Engineered Kir6.2 mutations that correct the trafficking defect of K(ATP) channels caused by specific SUR1 mutations.

KATP channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. The molecular interactions between SUR1 and Kir6.2 that govern channel gating and biogenesis are incompletely understood. In a recent study, we showed that a SUR1 and Kir6.2 mutation pair, E203K-SUR1 and Q52E-Kir6.2, at the SUR1/Kir6.2 interface near the plasma membrane increases the ATP-sensitivity of the channel by nearly 100-fold. Here, we report the finding that the same mutation pair also suppresses channel folding/trafficking defects caused by select SUR1 mutations in the first transmembrane domain of SUR1. Analysis of the contributions from individual mutations, however, revealed that the correction effect is attributed largely to Q52E-Kir6.2 alone. Moreover, the correction is dependent on the negative charge of the substituting amino acid at the Q52 position in Kir6.2. Our study demonstrates for the first time that engineered mutations in Kir6.2 can correct the biogenesis defect caused by specific mutations in the SUR1 subunit.

Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels.

The ATP-sensitive potassium (K(ATP)) channel consisting of the inward rectifier Kir6.2 and SUR1 (sulfonylurea receptor 1) couples cell metabolism to membrane excitability and regulates insulin secretion. Inhibition by intracellular ATP is a hallmark feature of the channel. ATP sensitivity is conferred by Kir6.2 but enhanced by SUR1. The mechanism by which SUR1 increases channel ATP sensitivity is not understood. In this study, we report molecular interactions between SUR1 and Kir6.2 that markedly alter channel ATP sensitivity. Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity ∼100-fold higher than wild-type channels. Cross-linking of E203C in SUR1 and Q52C in Kir6.2 locks the channel in a closed state and is reversible by reducing agents, demonstrating close proximity of the two residues. Our results reveal that ATP sensitivity in K(ATP) channels is a dynamic parameter dictated by interactions between SUR1 and Kir6.2.

ATP activates ATP-sensitive potassium channels composed of mutant sulfonylurea receptor 1 and Kir6.2 with diminished PIP2 sensitivity.

ATP-sensitive potassium (K(ATP)) channels are inhibited by ATP and activated by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Both channel subunits Kir6.2 and sulfonylurea receptor 1 (SUR1) contribute to gating: while Kir6.2 interacts with ATP and PIP(2), SUR1 enhances sensitivity to both ligands. Recently, we showed that a mutation, E128K, in the N-terminal transmembrane domain of SUR1 disrupts functional coupling between SUR1 and Kir6.2, leading to reduced ATP and PIP(2) sensitivities resembling channels formed by Kir6.2 alone. We show here that when E128K SUR1 was co-expressed with Kir6.2 mutants known to disrupt PIP(2) gating, the resulting channels were surprisingly stimulated rather than inhibited by ATP. To explain this paradoxical gating behavior, we propose a model in which the open state of doubly mutant channels is highly unstable; ATP binding induces a conformational change in ATP-unbound closed channels that is conducive to brief opening when ATP unbinds, giving rise to the appearance of ATP-induced stimulation.

N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2.

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 cell giveth and the cell taketh away: an overview of Notch pathway activation by endocytic trafficking of ligands and receptors.

Notch signaling is firmly established as a form of cell-to-cell communication that is critical throughout development. Dysregulation of Notch has been linked to cancer and developmental disorders, making it an important target for therapeutic intervention. One aspect of this pathway that sets it apart from others is its apparent reliance on endocytosis by signal-sending and signal-receiving cells. The subtle details of endocytosis-mediated molecular processing within both ligand- and receptor-presenting cells that are required for the Notch signal to maintain fidelity remain unclear. The endosomal system has long been known to play an important role in terminating signal transduction by directing lysosomal trafficking and degradation of cell surface receptors. More recently, endocytic trafficking has also been shown to be critical for activation of signaling. This review highlights four models of endocytic processing in the context of the Notch pathway. In ligand-presenting cells, endocytosis may be required for pre-processing of ligands to make them competent for interaction with Notch receptors and/or for exerting a pulling force on the ligand/Notch complex. In receptor-presenting cells, endocytosis may be a prerequisite for Notch cleavage and thus activation and/or it could be a means of limiting ligand-independent Notch activation. Recent advances in our understanding of how and why endocytosis of Notch receptors and ligands is required for activation and termination of signaling during normal development and in disease states are discussed.

Role of Hsp90 in biogenesis of the beta-cell ATP-sensitive potassium channel complex.

The pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. K(ATP) channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing K(ATP) channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with K(ATP) channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with beta-cell K(ATP) channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type K(ATP) channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric K(ATP) channels via SUR1, thereby affecting functional expression of the channel in beta-cell membrane.

Down-regulation of ZnT8 expression in INS-1 rat pancreatic beta cells reduces insulin content and glucose-inducible insulin secretion.

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.

Sulfonylurea receptor 1 mutations that cause opposite insulin secretion defects with chemical chaperone exposure.

The beta-cell ATP-sensitive potassium (K(ATP)) channel composed of sulfonylurea receptor SUR1 and potassium channel Kir6.2 serves a key role in insulin secretion regulation by linking glucose metabolism to cell excitability. Mutations in SUR1 or Kir6.2 that decrease channel function are typically associated with congenital hyperinsulinism, whereas those that increase channel function are associated with neonatal diabetes. Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Under normal conditions, both mutant channels showed poor surface expression due to retention in the endoplasmic reticulum, accounting for the loss of channel function phenotype in the congenital hyperinsulinism patients. This trafficking defect, however, could be corrected by treating cells with the oral hypoglycemic drugs sulfonylureas, which we have shown previously to act as small molecule chemical chaperones for K(ATP) channels. The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6- and 12-fold lower than wild-type channels, respectively. Further analyses revealed a nucleotide-independent decrease in mutant channel intrinsic open probability, suggesting the mutations may reduce ATP sensitivity by causing functional uncoupling between SUR1 and Kir6.2. In insulin-secreting cells, rescue of both mutant channels to the cell surface led to hyperpolarized membrane potentials and reduced insulin secretion upon glucose stimulation. Our results show that sulfonylureas, as chemical chaperones, can dictate manifestation of the two opposite insulin secretion defects by altering the expression levels of the disease mutants.

Kir6.2 mutations associated with neonatal diabetes reduce expression of ATP-sensitive K+ channels: implications in disease mechanism and sulfonylurea therapy.

Heterozygous missense mutations in the pore-forming subunit Kir6.2 of ATP-sensitive K(+) channels (K(ATP) channels) have recently been shown to cause permanent neonatal diabetes mellitus (PNDM). Functional studies demonstrated that PNDM mutations reduce K(ATP) channel sensitivity to ATP inhibition, resulting in gain of channel function. However, the impact of these mutations on channel expression has not been examined. Here, we show that PNDM mutations, including Q52R, V59G, V59M, R201C, R201H, and I296L, not only reduce channel ATP sensitivity but also impair channel expression at the cell surface to varying degrees. By tagging the PNDM Kir6.2 mutant V59G or R201H with an additional mutation, N160D, that confers voltage-dependent polyamine block of K(ATP) channels, we demonstrate that in simulated heterozygous state, all surface channels are either wild-type or heteromeric channels containing both wild-type and mutant Kir6.2 subunits. Comparison of the various PNDM mutations in their effects on channel nucleotide sensitivity and expression, as well as disease phenotype, suggests that both channel-gating defect and expression level may play a role in determining disease severity. Interestingly, sulfonylureas significantly increase surface expression of certain PNDM mutants, suggesting that the efficacy of sulfonylurea therapy may be compromised by the effect of these drugs on channel expression.

Use-dependent inhibition of P2X3 receptors by nanomolar agonist.

P2X3 receptors desensitize within 100 ms of channel activation, yet recovery from desensitization requires several minutes. The molecular basis for this slow rate of recovery is unknown. We designed experiments to test the hypothesis that this slow recovery is attributable to the high affinity (< 1 nM) of desensitized P2X3 receptors for agonist. We found that agonist binding to the desensitized state provided a mechanism for potent inhibition of P2X3 current. Sustained applications of 0.5 nM ATP inhibited > 50% of current to repetitive applications of P2X3 agonist. Inhibition occurred at 1000-fold lower agonist concentrations than required for channel activation and showed strong use dependence. No inhibition occurred without previous activation and desensitization. Our data are consistent with a model whereby inhibition of P2X3 by nanomolar [agonist] occurs by the rebinding of agonist to desensitized channels before recovery from desensitization. For several ATP analogs, the concentration required to inhibit P2X3 current inversely correlated with the rate of recovery from desensitization. This indicates that the affinity of the desensitized state and recovery rate primarily depend on the rate of agonist unbinding. Consistent with this hypothesis, unbinding of [32P]ATP from desensitized P2X3 receptors mirrored the rate of recovery from desensitization. As expected, disruption of agonist binding by site-directed mutagenesis increased the IC50 for inhibition and increased the rate of recovery.