Show-ling Shyng - Academia.edu (original) (raw)

Papers by Show-ling Shyng

The Journal of biological chemistry, Jan 9, 2015

In pancreatic β-cells, Kv2.1 channels are the dominant delayed rectifier potassium channels respo... more In pancreatic β-cells, Kv2.1 channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β -cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase AMPK. Activation of AMPK mimics, whereas inhibition of AMPK occludes the effect of leptin. Inhibition of CaMKKβ;, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase PKA is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, while stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization; and pharmacol...

Biophysical Journal, 2014

American journal of physiology. Endocrinology and metabolism, 2002

ATP-sensitive potassium (K(ATP)) channels are inhibited by intracellular ATP and activated by ADP... more ATP-sensitive potassium (K(ATP)) channels are inhibited by intracellular ATP and activated by ADP. Nutrient oxidation in beta-cells leads to a rise in [ATP]-to-[ADP] ratios, which in turn leads to reduced K(ATP) channel activity, depolarization, voltage-dependent Ca(2+) channel activation, Ca(2+) entry, and exocytosis. Persistent hyperinsulinemic hypoglycemia of infancy (HI) is a genetic disorder characterized by dysregulated insulin secretion and, although rare, causes severe mental retardation and epilepsy if left untreated. The last five or six years have seen rapid advance in understanding the molecular basis of K(ATP) channel activity and the molecular genetics of HI. In the majority of cases for which a genotype has been uncovered, causal HI mutations are found in one or the other of the two genes, SUR1 and Kir6.2, that encode the K(ATP) channel. This article will review studies that have defined the link between channel activity and defective insulin release and will consider...

Science (New York, N.Y.), Jan 6, 1998

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple cell metabolism to electr... more Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple cell metabolism to electrical activity. Phosphatidylinositol phosphates (PIPs) profoundly antagonized ATP inhibition of KATP channels when applied to inside-out membrane patches. It is proposed that membrane-incorporated PIPs can bind to positive charges in the cytoplasmic region of the channel's Kir6.2 subunit, stabilizing the open state of the channel and antagonizing the inhibitory effect of ATP. The tremendous effect of PIPs on ATP sensitivity suggests that in vivo alterations of membrane PIP levels will have substantial effects on KATP channel activity and hence on the gain of metabolism-excitation coupling.

Journal of Biological Chemistry, 2015

Small molecules that correct protein misfolding and misprocessing defects offer a potential thera... more Small molecules that correct protein misfolding and misprocessing defects offer a potential therapy for numerous human diseases. However, mechanisms underlying pharmacological correction of such defects, especially in heteromeric complexes with structurally diverse constituent proteins, are not well understood. Here we investigate how two chemically distinct compounds, glibenclamide and carbamazepine, correct biogenesis defects in ATP-sensitive potassium (KATP) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2. We present evidence that despite structural differences, carbamazepine and glibenclamide compete for binding to KATP channels and both drugs share a binding pocket in SUR1 to exert their effects. Moreover, both compounds engage Kir6.2, in particular the distal N-terminus of Kir6.2 which is involved in normal channel biogenesis, for their chaperoning effects on SUR1 mutants. Conversely, both drugs can correct channel biogenesis defects caused by Kir6.2 mutations in a SUR1-dependent manner. Using an unnatural, photocrosslinkable amino acid, azido-phenylalanine, genetically encoded in Kir6.2, we demonstrate in living cells that both drugs promote interactions between the distal N-terminus of Kir6.2 and SUR1. These findings reveal a converging pharmacological chaperoning mechanism wherein glibenclamide and carbamazepine stabilize the heteromeric subunit interface critical for channel biogenesis to overcome defective biogenesis caused by mutations in individual subunits.

