Genotype-phenotype correlation in Taiwanese children with diazoxide-unresponsive congenital hyperinsulinism.

Objective: Congenital hyperinsulinism (CHI) is a group of clinically and genetically heterogeneous disorders characterized by dysregulated insulin secretion. The aim of the study was to elucidate genetic etiologies of Taiwanese children with the most severe diazoxide-unresponsive CHI and analyze their genotype-phenotype correlations. Methods: We combined Sanger with whole exome sequencing (WES) to analyze CHI-related genes. The allele frequency of the most common variant was estimated by single-nucleotide polymorphism haplotype analysis. The functional effects of the ATP-sensitive potassium (KATP) channel variants were assessed using patch clamp recording and Western blot. Results: Nine of 13 (69%) patients with ten different pathogenic variants (7 in ABCC8, 2 in KCNJ11 and 1 in GCK) were identified by the combined sequencing. The variant ABCC8 p.T1042QfsX75 identified in three probands was located in a specific haplotype. Functional study revealed the human SUR1 (hSUR1)-L366F KATP channels failed to respond to intracellular MgADP and diazoxide while hSUR1-R797Q and hSUR1-R1393C KATP channels were defective in trafficking. One patient had a de novo dominant mutation in the GCK gene (p.I211F), and WES revealed mosaicism of this variant from another patient. Conclusion: Pathogenic variants in KATP channels are the most common underlying cause of diazoxide-unresponsive CHI in the Taiwanese cohort. The p.T1042QfsX75 variant in the ABCC8 gene is highly suggestive of a founder effect. The I211F mutation in the GCK gene and three rare SUR1 variants associated with defective gating (p.L366F) or traffic (p.R797Q and p.R1393C) KATP channels are also associated with the diazoxide-unresponsive phenotype.

Lack of the peroxiredoxin 6 gene causes impaired spatial memory and abnormal synaptic plasticity.

Peroxiredoxin 6 (PRDX6) is expressed dominantly in the astrocytes and exerts either neuroprotective or neurotoxic effects in the brain. Although PRDX6 can modulate several signaling cascades involving cognitive functions, its physiological role in spatial memory has not been investigated yet. This study aims to explore the function of the Prdx6 gene in spatial memory formation and synaptic plasticity. We first tested Prdx6-/- mice on a Morris water maze task and found that their memory performance was defective, along with reduced long-term potentiation (LTP) in CA3-CA1 hippocampal synapses recorded from hippocampal sections of home-caged mice. Surprisingly, after the probe test, these knockout mice exhibited elevated hippocampal LTP, higher phosphorylated ERK1/2 level, and decreased reactive astrocyte markers. We further reduced ERK1/2 phosphorylation by administering MEK inhibitor, U0126, into Prdx6-/- mice before the probe test, which reversed their spatial memory deficit. This study is the first one to report the role of PRDX6 in spatial memory and synaptic plasticity. Our results revealed that PRDX6 is necessary for maintaining spatial memory by modulating ERK1/2 phosphorylation and astrocyte activation.

Cellular prion protein transcriptionally regulated by NFIL3 enhances lung cancer cell lamellipodium formation and migration through JNK signaling.

Tumor invasion and metastasis are the major causes of treatment failure and mortality in lung cancer patients. In this study, we identified a group of genes with differential expression in in situ and invasive lung adenocarcinoma tissues by expression profiling; among these genes we further characterized the association of the upregulation of PRNP, the gene encoding cellular Prion protein (PrPc), with lung adenocarcinoma invasiveness. Immunohistochemistry on clinical specimens showed an association of PrPc expression with invasive but not in situ lung adenocarcinoma. Consistently, the expression of PrPc was higher in the highly invasive than in the lowly invasive lung adenocarcinoma cell lines. Knockdown of PrPc expression in cultured lung adenocarcinoma cells decreased their lamellipodium formation, in vitro migration and invasion, and in vivo experimental lung metastasis. Phosphorylation of JNKs was found to correlate with PrPc expression and the inhibition of JNKs suppressed the PrPc-induced up-regulation of lamellipodium formation, cell migration, and invasion. Moreover, we identified the nuclear factor, interleukin 3 regulated (NFIL3) protein as a transcriptional activator of the PRNP promoter. Accordingly, NFIL3 promoted lung cancer cell migration and invasion in a PrPc-dependent manner. High NFIL3 expression in clinical specimens of lung adenocarcinoma was also associated with tumor invasiveness. Overall, our observations suggest that the NFIL3/PrPc axis, through regulating lamellipodium formation and cell mobility via JNK signaling, plays a critical role in lung cancer invasiveness and metastasis.

