Functional characterisation of human SGLT-5 as a novel kidney-specific sodium-dependent sugar transporter

Sodium glucose cotransporters (SGLT) actively catalyse carbohydrate transport across cellular membranes. Six of the 12 known SGLT family members have the capacity to bind and/or transport monosaccharides (SGLT-1 to 6); of these, all but SGLT-5 have been characterised. Here we demonstrate that human SGLT-5 is exclusively expressed in the kidney. Four splice variants were detected and the most abundant SGLT-5-mRNA was functionally characterised. SGLT-5 mediates sodium-dependent [(14)C]-α-methyl-D-glucose (AMG) transport that can be inhibited by mannose, fructose, glucose, and galactose. Uptake studies using demonstrated high capacity transport for mannose and fructose and, to a lesser extent, glucose, AMG, and galactose. SGLT-5 mediated mannose, fructose and AMG transport was weakly (μM potency) inhibited by SGLT-2 inhibitors. In summary, we have characterised SGLT-5 as a kidney mannose transporter. Further studies are warranted to explore the physiological role of SGLT-5.     

Dephosphorylation of beta2-syntrophin and Ca2+/mu-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca2+-dependent proteolysis of ICA512. In vitro mu-calpain cleaves ICA512 between a putative PEST domain and the beta2-syntrophin binding site, whereas binding of ICA512 to beta2-syntrophin protects the former from cleavage. beta2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-beta2-syntrophin and stimulation of insulin secretion induces the Ca2+-dependent, okadaic acid-sensitive dephosphorylation of beta2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from beta2-syntrophin-utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by mu-calpain.     

Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins

Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.     

Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.