Channels, 2014

In pancreatic β-cells, K(ATP) channels consisting of Kir6.2 and SUR1 couple cell metabolism to me... more In pancreatic β-cells, K(ATP) channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. Sulfonylureas, insulin secretagogues used to treat type II diabetes, inhibit K(ATP) channel activity primarily by abolishing the stimulatory effect of MgADP endowed by SUR1. In addition, sulfonylureas have been shown to function as pharmacological chaperones to correct channel biogenesis and trafficking defects. Recently, we reported that carbamazepine, an anticonvulsant known to inhibit voltage-gated sodium channels, has profound effects on K(ATP) channels. Like sulfonylureas, carbamazepine corrects trafficking defects in channels bearing mutations in the first transmembrane domain of SUR1. Moreover, carbamazepine inhibits the activity of K(ATP) channels such that rescued mutant channels are unable to open when the intracellular ATP/ADP ratio is lowered by metabolic inhibition. Here, we investigated the mechanism by which carbamazepine inhibits K(ATP) channel activity. We show that carbamazepine specifically blocks channel response to MgADP. This gating effect resembles that of sulfonylureas. Our results reveal striking similarities between carbamazepine and sulfonylureas in their effects on K(ATP) channel biogenesis and gating and suggest that the 2 classes of drugs may act via a converging mechanism.

Frontiers in Physiology, 2013

ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are inv... more ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic β-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The β-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed.

Orphanet journal of rare diseases, 2013

Many genetic diseases are due to defects in protein trafficking where the mutant protein is recog... more Many genetic diseases are due to defects in protein trafficking where the mutant protein is recognized by the quality control systems, retained in the endoplasmic reticulum (ER), and degraded by the proteasome. In many cases, the mutant protein retains function if it can be trafficked to its proper cellular location. We have identified structurally diverse correctors that restore the trafficking and function of the most common mutation causing cystic fibrosis, F508del-CFTR. Most of these correctors do not act directly as ligands of CFTR, but indirectly on other pathways to promote folding and correction. We hypothesize that these proteostasis regulators may also correct other protein trafficking diseases. To test our hypothesis, we used stable cell lines or transient transfection to express 2 well-studied trafficking disease mutations in each of 3 different proteins: the arginine-vasopressin receptor 2 (AVPR2, also known as V2R), the human ether-a-go-go-related gene (KCNH2, also kno...

The Journal of Physiology, 1999

ATP-sensitive potassium channels (KATP) are unique among K¤ channels in being rapidly and reversi... more ATP-sensitive potassium channels (KATP) are unique among K¤ channels in being rapidly and reversibly inhibited by the non-hydrolytic binding of cytoplasmic adenine nucleotides . The realization that these channels are encoded by an ATP binding cassette (ABC) protein, the sulphonylurea receptor (SUR1 or SUR2), in addition to an inward rectifier K¤ channel subunit (Kir6.1 or Kir6.2; led to the natural presumption that inhibitory ATP binding should occur at the nucleotide binding folds (NBFs) of the SUR subunit. Mutagenic studies have now demonstrated that the NBFs of SUR1 control activation of the channel by Mg¤-bound nucleotides and diazoxide, probably as a consequence of these agents mimicking, or enhancing, an effect of nucleotide hydrolysis ). However, none of these studies has demonstrated an effect of SUR1 mutations on the intrinsic sensitivity of expressed channels to ATP inhibition. On the other hand, tandem dimeric constructs of SUR1 and Kir6.2 express channels with reduced ATP sensitivity , and, moreover, mutations of Kir6.2 can alter the inhibitory effect of ATP on channels formed by co-expression with SUR1 (Shyng et al. 1997a;. In the study of , mutations of asparagine 160 in the second transmembrane segment alter sensitivity to polyamine-induced rectification, as well as decreasing the apparent ATP sensitivity approximately 5-fold. reported that a C-terminally truncated Kir6.2 subunit (Kir6.2ÄC36) could express ATPsensitive channels in the absence of SUR1, and reported a significant reduction of ATP sensitivity for Kir6.2ÄC36 channels with an additional mutation of lysine 185 in the truncated C-terminus. have confirmed these findings, and John et al. (1998), as well as Mikhailov et al. (1998), have most recently demonstrated that even full length Kir6.2 can form ATP-sensitive channels in the absence of SUR1. These results are fully consistent with the alternative hypothesis that ATP inhibits the channel through a direct interaction with the Kir6.2 subunit. Shyng et al. (1997a) demonstrated a correlation between the shift in apparent Ké for ATP inhibition that results from mutations of amino acid 160 and changes in the channel open probability in the absence of ATP. This suggested that 1. To gain insight into the role of the cytoplasmic regions of the Kir6.2 subunit in regulating