Linkage analysis reveals allosteric coupling in Kir2.1 channels.

Potassium-selective inward rectifier (Kir) channels are a class of membrane proteins necessary for maintaining stable resting membrane potentials, controlling excitability, and shaping the final repolarization of action potentials in excitable cells. In addition to the strong inward rectification of the ionic current caused by intracellular blockers, Kir2.1 channels possess "weak" inward rectification observed in inside-out patches after prolonged washout of intracellular blockers. The mechanisms underlying strong inward rectification have been attributed to voltage-dependent block by intracellular Mg2+ and polyamines; however, the mechanism responsible for weak rectification remains elusive. Hypotheses include weak voltage-dependent block and intrinsic voltage-dependent gating. Here, we performed a conductance Hill analysis of currents recorded with a double-ramp protocol to evaluate different mechanisms proposed for weak inward rectification of Kir2.1 channels. Linkage analysis in the form of a Hill plot revealed that the ramp currents could be best explained by allosteric coupling between a mildly voltage-dependent pore gate (gating charge ∼0.18 eo) and a voltage sensor (gating charge ∼1.7 eo). The proposed voltage sensor stabilized the closing of the pore gate (coupling factor ∼31). We anticipate that the use of linkage analysis will broaden understanding of functional coupling in ion channels and proteins in general.

Activation of the Ca2+-sensing receptors increases currents through inward rectifier K+ channels via activation of phosphatidylinositol 4-kinase.

Inward rectifier K+ channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP2). Stimulation of the Ca2+-sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both Gq/11, which decreases PIP2, and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP2. How membrane PIP2 levels are regulated by CaR activation and whether these changes modulate inward rectifier K+ are unknown. In this study, we found that activation of CaR by the allosteric agonist, NPSR568, increased inward rectifier K+ current (I K1) in guinea pig ventricular myocytes and currents mediated by Kir2.1 channels exogenously expressed in HEK293T cells with a similar sensitivity. Moreover, using the fluorescent PIP2 reporter tubby-R332H-cYFP to monitor PIP2 levels, we found that CaR activation in HEK293T cells increased membrane PIP2 concentrations. Pharmacological studies showed that both phospholipase C (PLC) and PI-4-K are activated by CaR stimulation with the latter played a dominant role in regulating membrane PIP2 and, thus, Kir currents. These results provide the first direct evidence that CaR activation upregulates currents through inward rectifier K+ channels by accelerating PIP2 synthesis. The regulation of I K1 plays a critical role in the stability of the electrical properties of many excitable cells, including cardiac myocytes and neurons. Further, synthetic allosteric modulators that increase CaR activity have been used to treat hyperparathyroidism, and negative CaR modulators are of potential importance in the treatment of osteoporosis. Thus, our results provide further insight into the roles played by CaR in the cardiovascular system and are potentially valuable for heart disease treatment and drug safety.

Mechanism for attenuated outward conductance induced by mutations in the cytoplasmic pore of Kir2.1 channels.

Outward currents through Kir2.1 channels regulate the electrical properties of excitable cells. These currents are subject to voltage-dependent attenuation by the binding of polyamines to high- and low-affinity sites, which leads to inward rectification, thereby controlling cell excitability. To examine the effects of positive charges at the low-affinity site in the cytoplasmic pore on inward rectification, we studied a mutant Kir channel (E224K/H226E) and measured single-channel currents and streaming potentials (Vstream), the latter provide the ratio of water to ions queued in a single-file permeation process in the selectivity filter. The water-ion coupling ratio was near one at a high K(+) concentration ([K(+)]) for the wild-type channel and increased substantially as [K(+)] decreased. On the other hand, fewer ions occupied the selectivity filter in the mutant at all [K(+)]. A model for the Kir channel involving a K(+) binding site in the wide pore was introduced. Model analyses revealed that the rate constants associated with the binding and release to and from the wide-pore K(+) binding site was modified in the mutant. These effects lead to the reduced contribution of a conventional two-ion permeation mode to total conductance, especially at positive potentials, thereby inward rectification.

