Gating of inward rectifier K+ channels by proton-mediated interactions of N- and C-terminal domains

Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO(2)/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO(2) and pH(i). These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K(+) channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator.     

IL-1 beta attenuates IFN-alpha beta-induced antiviral activity and STAT1 activation in the liver: involvement of proteasome-dependent pathway

IFN-alphabeta is the only established treatment for viral hepatitis; however, more than 60% of patients are poorly responsive. Because viral hepatitis is associated with inflammation, we hypothesized that inflammation may attenuate the efficacy of IFN therapy. To test this hypothesis, the effect of IL-1beta, one of the major proinflammatory cytokines, on IFN signaling pathway in the liver was examined. Administration of IL-1beta in vivo attenuated IFN-alphabeta-induced STAT1 tyrosine phosphorylation in the liver but not in the spleen. The inhibitory action of IL-1beta in vivo was not affected by depleting hepatic Kupffer cells, suggesting that IL-1beta may directly target IFN-alphabeta signaling in hepatocytes. Indeed, pretreatment of human hepatocellular carcinoma HepG2 cells with IL-1beta suppressed IFN-alphabeta-induced antiviral activity and antiviral protein MxA mRNA expression. Furthermore, IL-1beta attenuated IFN-alphabeta-induced STAT1 binding and tyrosine phosphorylation without affecting the level of STAT1 protein. This inhibitory effect can be reversed by pretreatment with either proteasome inhibitors or transfection of dominant negative NF-kappaB inducing kinase mutants. Taken together, these findings suggest that IL-1beta attenuates IFN-alphabeta-induced STAT1 activation by a proteasome-dependent mechanism. In view of high levels of IL-1beta in the serum or within the liver of patients with chronic liver diseases, attenuation of IFN-alphabeta signaling in the liver by IL-1beta could be one of the mechanisms underlying the resistance to IFN therapy in chronic hepatitis C, and IL-1beta could be a potential therapeutic target for improving the efficacy of IFN therapy.     

B7 requirements for primary and secondary protein- and polysaccharide-specific Ig isotype responses to Streptococcus pneumoniae

The requirements for B7 costimulation during an in vivo humoral response to an intact extracellular bacteria have not been reported. In this study we immunized mice with Streptococcus pneumoniae (R36A) to determine the B7 requirements for induction of Ig, specific for two determinants on R36A, the phosphorylcholine (PC) determinant of C-polysaccharide and pneumococcal surface protein A (PspA). We show that the primary anti-PspA response, the development of PspA-specific memory, and the induction of the secondary anti-PspA response in primed mice were completely dependent upon B7 costimulation. Of note, costimulation was required only briefly after the secondary immunization compared with after the primary immunization for optimal induction of Ig. Blockade of B7 costimulation at the time of secondary immunization also completely abrogated the established state of memory, but did not induce tolerance. In contrast to the anti-PspA response, the primary anti-PC response involved only a very short period of B7 costimulation. Whereas B7-2 alone was required for induction of the primary anti-PspA and anti-PC responses, a redundant role for B7-1 and B7-2 was noted for the PspA-specific secondary response. CTLA4Ig blocked both the anti-PC and anti-PspA responses equally well over a wide range of bacterial doses. These studies demonstrate a critical, but variable, role for B7-dependent costimulation during an Ig response to an extracellular bacteria.     

Significant role for Fas in the pathogenesis of autoimmune diabetes

Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the beta cells of the islets of Langerhans. beta cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in beta cell destruction, including Fas, perforin, and TNF-alpha. Evidence for Fas-mediated lysis of beta cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on beta cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.     

Biophysical and molecular mechanisms underlying the modulation of heteromeric Kir4.1-Kir5.1 channels by CO2 and pH

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4. 1-Kir5.1 were studied in inside-out patches. These Kir4.1-Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P(open)) approximately 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P(open) without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at approximately 1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1-Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P(open) and reduced channel sensitivity to intracellular protons. In the presence of 10 microM PIP2, the Kir4.1-Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1-Kir5.1. In excised patches, interestingly, the Kir4.1-Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.     

