Tohru Gonoi

Reconstitution of IKATP: An Inward Rectifier Subunit Plus the Sulfonylurea Receptor

A member of the inwardly rectifying potassium channel family was cloned here. The channel, called BIR (Kir6.2), was expressed in large amounts in rat pancreatic islets and glucose-responsive insulin-secreting cell lines. Coexpression with the sulfonylurea receptor SUR reconstituted an inwardly rectifying potassium conductance of 76 picosiemens that was sensitive to adenosine triphosphate (ATP) (IKATP) and was inhibited by sulfonylureas and activated by diazoxide. The data indicate that these pancreatic β cell potassium channels are a complex composed of at least two subunits-BIR, a member of the inward rectifier potassium channel family, and SUR, a member of the ATP-binding cassette superfamily. Gene mapping data show that these two potassium channel subunit genes are clustered on human chromosome 11 at position 11p15.1.

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.

Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem.

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the β-subunit. The reduced channel activities are due to a decrease in α1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with β-subunits, regulates Ca2+ channel expression at the cell surface.

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)

PKA‐mediated phosphorylation of the human KATP channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation

ATP‐sensitive potassium (KATP) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP‐sensitive potassium (KATP) channels are heteromultimeric membrane proteins comprising the pore‐forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2). The molecular mechanism by which hormones and neurotransmitters modulate KATP channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2 subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C‐terminus of Kir6.2 (S372) and SUR1 (S1571). Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G‐protein (Gs)‐coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface. Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the KATP channel, and may explain the mechanism by which Gs‐coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.

Identification of Distinct Ligands for the C-type Lectin Receptors Mincle and Dectin-2 in the Pathogenic Fungus Malassezia

Various C-type lectin receptors (CLRs), including Mincle and Dectin-2, function as pattern recognition receptors and play a central role in immunity to fungal pathogens. However, the precise structures of the CLR ligands in various pathogenic fungi have yet to be completely defined. Here we report that Malassezia, an opportunistic skin fungal pathogen, is cooperatively recognized by Mincle and Dectin-2 through distinct ligands. Solvent-based fractionation revealed that Mincle and Dectin-2 recognize lipophilic and hydrophilic components of Malassezia, respectively. Mass spectrometry and nuclear magnetic resonance (NMR) revealed glyceroglycolipid and unique mannosyl fatty acids linked to mannitol as two Mincle ligands. An O-linked mannobiose-rich glycoprotein was identified as a Malassezia ligand for Dectin-2. Cytokine production in response to the Mincle ligands and the Dectin-2 ligand was abrogated in Mincle(-/-) and Dectin-2(-/-) dendritic cells, respectively. These results demonstrate that Mincle and Dectin-2 recognize distinct ligands in Malassezia to induce host immune responses.

Taste Buds Have a Cyclic Nucleotide-activated Channel, CNGgust

Cyclic nucleotide-gated (CNG) channels have been characterized as important factors involved in physiological processes including sensory reception for vision and olfaction. The possibility thus exists that a certain CNG channel functions in gustation as well. In the present study, we carried out reverse transcription-polymerase chain reaction and genomic DNA cloning and characterized a CNG channel (CNGgust) as a cyclic nucleotide-activated species expressed in rat tongue epithelial tissues where taste reception takes place. Several types of 5′-rapid amplification of cDNA ends clones of CNGgust cDNA were obtained with various 5′-terminal sequences. As the CNGgust gene was a single copy, the formation of such CNGgust variants should result from alternative splicing. The encoded protein was homologous to known vertebrate CNG channels with 50–80% similarities in amino acid sequence, and particularly homologous to bovine testis CNG channel and human cone CNG channel with 82% similarities. CNGgust was functional when expressed in human embryonic kidney cells, where it opened upon the addition of cGMP or cAMP. Immunohistochemical analysis using an antibody raised against a CNGgust peptide demonstrated the channel to be localized on the pore side of each taste bud in the circumvallate papillae, with no signal observed for degenerated taste buds after denervation of the glossopharyngeal nerve. All these results, together with the indication that cyclic nucleotides play a role gustatory signaling pathway(s), strongly suggest the involvement of CNGgust in taste signal transduction.

Post-natal disappearance of transient calcium channels in mouse skeletal muscle: effects of denervation and culture.

1. The whole‐cell voltage clamp technique was used to record Ba2+ currents in voltage‐sensitive Ca2+ channels in mouse flexor digitorum brevis muscles developing in situ from day 1 to 30 after birth. Effects of denervation and tissue culture on the Ca2+ channel currents were also studied. 2. The muscle fibres in newborn mice showed two distinct types of Ca2+ channel currents, a low‐threshold transient current and a high‐threshold sustained current. 3. The specific amplitude of the transient current was 2.7 +/‐ 1.7 (S.D.) A/F in response to ‐30 mV test pulses in medium containing 30 mM‐Ba2+ on day 1 after birth. The transient current decreased progressively in the post‐natal days and became undetectable by day 17. In contrast, the specific amplitude of the sustained current in response to +20 mV test pulses increased 4‐fold from 6.9 A/F on day 1 to 27.7 A/F on day 30. 4. The disappearance of the transient current could not be accounted for by either shifts in voltage dependence of activation and inactivation or changes in activation and inactivation times of the two types of current during development. 5. Denervating muscle fibres on day 8 after birth did not prevent the disappearance of the transient current. Denervating them on day 17 did not allow reappearance of the transient current. However, the increase of the sustained current was suppressed by the denervation either on day 8 or day 17. 6. In muscle fibres isolated on day 8 after birth and cultured thereafter, the transient current did not disappear until day 19 in culture (27 days after birth), while the sustained current was maintained at the level on day 8. 7. In muscle fibres isolated on day 17, when the transient current had become undetectable, and cultured thereafter, the transient current did not reappear until day 15 in culture (32 days after birth), while the sustained current was maintained at a level similar to that on day 17. 8. We conclude that innervation has little influence on the developmental disappearance of the transient Ca2+ channel current in mouse muscle fibres, and suggest that some influencing factors from surroundings other than the nerve may be required for the disappearance of the functional transient channels.