Voltage-dependent inhibition of outward Kir2.1 currents by extracellular spermine.

Outward currents through inward rectifier Kir2.1 channels play crucial roles in controlling the electrical properties of excitable cells. Extracellular monovalent and divalent cations have been shown to reduce outward K(+) conductance. In the present study, we examined whether spermine, with four positive charges, also inhibits outward Kir2.1 currents. We found that extracellular spermine inhibits steady-state outward Kir2.1 currents, an effect that increases as the voltage becomes more depolarizing, similar to that observed for intracellular spermine. However, several lines of evidence suggest that extracellular spermine does not inhibit outward currents by entering the cytoplasmic pore. Site-directed mutagenesis studies support that extracellular spermine directly interacts with the extracellular domain. In addition, we found that the voltage-dependent decay of outward Kir2.1 currents was necessary for inhibition by extracellular spermine. Further, a region at or near the selectivity filter and the cytoplasmic pore are involved in the voltage-dependent decay and thus in the inhibition of outward currents by extracellular spermine. Taken together, the data suggest that extracellular spermine bound to the mouth of the extracellular pore may induce an allosteric effect on voltage-dependent decay of outward currents, a process in which a region in the vicinity of the selectivity filter and cytoplasmic pore are involved. This study reveals that the extracellular pore domain, the selectivity filter and the cytoplasmic pore are in communication and this coupling is involved in modulating K(+) conduction in the Kir2.1 channel.

Revisiting inward rectification: K ions permeate through Kir2.1 channels during high-affinity block by spermidine.

Outward currents through Kir2.1 channels play crucial roles in controlling the electrical properties of excitable cells, and such currents are subjected to voltage-dependent block by intracellular Mg(2+) and polyamines that bind to both high- and low-affinity sites on the channels. Under physiological conditions, high-affinity block is saturated and yet outward Kir2.1 currents can still occur, implying that high-affinity polyamine block cannot completely eliminate outward Kir2.1 currents. However, the underlying molecular mechanism remains unknown. Here, we show that high-affinity spermidine block, rather than completely occluding the single-channel pore, induces a subconducting state in which conductance is 20% that of the fully open channel. In a D172N mutant lacking the high-affinity polyamine-binding site, spermidine does not induce such a substate. However, the kinetics for the transitions between the substate and zero-current state in wild-type channels is the same as that of low-affinity block in the D172N mutant, supporting the notion that these are identical molecular events. Thus, the residual outward current after high-affinity spermidine block is susceptible to low-affinity block, which determines the final amplitude of the outward current. This study provides a detailed insight into the mechanism underlying the emergence of outward Kir2.1 currents regulated by inward rectification attributed to high- and low-affinity polyamine blocks.

Extracellular K+ elevates outward currents through Kir2.1 channels by increasing single-channel conductance.

Outward currents through inward rectifier K+ channels (Kir) play a pivotal role in determining resting membrane potential and in controlling excitability in many cell types. Thus, the regulation of outward Kir current (IK1) is important for appropriate physiological functions. It is known that outward IK1 increases with increasing extracellular K+ concentration ([K+]o), but the underlying mechanism is not fully understood. A "K+-activation of K+-channel" hypothesis and a "blocking-particle" model have been proposed to explain the [K+]o-dependence of outward IK1. Yet, these mechanisms have not been examined at the single-channel level. In the present study, we explored the mechanisms that determine the amplitudes of outward IK1 at constant driving forces [membrane potential (Vm) minus reversal potential (EK)]. We found that increases in [K+]o elevated the single-channel current to the same extent as macroscopic IK1 but did not affect the channel open probability at a constant driving force. In addition, spermine-binding kinetics remained unchanged when [K+]o ranged from 1 to 150 mM at a constant driving force. We suggest the regulation of K+ permeation by [K+]o as a new mechanism for the [K+]o-dependence of outward IK1.

The extracellular K+ concentration dependence of outward currents through Kir2.1 channels is regulated by extracellular Na+ and Ca2+.