The Journal of Cell Biology, 1994

The cellular prion protein (PrPc) is a glycolipid-anchored, cell surface protein of unknown funct... more The cellular prion protein (PrPc) is a glycolipid-anchored, cell surface protein of unknown function, a posttranslationally modified isoform of which PrPSc is involved in the pathogenesis of Creutzfeldt-Jakob disease, scrapie, and other spongiform encephalopathies. We have shown previously that chPrP, a chicken homologue of mammalian PrPC, constitutively cycles between the cell surface and an endocytic compartment, with a transit time of approximately 60 min in cultured neuroblastoma cells. We now report that endocytosis of chPrP is mediated by clathrin-coated pits. Immunogold labeling of neuroblastoma cells demonstrates that the concentration of chPrP within 0.05 microns of coated pits is 3-5 times higher than over other areas of the plasma membrane. Moreover, gold particles can be seen within coated vesicles and deeply invaginated coated pits that are in the process of pinching off from the plasma membrane. ChPrP is also localized to coated pits in primary cultures of neurons and glia, and is found in coated vesicles purified from chicken brain. Finally, internalization of chPrP is reduced by 70% after neuroblastoma cells are incubated in hypertonic medium, a treatment that inhibits endocytosis by disrupting clathrin lattices. Caveolae, plasmalemmal invaginations in which several other glycolipid-anchored proteins are concentrated, are not seen in neuroblastoma cells analyzed by thin-section or deep-etch electron microscopy. Moreover, these cells do not express detectable levels of caveolin, a caveolar coat protein. Since chPrP lacks a cytoplasmic domain that could interact directly with the intracellular components of clathrin-coated pits, we propose that the polypeptide chain of chPrP associates with the extracellular domain of a transmembrane protein that contains a coated pit internalization signal.

Science, 1996

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple the cellular metabolic st... more Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple the cellular metabolic state to electrical activity and are a critical link between blood glucose concentration and pancreatic insulin secretion. A mutation in the second nucleotide-binding fold (NBF2) of the sulfonylurea receptor (SUR) of an individual diagnosed with persistent hyperinsulinemic hypoglycemia of infancy generated KATP channels that could be opened by diazoxide but not in response to metabolic inhibition. The hamster SUR, containing the analogous mutation, had normal ATP sensitivity, but unlike wild-type channels, inhibition by ATP was not antagonized by adenosine diphosphate (ADP). Additional mutations in NBF2 resulted in the same phenotype, whereas an equivalent mutation in NBF1 showed normal sensitivity to MgADP. Thus, by binding to SUR NBF2 and antagonizing ATP inhibition of KATP++ channels, intracellular MgADP may regulate insulin secretion.

Proceedings of the National Academy of Sciences, 2001

The ATP-sensitive potassium channel (KATP) regulates insulin secretion in pancreatic ␤ cells. Los... more The ATP-sensitive potassium channel (KATP) regulates insulin secretion in pancreatic ␤ cells. Loss of functional KATP channels because of mutations in either the SUR1 or Kir6.2 channel subunit causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We investigated the molecular mechanism by which a single phenylalanine deletion in SUR1 (⌬F1388) causes PHHI. Previous studies have shown that coexpression of ⌬F1388 SUR1 with Kir6.2 results in no channel activity. We demonstrate here that the lack of functional expression is due to failure of the mutant channel to traffic to the cell surface. Trafficking of K ATP channels requires that the endoplasmic reticulum-retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during channel assembly. To ask whether ⌬F1388 SUR1 forms functional channels with Kir6.2, we inactivated the RKR signal in ⌬F1388 SUR1 by mutation to AAA (⌬F1388 SUR1 AAA). Inactivation of similar endoplasmic reticulumretention signals in the cystic fibrosis transmembrane conductance regulator has been shown to partially overcome the trafficking defect of a cystic fibrosis transmembrane conductance regulator mutation, ⌬F508. We found that coexpression of ⌬F1388 SUR1 AAA with Kir6.2 led to partial surface expression of the mutant channel. Moreover, mutant channels were active. Compared with wild-type channels, the mutant channels have reduced ATP sensitivity and do not respond to stimulation by MgADP or diazoxide. The RKR 3 AAA mutation alone has no effect on channel properties. Our results establish defective trafficking of K ATP channels as a molecular basis of PHHI and show that F1388 in SUR1 is critical for normal trafficking and function of KATP channels.

