Jocelyn M. Baldwin

Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants

OBJECTIVE Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS ZnT8−/− mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8−/− islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn2+ transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

The human concentrative and equilibrative nucleoside transporter families, SLC28 and SLC29.

Nucleoside transport in humans is mediated by members of two unrelated protein families, the SLC28 family of cation-linked concentrative nucleoside transporters (CNTs) and the SLC29 family of energy-independent, equilibrative nucleoside transporters (ENTs). These families contain three and four members, respectively, which differ both in the stoichiometry of cation coupling and in permeant selectivity. Together, they play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis. Moreover, they facilitate cellular uptake of several nucleoside and nucleobase drugs used in cancer chemotherapy and treatment of viral infections. Thus, the transporter content of target cells can represent a key determinant of the response to treatment. In addition, by regulating the concentration of adenosine available to cell surface receptors, nucleoside transporters modulate many physiological processes ranging from neurotransmission to cardiovascular activity. This review describes the molecular and functional properties of the two transporter families, with a particular focus on their physiological roles in humans and relevance to disease treatment.

Single nucleotide polymorphisms that were identified in affective mood disorders affect ATP-activated P2X7 receptor functions.

Genetic linkage studies have previously identified many single non-synonymous nucleotide polymorphisms (SNPs) in the human P2RX7 gene in individuals with affective mood disorders. The P2RX7 gene encodes the P2X(7) receptor (P2X(7)R) that operates as an ATP-activated Ca(2+)-permeable cationic channel and induces formation of a large pore, the two functional properties that are critical for the physiological and pathological roles of the receptor. The current knowledge regarding the effects of SNPs on the P2X(7)R functional properties, which is indispensable to help elucidate the disease mechanism, is limited. In this study, we introduced by site-directed mutagenesis twelve SNP mutations in the human P2X(7) receptor that were previously identified in or associated with affective mood disorders, expressed the resultant mutants in human embryonic kidney cells, and characterized their functional properties by electrophysiology. All mutations except Q460R gave rise to profound effects on the P2X(7)R function. G150R, E186K and I568N conferred complete loss of function. V76A, R117W, L191P, T357S and E496A resulted in strong impairment of, whereas H155Y and A348T caused significant increase in, both ATP-activated ion channel function and pore formation. Q521H reduced the receptors sensitivity to extracellular Ca(2+) inhibition. An atomic structure model of the human P2X(7)R, based on the crystal structure of the zebrafish P2X(4) receptor, suggests that the SNP mutational effects may result from changes in subunit interaction, agonist binding and/or channel gating. These results provide essential knowledge for a better understanding of the relationships between human P2RX7 SNPs and associated pathologies as well as the receptor structure-function relationships.

A Rare Mutation in ABCC8/SUR1 Leading to Altered ATP-Sensitive K+ Channel Activity and β-Cell Glucose Sensing Is Associated With Type 2 Diabetes in Adults

OBJECTIVE— ATP-sensitive K+ channels (KATP channels) link glucose metabolism to the electrical activity of the pancreatic β-cell to regulate insulin secretion. Mutations in either the Kir6.2 or sulfonylurea receptor (SUR) 1 subunit of the channel have previously been shown to cause neonatal diabetes. We describe here an activating mutation in the ABCC8 gene, encoding SUR1, that is associated with the development of type 2 diabetes only in adults. RESEARCH DESIGN AND METHODS— Recombinant KATP channel subunits were expressed using pIRES2-based vectors in human embryonic kidney (HEK) 293 or INS1(832/13) cells and the subcellular distribution of c-myc–tagged SUR1 channels analyzed by confocal microscopy. KATP channel activity was measured in inside-out patches and plasma membrane potential in perforated whole-cell patches. Cytoplasmic [Ca2+] was imaged using Fura-Red. RESULTS— A mutation in ABCC8/SUR1, leading to a Y356C substitution in the seventh membrane-spanning α-helix, was observed in a patient diagnosed with hyperglycemia at age 39 years and in two adult offspring with impaired insulin secretion. Single KATP channels incorporating SUR1-Y356C displayed lower sensitivity to MgATP (IC50 = 24 and 95 μmol/l for wild-type and mutant channels, respectively). Similar effects were observed in the absence of Mg2+, suggesting an allosteric effect via associated Kir6.2 subunits. Overexpression of SUR1-Y356C in INS1(832/13) cells impaired glucose-induced cell depolarization and increased in intracellular free Ca2+ concentration, albeit more weakly than neonatal diabetes–associated SUR1 mutants. CONCLUSIONS— An ABCC8/SUR1 mutation with relatively minor effects on KATP channel activity and β-cell glucose sensing causes diabetes in adulthood. These data suggest a close correlation between altered SUR1 properties and clinical phenotype.