It has been known for more than three decades that outward Kir currents (I(K1)) increase with increasing extracellular K(+) concentration ([K(+)](o)). Although this increase in I(K1) can have significant impacts under pathophysiological cardiac conditions, where [K(+)](o) can be as high as 18 mm and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K(+)](o) dependence of Kir2.1-mediated outward I(K1) was due to [K(+)](o)-dependent inhibition of outward I(K1) by extracellular Na(+) and Ca(2+). This could be accounted for by Na(+)/Ca(2+) inhibition of I(K1) through screening of local negative surface charges. Consistent with this, extracellular Na(+) and Ca(2+) reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward I(K1) in a similar [K(+)](o)-dependent manner as Na(+)/Ca(2+). Site-directed mutagenesis studies identified Asp(114) and Glu(153) as the source of surface charges. Reducing K(+) activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na(+) and Ca(2+), suggesting that in addition to exerting a surface electrostatic effect, Na(+) and Ca(2+) might inhibit outward I(K1) by inhibiting K(+) activation. This study identified interactions of K(+) with Na(+) and Ca(2+) that are important for the [K(+)](o) dependence of Kir2.1-mediated outward I(K1).

K+ binding in the G-loop and water cavity facilitates Ba2+ movement in the Kir2.1 channel.

K+ are selectively coordinated in the selectivity filter and concerted K+ and water movements in this region ensure high conduction rates in K+ channels. In channels with long pores many K+ binding sites are located intracellular to the selectivity filter (inner vestibule), but their contribution to permeation has not been well studied. We investigated this phenomenon by slowing the ion permeation process via blocking inwardly rectifying Kir2.1 channels with Ba2+ in the selectivity filter and observing the effect of K+ in the inner vestibule on Ba2+ exit. The dose-response effect of the intracellular K+ concentration ([K+]i) on Ba2+ exit was recorded with and without intracellular polyamines, which compete with K+ for binding sites. Ba2+ exit was facilitated by the cooperative binding of at least three K+. Site-directed mutagenesis studies suggest that K+ interacting with Ba2+ bound in the selectivity filter were located in the region between selectivity filter and cytoplasmic pore, i.e. the water cavity and G-loop. One of the K+ binding sites was located at residue D172 and another was possibly at M301. This study provides functional evidence for the three K+ binding sites in the inner vestibule previously identified by crystal structure study.

Charges in the cytoplasmic pore control intrinsic inward rectification and single-channel properties in Kir1.1 and Kir2.1 channels.

An E224G mutation of the Kir2.1 channel generates intrinsic inward rectification and single-channel fluctuations in the absence of intracellular blockers. In this study, we showed that positively charged residues H226, R228 and R260, near site 224, regulated the intrinsic inward rectification and single-channel properties of the E224G mutant. By carrying out systematic mutations, we found that the charge effect on the intrinsic inward rectification and single-channel conductance is consistent with a long-range electrostatic mechanism. A Kir1.1 channel where the site equivalent to E224 in the Kir2.1 channel is a glycine residue does not show inward rectification or single-channel fluctuations. The G223K and N259R mutations of the Kir1.1 channel induced intrinsic inward rectification and reduced the single-channel conductance but did not generate large open-channel fluctuations. Substituting the cytoplasmic pore of the E224G mutant into the Kir1.1 channel induced open-channel fluctuations and intrinsic inward rectification. The single-channel conductance of the E224G mutant showed inward rectification. Also, a voltage-dependent gating mechanism decreased open probability during depolarization and contributed to the intrinsic inward rectification in the E224G mutant. In addition to an electrostatic effect, a close interaction of K(+) with channel pore may be required for generating open-channel fluctuations in the E224G mutant.

Electrostatics in the cytoplasmic pore produce intrinsic inward rectification in kir2.1 channels.

Inward rectifier K+ channels are important in regulating membrane excitability in many cell types. The physiological functions of these channels are related to their unique inward rectification, which has been attributed to voltage-dependent block. Here, we show that inward rectification can also be induced by neutral and positively charged residues at site 224 in the internal vestibule of tetrameric Kir2.1 channels. The order of extent of inward rectification is E224K mutant > E224G mutant > wild type in the absence of internal blockers. Mutating the glycines at the equivalent sites to lysines also rendered weak inward rectifier Kir1.1 channels more inwardly rectifying. Also, conjugating positively charged methanethiosulfonate to the cysteines at site 224 induced strong inward rectification, whereas negatively charged methanethiosulfonate alleviated inward rectification in the E224C mutant. These results suggest that charges at site 224 may control inward rectification in the Kir2.1 channel. In a D172N mutant, spermine interacting with E224 and E299 induced channel inhibition during depolarization but did not occlude the pore, further suggesting that a mechanism other than channel block is involved in the inward rectification of the Kir2.1 channel. In this and our previous studies we showed that the M2 bundle crossing and selectivity filter were not involved in the inward rectification induced by spermine interacting with E224 and E299. We propose that neutral and positively charged residues at site 224 increase a local energy barrier, which reduces K+ efflux more than K+ influx, thereby producing inward rectification.