Proceedings of the National Academy of Sciences, 2000

ATP-sensitive potassium channels (KATP channels) regulate cell excitability in response to metabo... more ATP-sensitive potassium channels (KATP channels) regulate cell excitability in response to metabolic changes. KATP channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K ؉ channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), activate KATP channels and antagonize ATP inhibition of KATP channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP2 levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed K ATP channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K 1͞2, the half maximal inhibitory concentration, Ϸ 60 M) than the sensitivities from control cells (K1͞2 Ϸ 10 M). An inactive form of the PIP5K had little effect on the K1͞2 of wild-type channels but increased the ATP-sensitivity of a mutant KATP channel that has an intrinsically lower ATP sensitivity (from K 1͞2 Ϸ 450 M to K1͞2 Ϸ 100 M), suggesting a decrease in membrane PIP2 levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP 2 and PI-3,4,5-P3 levels, is a significant determinant of the physiological nucleotide sensitivity of KATP channels.

PLoS ONE, 2009

The SLC30A8 gene codes for a pancreatic beta-cell-expressed zinc transporter, ZnT8. A polymorphis... more The SLC30A8 gene codes for a pancreatic beta-cell-expressed zinc transporter, ZnT8. A polymorphism in the SLC30A8 gene is associated with susceptibility to type 2 diabetes, although the molecular mechanism through which this phenotype is manifest is incompletely understood. Such polymorphisms may exert their effect via impacting expression level of the gene product. We used an shRNA-mediated approach to reproducibly downregulate ZnT8 mRNA expression by .90% in the INS-1 pancreatic beta cell line. The ZnT8-downregulated cells exhibited diminished uptake of exogenous zinc, as determined using the zinc-sensitive reporter dye, zinquin. ZnT8-downregulated cells showed reduced insulin content and decreased insulin secretion (expressed as percent of total insulin content) in response to hyperglycemic stimulus, as determined by insulin immunoassay. ZnT8-depleted cells also showed fewer dense-core vesicles via electron microscopy. These data indicate that reduced ZnT8 expression in cultured pancreatic beta cells gives rise to a reduced insulin response to hyperglycemia. In addition, although we provide no direct evidence, these data suggest that an SLC30A8 expression-level polymorphism could affect insulin secretion and the glycemic response in vivo.

