Susumu Seino

A FAMILY OF SULFONYLUREA RECEPTORS DETERMINES THE PHARMACOLOGICAL PROPERTIES OF ATP-SENSITIVE K+ CHANNELS

We have cloned an isoform of the sulfonylurea receptor (SUR), designated SUR2. Coexpression of SUR2 and the inward rectifier K+ channel subunit Kir6.2 in COS1 cells reconstitutes the properties of K(ATP) channels described in cardiac and skeletal muscle. The SUR2/Kir6.2 channel is less sensitive than the SUR/Kir6.2 channel (the pancreatic beta cell KATP channel) to both ATP and the sulfonylurea glibenclamide and is activated by the cardiac K(ATP) channel openers, cromakalim and pinacidil, but not by diazoxide. In addition, SUR2 binds glibenclamide with lower affinity. The present study shows that the ATP sensitivity and pharmacological properties of K(ATP) channels are determined by a family of structurally related but functionally distinct sulfonylurea receptors.

Subunit stoichiometry of the pancreatic β-cell ATP-sensitive K+ channel

We have investigated the subunit stoichiometry of the pancreatic β‐cell ATP‐sensitive K+ (KATP) channel (SUR1/Kir6.2 channel) by constructing cDNA encoding a single polypeptide (βα polypeptide) consisting of a SUR1 (β) subunit and a Kir6.2 (α) subunit. 86Rb+ efflux and single‐channel properties of COS1 cells expressing βα polypeptides were similar to those of COS1 cells coexpressing α monomers and β monomers. Coexpression of βα polypeptides with α monomers inhibited the K+ currents, while coexpression with β monomers did not. We then constructed another single polypeptide (βα2) consisting of a β subunit and a dimeric repeat of the α subunit. 86Rb+ efflux from COS1 cells expressing βα2 polypeptides was small, but was restored by supplementation with β monomers. These results indicate that the activity of KATP channels is optimized when the α and β subunits are coexpressed with a molar ratio of 1:1. Since inward rectifier K+ channels are thought to function as homo‐ or hetero‐tetramers, this suggests that the KATP channel functions as a multimeric protein, most likely a hetero‐octamer composed of a tetramer of the Kir6.2 subunit and a tetramer of the SUR1 subunit.

A Nonsense Mutation in the Inward Rectifier Potassium Channel Gene, Kir6.2, Is Associated With Familial Hyperinsulinism

ATP-sensitive potassium (KATP) channels are an essential component of glucose-dependent insulin secretion in pancreatic islet β-cells. These channels comprise the sulfonylurea receptor (SUR1) and Kir6.2, a member of the inward rectifier K+ channel family. Mutations in the SUR1 subunit are associated with familial hyperinsulinism (HI) (MIM:256450), an inherited disorder characterized by hyperinsulinism in the neonate. Since the Kir6.2 gene maps to human chromosome 11p15.1 (1,2), which also encompasses a locus for HI, we screened the Kir6.2 gene for the presence of mutations in 78 HI probands by single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses. A nonsense mutation, Tyr→Stop at codon 12 (designated Y12X) was observed in the homozygous state in a single proband. 86Rb+ efflux measurements and single-channel recordings of COS-1 cells co-expressing SUR1 and either wild-type or Y12X mutant Kir6.2 proteins confirmed that KATP channel activity was abolished by this nonsense mutation. The identification of an HI patient homozygous for the Kir6.2/Y12X allele affords an opportunity to observe clinical features associated with mutations resulting in an absence of Kir6.2. These data provide evidence that mutations in the Kir6.2 sub-unit of the islet β-cell KATP channel are associated with the HI phenotype and also suggest that the majority of HI cases are not attributable to mutations in the coding region of the Kir6.2 gene.

Expression and role of ionotropic glutamate receptors in pancreatic islet cells.