A ring of negative charges in the intracellular vestibule of Kir2.1 channel modulates K+ permeation.

The glutamate at site 224 of a Kir2.1 channel plays an important role in K+ permeation. The single-channel inward current flickers with reduced conductance in an E224G mutant. We show that open-channel fluctuations can also be observed in E224C, E224K, and E224Q mutants. Yet, open-channel fluctuations were not observed in either the wild-type or an E224D mutant. Introducing a negatively charged methanethiosulfonate reagent to the E224C mutant irreversibly increased channel conductance and eliminated open-channel fluctuations. These results suggest that although the negatively charged residue 224 is located at the internal vestibule, it is important for smooth inward K+ conduction. We identified a substate in the E224G mutant and showed that open-channel fluctuations are mainly attributed to rapid transitions between the substate and the main state. Also, we characterized the voltage- and ion-dependence of the substate kinetics. The open-channel fluctuations decreased in internal NH4+ or Tl+ as compared to internal K+. These results suggest that NH4+ and Tl+ gate the E224G mutant in a more stable state. Based on an ion-conduction model, we propose that the appearance of the substate in the E224G mutant is due to changes of ion gating in association with variations of ion-ion interaction in the permeation pathway.

The effects of spermine on the accessibility of residues in the M2 segment of Kir2.1 channels expressed in Xenopus oocytes.

We examined the effects of spermine binding to aspartate at site 172 on the accessibility of internal trimethylammonioethylmethane thiosulphonate (MTSET) to substituted cysteines within the pore of a Kir2.1 channel. Spermine prevented MTSET modification in Q164C and G168C mutants, indicating that sites 164 and 168 are located externally to the spermine binding site. The rates of MTSET modification were significantly reduced by spermine in I176C mutants, indicating that site 176 is located internally to D172 and that the bound spermine hinders the reaction of MTSET with cysteine at site 176. Spermidine, putrescine and Mg2+ also decreased MTSET modification at site 176. The order of effect is putrescine > spermidine approximately = spermine approximately = Mg2+. To account for the electrostatic and physical repulsion between MTSET and polyamines, possible locations of polyamines in the pore are discussed. In D172C mutants, the spermine that bound to sites 224 and 299 completely inhibited channels at +40 mV, yet MTSET remained accessible to site 172. In addition, in the D172C mutant, spermine did not affect the exit rate of Ba2+ bound to the threonine at the site 141. These results indicate that spermine bound at the cytoplasmic pore induces channel closure at positions 141-172. The effects of spermine on the accessibility of amino acids in the pore may shed light on the structural and functional relationships of the Kir2.1 channels during inward rectification.

Conformational changes in Kir2.1 channels during NH4+-induced inactivation.

We have shown previously that NH(4)(+) binding to the external pore of a Kir2.1 channel induces channel inactivation possibly through conformational changes. In this study, we performed further biophysical analyses of the NH(4)(+)-induced inactivation modeled by a refined kinetic scheme. Also, we investigated the conformational change hypothesis by examining whether the chemical modification of single-cysteine substitution of amino acids located at the internal pore alters the kinetics of the NH(4)(+)-induced inactivation. In addition, we examined whether the mutation of amino acids located at various parts of a Kir2.1 channel influences the NH(4)(+)-induced inactivation. Kir2.1 channels were expressed in Xenopus oocytes and studied using patch-clamp techniques. The gating of the NH(4)(+)-induced inactivation was affected by mutation of several amino acids located at various regions of the Kir2.1 channel. These results suggest that amino acids from different parts of a Kir2.1 channel are involved in the channel closure. Furthermore, internal chemical modification of several cysteine mutants resulted in the block of inward currents and changes in the on and off rate for the NH(4)(+)-induced inactivation, suggesting that the internal pore mouth is involved in the closure of a Kir2.1 channel. Taken together these results provide new evidence for conformational changes affecting the NH(4)(+)-induced inactivation in the Kir2.1 channel.

Solution structure of a K(+)-channel blocker from the scorpion Tityus cambridgei.

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.