Cloning and characterization of the glutamate dehydrogenase gene inBacillus licheniformis

The gdhA genes of IRC-3 GDH strain and IRC-8 GDH strain were cloned, and they both successfully complemented the nutritional lesion of an E. coli glutamate auxotroph, Q100 GDH". However, the gdhA gene from the mutant IRC-8 GDH strain failed to complement the glutamate deficiency of the wild type strain IRC-3. The gdhA genes of the wild type and mutant origin were sequenced separately. No nucleotide difference was detected between them. Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant. Additionally, no GDH inhibitor was found in the wild type strain IRC-3. It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression. Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the family I -type hexameric protein, while the GDH of Bacillus subtilis belongs to family II.     

Evaluation of a new apolipoprotein(a) isoform-independent assay for serum Lipoprotein(a)

The risk factor, Lipoprotein(a), [(Lp(a)], has been measured in numerous clinical studies by a variety of immunochemical assay methods. It is becoming apparent that for many of these assays antibody specificity towards the apolipoprotein(a) [apo(a)] repetitive component [the kringle 4 - type 2 repeats] and apo(a) size heterogeneity can significantly affect the accuracy of serum Lp(a) measurements. To address this issue, we investigated whether our current in house Lp(a) [Mercodia] assay showed such bias compared to a recently available assay [Apo-Tek], claiming to possess superior capability for isoform-independent measurement of Lp(a). Levels of Lipoprotein(a) by both Apo-Tek and Mercodia assays correlated inversely with apo(a) isoform sizes. No significant differences were observed between assays in ranges of Lp(a) concentration within each isoform group. The Mercodia assay exhibited similar isoform-independent behaviour to that of Apo-Tek for e quantitation of serum Lipoprotein(a). Essentially identical results were obtained by the two methods, suggesting that Mercodia assay's capture monoclonal antibody also (as is the case for Apo-Tek) does not recognize the kringle 4-type 2 repetitive domain of apo(a). Correlation of Lp(a) concentrations in patient specimens between Apo-Tek and Mercodia assays showed good agreement, although an overall higher degree of imprecision and non-linearity was noted for the Apo-Tek procedure. A change-over to the Apo-Tek assay would therefore not improve on our current assessment of risk contribution from Lp(a) for atherosclerotic vascular disease in individuals with measurable levels of circulating Lipoprotein(a).     

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.     

UDP-N-acetylglucosamine pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically regulated

Giardia synthesizes UDP-GalNAc during cyst wall formation (encystment) via a pathway of inducible enzymes similar to that used to synthesize chitin or peptidoglycan and that includes the UTP-requiring UDP-N-acetylglucosamine pyrophosphorylase. Although it has never been reported as a regulatory enzyme in any system studied to date, kinetic data including Hill plots demonstrate clearly that UDP-N-acetylglucosamine pyrophosphorylase activity, purified from encysting Giardia, is allosterically activated anabolically by physiological levels of glucosamine 6-phosphate (3 microm). Capillary electrophoresis demonstrates that within 24 h after trophozoites are induced to encyst, the level of glucosamine 6-phosphate increases 3-fold over that of non-encysting cells and that by 48 h into encystment the level of glucosamine 6-phosphate has decreased to non-encysting levels or below. UDP-N-acetylglucosamine pyrophosphorylase protein is present constitutively in encysting as well as non-encysting cells. UDP-N-acetylglucosamine pyrophosphorylase immunoaffinity purified from encysting and non-encysting cells exhibited the same molecular weight, amino acid composition, and circular dichroism spectra. Moreover, regardless of whether the enzyme came from encysting or non-encysting cells, the change in its circular dichroism spectra and up to a 6-fold increase in its specific activity anabolically were due to its activation with glucosamine 6-phosphate. Thus, the data support the idea that UDP-N-acetylglucosamine pyrophosphorylase is a major regulatory point in amino sugar synthesis in encysting Giardia and that its allosteric anabolic activation may shift the equilibrium of this pathway toward UDP-GalNAc synthesis.