Although the excitatory amino acid glutamate and its receptors play crucial roles in many functions of the central nervous system (CNS), their presence in the peripheral tissues has remained unclear. In the present study, we have identified kainate, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA), and N‐methyl‐D‐aspartate (NMDA) receptor subtype mRNAs in pancreatic islets, using reverse transcriptase polymerase chain reaction (RT‐PCR). Intracellular calcium ([Ca2+]i) measurements and electrophysiological recordings indicate that kainate, AMPA, and NMDA all elicit increases of [Ca2+]i in single pancreatic β‐cells and depolarize them. In addition, kainate and AMPA stimulate insulin secretion from isolated pancreatic islets, whereas NMDA does not. Also, immunocytochemical study shows the presence of intense glutaminase immunoreactivity in pancreatic α‐cells and intrapancreatic ganglia, a finding compatible with the possibility that glutamate is released from α‐cells as well as from neurons. Because the inhibitory amino acid γ‐amino butyric acid (GABA) is present in β‐cells as well as in neurons and inhibits glucagon secretion from α‐cells, the present study suggests that glutamate and GABA are coordinated in the regulation of hormone secretion in pancreatic islets.—Inagaki, N., Kuromi, H., Gonoi, T., Okamoto, Y., Ishida, H., Seino, Y., Kaneko, T., Iwanaga, T., Seino, S. Expression and role of ionotropic glutamate receptors in pancreatic islet cells. FASEB J. 9, 686‐691 (1995)

Sequence Variants in the Pancreatic Islet β-Cell Inwardly Rectifying K+ Channel Kir6.2 (Bir) Gene: Identification and Lack of Role in Caucasian Patients with NIDDM

Signals derived from the metabolism of glucose in pancreatic β-cells lead to insulin secretion via the closure of ATP-sensitive K+ channels (KATP). The cloning of the gene encoding the (β-cell inward rectifier Kir6.2 (Bir), a subunit of the β-cell KATP channel, provided the opportunity to look for mutations in this gene that might contribute to the impaired insulin secretion of NIDDM. By single-strand conformational polymorphism (SSCP) analysis on 35 Northern-European Caucasian patients with NIDDM, six sequence variants were detected: Glu10gag→Lys10aag (E10K), Glu23gag→Lys23aag (E23K), Leu270ctg→Val270gtg (L270V), Ile337atc→Val337gtc (I337V), and two silent mutations. Allelic frequencies for the missense variants were compared between the NIDDM group (n = 306) and nondiabetic control subjects (n = 175) and did not differ between the two groups. Pairwise allelic associations indicated significant linkage disequilibrium between the variants in Kir6.2 and between them and a nearby pancreatic β-cell sulfonylurea receptor (SUR1) missense variant (S1370A), but these linkage disequilibria did not differ between the NIDDM and control groups. The results of these studies thus revealed that mutations in the coding region of Kir6.2 1) were not responsible for the previously noted association of the SUR1 variants with NIDDM (Inoue H et al., Diabetes 45:825–831, 1996) and 2) did not contribute to the impaired insulin secretion characteristic of NIDDM in Caucasian patients.

Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells

We have cloned a cDNA encoding a novel protein of 302 amino acids (designated Noc2, no C2 domain) that has 40.7% amino acid identity with and 77.9% similarity to the N-terminal region of rabphilin-3A, a target molecule of Rab3A. However, unlike rabphilin-3A, Noc2 lacks two C2 domains that are thought to interact with Ca2+ and phospholipids. Noc2 is expressed predominantly in endocrine tissues and hormone-secreting cell lines and at very low levels in brain. Immunoblot analysis of subcellular fractions of the insulin-secreting cell line MIN6 and immunocytochemistry reveal that Noc2 is a 38-kDa protein present in the cytoplasm. Overexpression of Noc2 in PC12 cells cotransfected with growth hormone enhances high K+-induced growth hormone secretion. Screening a mouse embryonic cDNA library with the yeast two-hybrid system shows that Noc2 interacts with the LIM domain-containing protein zyxin, a component of the cytoskeleton, and this interaction is further confirmed by the coimmunoprecipitation experiment. Accordingly, Noc2 is probably involved in regulated exocytosis in endocrine cells by interacting with the cytoskeleton.