Insights into the Molecular Mechanisms Underlying Mammalian P2X7 Receptor Functions and Contributions in Diseases, Revealed by Structural Modeling and Single Nucleotide Polymorphisms

The mammalian P2X7 receptors (P2X7Rs), a member of the ionotropic P2X receptor family with distinctive functional properties, play an important part in mediating extracellular ATP signaling in health and disease. A clear delineation of the molecular mechanisms underlying the key receptor properties, such as ATP-binding, ion permeation, and large pore formation of the mammalian P2X7Rs, is still lacking, but such knowledge is crucial for a better understanding of their physiological functions and contributions in diseases and for development of therapeutics. The recent breakthroughs in determining the atomic structures of the zebrafish P2X4.1R in the closed and ATP-bound open states have provided the long-awaited structural information. The human P2RX7 gene is abundant with non-synonymous single nucleotide polymorphisms (NS-SNPs), which generate a repertoire of human P2X7Rs with point mutations. Characterizations of the NS-SNPs identified in patients of various disease conditions and the resulting mutations have informed previously unknown molecular mechanisms determining the mammalian P2X7R functions and diseases. In this review, we will discuss the new insights into such mechanisms provided by structural modeling and recent functional and genetic linkage studies of NS-SNPs.

A rare mutation in ABCC8/SUR1 leading to altered KATP channel activity and β-cell glucose sensing is associated with type 2 diabetes mellitus in adults

OBJECTIVE— ATP-sensitive K+ channels (KATP channels) link glucose metabolism to the electrical activity of the pancreatic β-cell to regulate insulin secretion. Mutations in either the Kir6.2 or sulfonylurea receptor (SUR) 1 subunit of the channel have previously been shown to cause neonatal diabetes. We describe here an activating mutation in the ABCC8 gene, encoding SUR1, that is associated with the development of type 2 diabetes only in adults. RESEARCH DESIGN AND METHODS— Recombinant KATP channel subunits were expressed using pIRES2-based vectors in human embryonic kidney (HEK) 293 or INS1(832/13) cells and the subcellular distribution of c-myc–tagged SUR1 channels analyzed by confocal microscopy. KATP channel activity was measured in inside-out patches and plasma membrane potential in perforated whole-cell patches. Cytoplasmic [Ca2+] was imaged using Fura-Red. RESULTS— A mutation in ABCC8/SUR1, leading to a Y356C substitution in the seventh membrane-spanning α-helix, was observed in a patient diagnosed with hyperglycemia at age 39 years and in two adult offspring with impaired insulin secretion. Single KATP channels incorporating SUR1-Y356C displayed lower sensitivity to MgATP (IC50 = 24 and 95 μmol/l for wild-type and mutant channels, respectively). Similar effects were observed in the absence of Mg2+, suggesting an allosteric effect via associated Kir6.2 subunits. Overexpression of SUR1-Y356C in INS1(832/13) cells impaired glucose-induced cell depolarization and increased in intracellular free Ca2+ concentration, albeit more weakly than neonatal diabetes–associated SUR1 mutants. CONCLUSIONS— An ABCC8/SUR1 mutation with relatively minor effects on KATP channel activity and β-cell glucose sensing causes diabetes in adulthood. These data suggest a close correlation between altered SUR1 properties and clinical phenotype.

