilization, Adenosine Triphosphate (ATP)-linked respiration, and mitochondrial capacity in living cells. Decreased mitochondrial function was observed in the PWS patients compared to the healthy controls with significant differences in basal respiration, maximal respiratory capacity, and ATP-linked respiration. These results suggest disturbed mitochondrial bioenergetics in PWS although the low number of studied subjects will require a larger subject population before a general consensus can be reached to identify if mitochondrial dysfunction is a contributing factor in PWS.

Identification of a mammalian mitochondrial porphyrin transporter.

The movement of anionic porphyrins (for example, haem) across intracellular membranes is crucial to many biological processes, but their mitochondrial translocation and coordination with haem biosynthesis is not understood. Transport of porphyrins into isolated mitochondria is energy-dependent, as expected for the movement of anions into a negatively charged environment. ATP-binding cassette transporters actively facilitate the transmembrane movement of substances. We found that the mitochondrial ATP-binding cassette transporter ABCB6 is upregulated (messenger RNA and protein in human and mouse cells) by elevation of cellular porphyrins and postulated that ABCB6 has a function in porphyrin transport. We also predicted that ABCB6 is functionally linked to haem biosynthesis, because its mRNA is found in both human bone marrow and CD71+ early erythroid cells (by database searching), and because our results show that ABCB6 is highly expressed in human fetal liver, and Abcb6 in mouse embryonic liver. Here we demonstrate that ABCB6 is uniquely located in the outer mitochondrial membrane and is required for mitochondrial porphyrin uptake. After ABCB6 is upregulated in response to increased intracellular porphyrin, mitochondrial porphyrin uptake activates de novo porphyrin biosynthesis. This process is blocked when the Abcb6 gene is silenced. Our results challenge previous assumptions about the intracellular movement of porphyrins and the factors controlling haem biosynthesis.

Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.