Localization of the ATP-Sensitive K+ Channel Subunit Kir6.2 in Mouse Pancreas

Kir6.2, a member of the inward rectifier K+ channel family, is a component of the ATP-sensitive K+ (KATP) channel considered to play a key role in glucose-induced insulin secretion. We studied the distribution of Kir6.2 in mouse pancreas at the cellular level. The sites of Kir6.2 mRNA expression were determined by in situ hybridization histochemistry with a digoxigenin (DIG)-labeled antisense cRNA probe. The hybridization signal was unevenly present throughout the islets of Langerhans, while no distinct signal was detected in exocrine acinar cells. This distribution was confirmed by another cRNA probe complementary to a different region of Kir6.2 mRNA. In situ hybridization and immunofluorescence staining of serial sections with the anti-insulin, the anti-glucagon, and the anti-somato-statin antibodies showed Kir6.2 mRNA to be present in α-, β-, and δ-cells. Furthermore, immunofluorescence staining with antibody raised against Kir6.2 revealed that Kir6.2 protein is localized within the pancreatic islets and is not found in exocrine pancreas. Kir6.2 was further shown to be located together with insulin, glucagon, or somatostatin. The positive staining of Kir6.2 appeared concentrated along the contour of each islet cell, suggesting that Kir6.2 is at the plasma membrane of islet cells. These results suggest that Kir6.2, as a component of KATP channels, is an important molecule in the regulation of all the release of insulin, glucagon, and somatostatin.

Expression and complex formation of soluble N-ethyl-maleimide-sensitive factor attachment protein (SNAP) receptors in clonal rat endocrine cells

Syntaxin, synaptosome-associated protein 25 (SNAP-25) and synaptobrevin are neuronal receptors of N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (SNAP), cytosolic proteins essential for membrane transport. The expression of these SNAP receptors was investigated by immunoblotting in clonal endocrine cells; catecholamine-secreting adrenal medullary cells (PC12), growth hormone-secreting pituitary cells (GH3), and insulin-secreting pancreatic beta-cells (RINr and RINm5F). All of these receptors as well as NSF and SNAP are expressed in these cells and, co-immunoprecipitation experiments indicate that syntaxin is associated with SNAP-25 and synaptobrevin after solubilization. These results suggest that syntaxin, SNAP-25, and synaptobrevin form a complex and are involved in the secretion of hormones from endocrine cells by exocytosis.

Localization and Functional Role of Synaptotagmin III in Insulin Secretory Vesicles in Pancreatic β-Cells

Pancreatic β-cells secrete insulin by Ca2+-triggered exocytosis of insulin-containing large dense-core vesicles. Synaptotagmin is a Ca2+/phospholipid-binding protein and is a good candidate for the Ca2+ sensor for exocytosis of synaptic vesicles in neurons. In the present study, we generated a polyclonal antibody against synaptotagmin III, and found that synaptotagmin III immunoreactivity was present at high levels in insulincontaining pancreatic islet cells and insulin-secreting clonal MIN6 cells. In subcellular fractionations of MIN6 cells, synaptotagmin III was recovered in the vesicular fractions containing both insulin and vesicleassociated membrane protein-2 (VAMP-2), but not in synaptophysin-positive fractions. The secretory vesicles immunoprecipitated by anti-VAMP-2 antibody contained synaptotagmin III and insulin. In addition, treatment of streptolysin-O-permeabilized MIN6 cells with anti-synaptotagmin III antibody significantly inhibited Ca2+-triggered insulin secretion. These results indicate that synaptotagmin III is localized in insulin-containing dense-core vesicles in pancreatic β-cells, and further strongly suggest that synaptotagmin III is the Ca2+ sensor in the exocytosis of insulin secretory vesicles.

Somatostatin receptor subtype SSTR2 mediates the inhibition of high‐voltage‐activated calcium channels by somatostatin and its analogue SMS 201‐995

Somatostatin and its anologue SMS 201‐995 inhibit high voltage‐activated (HVA) Ca2+ currents in the rat insulinoma cell line RINm5F which stably express cloned human somatostatin receptor subtype 2 (hSSTR2). In contrast, neither somatostatin nor SMS 201‐995 suppresses the HVA Ca2+ currents in RINm5F which stably express cloned hSSTR1. These results suggest that somatostatin‐induced inhibition of HVA Ca2+ currents is mediated by a specific receptor subtype and that inhibition of calcium influx through HVA Ca2+ channels is one of the mechanisms of SMS 201‐995 action on inhibitory processes of hormone secretion and cell proliferation.