Residues 155 and 348 Contribute to the Determination of P2X7 Receptor Function via Distinct Mechanisms Revealed by Single-nucleotide Polymorphisms

P2X7 receptors are important in mediating the physiological functions of extracellular ATP, and altered receptor expression and function have a causative role in the disease pathogenesis. Here, we investigated the mechanisms determining the P2X7 receptor function by following two human single-nucleotide polymorphism (SNP) mutations that replace His-155 and Ala-348 in the human (h) P2X7 receptor with the corresponding residues, Tyr-155 and Thr-348, in the rat (r) P2X7 receptor. H155Y and A348T mutations in the hP2X7 receptor increased ATP-induced currents, whereas the reciprocal mutations, Y155H and T348A, in the rP2X7 receptor caused the opposite effects. Such a functional switch is a compelling indication that these residues are critical for P2X7 receptor function. Additional mutations of His-155 and Ala-348 in the hP2X7 receptor to residues with diverse side chains revealed a different dependence on the side chain properties, supporting the specificity of these two residues. Substitutions of the residues surrounding His-155 and Ala-348 in the hP2X7 receptor with the equivalent ones in the rP2X7 receptor also affected ATP-induced currents but were not fully reminiscent of the H155Y and A348T effects. Immunofluorescence imaging and biotin labeling assays showed that H155Y in the hP2X7 receptor increased and Y155H in the rP2X7 receptor decreased cell-surface expression. Such contrasting effects were not obvious with the reciprocal mutations of residue 348. Taken together, our results suggest that residues at positions 155 and 348 contribute to P2X7 receptor function via determining the surface expression and the single-channel function, respectively. Such interpretations are consistent with the locations of the residues in the structural model of the hP2X7 receptor.

The sodium-dependent D-glucose transport protein of Helicobacter pylori

Helicobacter pylori is a gram-negative pathogenic microaerophile with a particular tropism for the mucosal surface of the gastric epithelium. Despite its obligatory microaerophilic character, it can metabolize D-glucose and/or D-galactose in both oxidative and fermentative pathways via a Na(+)-dependent secondary active transport, a glucokinase and enzymes of the pentose phosphate pathway. We have assigned the Na(+)-dependent transport of glucose to the protein product of the H. pylori 1174 gene. The gene was heterologously expressed in a glucose transport-deficient Escherichia coli strain, where transport activities of radiolabelled D-glucose, D-galactose and 2-deoxy-D-glucose were restored, consistent with the expected specificity of the hexose uptake system in H. pylori. D-mannose was also identified as a substrate. The HP1174 transport protein was purified and reconstituted into proteoliposomes, where sodium dependence of sugar transport activity was demonstrated. Additionally the tryptophan/tyrosine fluorescence of the purified protein showed quenching by 2-deoxy-D-glucose, D-mannose, D-glucose or D-galactose in the presence of sodium ions. This is the first reported purification and characterization of an active glucose transport protein member of the TC 2.1.7 subgroup of the Major Facilitator Superfamily, constituting the route for entry of sugar nutrients into H. pylori. A model is derived of its three-dimensional structure as a paradigm of the family.

Pseudo half-molecules of the ABC transporter, COMATOSE, bind Pex19 and target to peroxisomes independently but are both required for activity

PEX19‐2 binds to CTS‐N by pull down (View Interaction)

Relative substrate affinities of wild-type and mutant forms of the Escherichia coli sugar transporter GalP determined by solid-state NMR

Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is used for the first time to examine the relative substrate-binding affinities of mutant forms of the Escherichia coli sugar transporter GalP in membrane preparations. The SSNMR method of 13C cross-polarization magic-angle spinning (CP-MAS) is applied to five site-specific mutants (W56F, W239F, R316W, T336Y and W434F), which have a range of different sugar-transport activities compared to the wild-type protein. It is shown that binding of the substrate D-glucose can be detected independently of sugar transport activity using SSNMR, and that the NMR peak intensities for uniformly 13C-labelled glucose are consistent with wild-type GalP and the mutants having different affinities for the substrate. The W239F and W434F mutants showed binding affinities similar to that of the wild-type protein, whereas the affinity of glucose-binding to the W56F mutant was reduced. The R316W mutant showed no detectable binding; this position corresponds to the second basic residue in the highly conserved (R/K)XGR(R/K) motif in the major facilitator superfamily of transport proteins and to a mutation in human GLUT1 found in individuals with GLUT1-deficiency syndrome. The T336Y mutant also showed no detectable binding; this mutation is likely to have perturbed helix structure or packing to an extent that conformational changes in the protein are hindered. The effects of the mutations on substrate-binding are discussed with reference to the putative positions of the residues in a 3D homology model of GalP based on the X-ray crystal structure of the E. coli glycerol-3-phosphate transporter GlpT.