PLoS ONE, 2013

In the absence of intracellular nucleotides, ATP-sensitive potassium (K ATP ) channels exhibit sp... more In the absence of intracellular nucleotides, ATP-sensitive potassium (K ATP ) channels exhibit spontaneous activity via a phosphatidylinositol-4,5-bisphosphate (PIP 2 )-dependent gating process. Previous studies show that stability of this activity requires subunit-subunit interactions in the cytoplasmic domain of Kir6.2; selective mutagenesis and disease mutations at the subunit interface result in time-dependent channel inactivation. Here, we report that mutation of the central glycine in the pore-lining second transmembrane segment (TM2) to proline in Kir6.2 causes K ATP channel inactivation. Unlike C-type inactivation, a consequence of selectivity filter closure, in many K + channels, the rate of inactivation in G156P channels was insensitive to changes in extracellular ion concentrations or ion species fluxing through the pore. Instead, the rate of G156P inactivation decreased with exogenous application of PIP 2 and increased when PIP 2 -channel interaction was inhibited with neomycin or poly-L-lysine. These findings indicate the G156P mutation reduces the ability of PIP 2 to stabilize the open state of K ATP channels, similar to mutations in the cytoplasmic domain that produce inactivation. Consistent with this notion, when PIP 2 -dependent open state stability was substantially increased by addition of a second gain-of-function mutation, G156P inactivation was abolished. Importantly, bath application and removal of Mg 2+ -free ATP or a nonhydrolyzable analog of ATP, which binds to the cytoplasmic domain of Kir6.2 and causes channel closure, recover G156P channel from inactivation, indicating crosstalk between cytoplasmic and transmembrane domains. The G156P mutation provides mechanistic insight into the structural and functional interactions between the pore and cytoplasmic domains of Kir6.2 during gating.

The Journal of General Physiology, 2003

ATP-sensitive potassium (K ATP ) channels are formed by the coassembly of four Kir6.2 subunits an... more ATP-sensitive potassium (K ATP ) channels are formed by the coassembly of four Kir6.2 subunits and four sulfonylurea receptor subunits (SUR). The cytoplasmic domains of Kir6.2 mediate channel gating by ATP, which closes the channel, and membrane phosphoinositides, which stabilize the open channel. Little is known, however, about the tertiary or quaternary structures of the domains that are responsible for these interactions. Here, we report that an ion pair between glutamate 229 and arginine 314 in the intracellular COOH terminus of Kir6.2 is critical for maintaining channel activity. Mutation of either residue to alanine induces inactivation, whereas charge reversal at positions 229 and 314 (E229R/R314E) abolishes inactivation and restores the wild-type channel phenotype. The close proximity of these two residues is demonstrated by disulfide bond formation between cysteine residues introduced at the two positions (E229C/R314C); disulfide bond formation abolishes inactivation and stabilizes the current. Using Kir6.2 tandem dimer constructs, we provide evidence that the ion pair likely forms by residues from two adjacent Kir6.2 subunits. We propose that the E229/R314 intersubunit ion pairs may contribute to a structural framework that facilitates the ability of other positively charged residues to interact with membrane phosphoinositides. Glutamate and arginine residues are found at homologous positions in many inward rectifier subunits, including the G-protein-activated inwardly rectifying potassium channel (GIRK), whose cytoplasmic domain structure has recently been solved. In the GIRK structure, the E229-and R314-corresponding residues are oriented in opposite directions in a single subunit such that in the tetramer model, the E229 equivalent residue from one subunit is in close proximity of the R314 equivalent residue from the adjacent subunit. The structure lends support to our findings in Kir6.2, and raises the possibility that a homologous ion pair may be involved in the gating of GIRKs.

The Journal of General Physiology, 2011

Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) chan... more Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (P(o)) and bursting patterns of activity observed in full-length K(ATP) channels. However, the nature of TMD0-Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in K(ATP) channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-K(ATP) channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced P(o), exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP(2). Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP(2). These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP(2). Moreover, they uncover a novel mechanism of K(ATP) channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2.

The Journal of General Physiology, 2000

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) activates K ATP and other inward rectifier (Kir) c... more Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) activates K ATP and other inward rectifier (Kir) channels. To determine residues important for PIP 2 regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of these mutations on channel function were examined using 86 Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues in two narrow regions (176-222 and 301-314) in the COOH terminus that are important for the maintenance of channel function and interaction with PIP 2 . Only one residue (R201A) simultaneously affected ATP and PIP 2 sensitivity, which is consistent with the notion that these ligands, while functionally competitive, are unlikely to bind to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315-390) of the COOH terminus affected channel function when neutralized. The data help to define the structural requirements for PIP 2 sensitivity of K ATP channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP 2 sensitivity in other inward rectifier channels, indicating a common structural basis for PIP 2 regulation. key words: potassium channel • ATP • PH domain • Kir6.2 • phospholipid