Probing ion permeation and gating in a K+ channel with backbone mutations in the selectivity filter

Potassium channels selectively conduct K+ ions across cell membranes, and use diverse mechanisms to control their gating. We studied ion permeation and gating of an inwardly rectifying K+ channel by individually changing the amide carbonyls of two conserved glycines lining the selectivity filter to ester carbonyls using nonsense suppression. Surprisingly, these backbone mutations do not significantly alter ion selectivity. However, they dramatically change the kinetics of single-channel gating and produce distinct subconductance levels. The mutation at the glycine closer to the inner mouth of the pore also abolishes high-affinity binding of Ba2+ to the channel, indicating the importance of this position in ion stabilization in the selectivity filter. Our results demonstrate that K+ ion selectivity can be retained even with significant reduction of electronegativity in the selectivity filter, and that conformational changes of the filter arising from interactions between permeant ions and the backbone carbonyls contribute directly to channel gating.     

Electrostatic charge at position 552 affects the activation and permeation of FMRFamide-gated Na+ channels

The FMRFamide-gated Na⁺ channel (FaNaC) is a unique peptide-gated sodium channel and a member of the epithelial sodium channel/degenerin family. Previous studies have shown that an aspartate residue (Asp⁵⁵²) in the second transmembrane domain is involved in activation of the FaNaC. To examine the significance of a negative charge at position 552, we used a cysteine-modification method. Macroscopic currents of a cysteine mutant (D552C) were potentiated or inhibited by use of positively or negatively charged sulfhydryl reagents ([2-(trimethylammonium)ethyl]methanethiosulfonate bromide, MTSET, and sodium (2-sulfonatoethyl)methanethiosulfonate, MTSES, respectively). Dose–response analysis showed that treatment with MTSET increased the potency of the FMRFamide in the FaNaC whereas treatment with MTSES reduced the maximum response. Negative charge at position 552 was necessary for the characteristic inward rectification of the FaNaC. These results suggest that negative electric charge at position 552 is important to the activation and permeation properties of the FaNaC.     

Concentration dependence of halide fluxes and selectivity of the anion pathway in toad skin

The isolated toad (Bufo bufo) skin was mounted under voltage-clamp conditions in a chamber shown to cause no significant edge damage. The serosal side of the skin was bathed with NaCl-Ringer's, and the passive voltage-sensitive anion conductance studied in its fully voltage activated state, V = -80 mV (apical bath negative). The active sodium currents were eliminated by replacing external Na+ with K+. With [Cl-]o varying between 1.45 mM and 110 mM (gluconate substitution) and [I-]o = 3 mM, the total clamping current (y) and the sum of halide currents (x), estimated from flux measurements, were related by y = 1.0x-3.7 microA cm-2 (r2 = 0.98, n = 50 preparations). The increase in [Cl-]o produced a sigmoidal increase in Cl- influx and clamping current, with the rate coefficient for the influx increasing with [Cl-]o for 1.45 less than [Cl-]o less than 60 mM, but decreasing slightly again as [Cl-]o was further raised to 110 mM. A similar relationship was obtained between the rate coefficient for the Br- influx and [Br-]o, and the I- influx and [Cl-]o, indicating that these three ions are transported by a pathway that is activated by Cl-o and Br-o. The rate coefficients for the influxes ranked as follows, I-:Cl-:Br- = 0.7:1:1.3. The I-/Cl- selectivity was shown to be independent of the degree of Cl-o activation of the anion pathway, and identical with the I-/Cl- selectivity of a furosemide-sensitive, conductive pathway. With [Cl-]o, [Br-]o, or [I-]o = 110 mM, the currents ranked as follows, Cl-:Br-:I- = 1:0.68:0.06, indicating that Cl-, to a lesser extent Br-, and I-, poorly activate the conductive anion pathway. External I- was a potent inhibitor of the Cl-o activation of the Cl- conductance. The unidirectional I- fluxes ([I-]o = [I-]i = 3 mM, [Cl-]o = [Cl-]i = 110 mM) revealed passive transport for V less than -50 mV, active transport for V = o mV, and exchange diffusion for V = 50 mV, confirming our previous finding that depending on the transepithelial potential, the toad skin exhibits three modes of anion transport. A model that shares some properties with that of the anion transport system of the red cell membrane accounts for our findings, and for an inwardly directed active Cl- flux in terms of Cl-/HCO3- exchange.     

The molecular charge and size of heparins determine their impact on the decidualization of human endometrial stromal cells

Heparin modulates the decidualization of human endometrial stromal cells (ESCs), but the molecular mechanisms behind these effects are still unknown. In the present study, we further specified this biological effect of heparin in human ESCs in vitro. ESCs were isolated from hysterectomy specimens, decidualized over 12 days using progesterone and 17β-estradiol and incubated with thrombin, factor Xa (FXa), unfractionated heparin, dextran sulfate, danaparoid or different low-molecular-weight heparins (LMWHs). Production of insulin-like growth factor (IGF)-I, prolactin (PRL) and IGF-binding protein (IGFBP)-1 by ESCs was measured using ELISAs. Like heparin, thrombin and FXa cause an increase in IGF-I in ESCs, suggesting an action of heparin independent from its anticoagulatory effects. This was supported by demonstrating the induction of the same effects on IGF-I, PRL and IGFBP-1 as heparin by dextran sulfate, a polysaccharide of similar size and charge as heparin, but without anticoagulatory properties. LMWHs with the same anti-FXa activity as heparin showed less pronounced effects on ESCs than heparin, whereas the very short pentasaccharide fondaparinux (17 kDa) had barely any effect, further supporting the primary role of molecular size and charge mediating these biological effects of heparin on ESCs. In conclusion, the effects of heparin on the decidualization of human ESCs seem to be independent of its anticoagulatory function, but rather depend on the charge and the size of this polysulfated glycosaminoglycan. Therefore, highly sulfated polysaccharides with a molecular weight >17 kDa might be an interesting pharmacological approach for the therapy of endometrial pathologies, e.g. the treatment of women suffering from recurrent miscarriage or repeated implantation failure.     

Activation of the interleukin-32 pro-inflammatory pathway in response to human papillomavirus infection and over-expression of interleukin-32 controls the expression of the human papillomavirus oncogene

High-risk variants of human papillomavirus (HPV) induce cervical cancer by persistent infection, and are regarded as the principal aetiological factor in this malignancy. The pro-inflammatory cytokine interleukin-32 (IL-32) is present at substantial levels in cervical cancer tissues and in HPV-positive cervical cancer cells. In this study, we identified the mechanism by which the high-risk HPV-16 E7 oncogene induces IL-32 expression in cervical cancer cells. We used antisense transfection, over-expression, or knock-down of IL-32 to assess the effects of the HPV-16 E7 oncogene on IL-32 expression in cervical cancer cells. Cyclo-oxygenase 2 (COX-2) inhibitor treatment was conducted, and the expression levels, as well as the promoter activities, of IL-32 and COX-2 were evaluated in human HPV-positive cervical cancer cell lines. E7 antisense treatment reduced the expression levels and promoter activities of COX-2, which is constitutively expressed in HPV-infected cells. Constitutively expressed IL-32 was also inhibited by E7 antisense treatment. Moreover, IL-32 expression was blocked by the application of the selective COX-2 inhibitor, NS398, whereas COX-2 over-expression resulted in increased IL-32 levels. These results show that the high-risk variant of HPV induces IL-32 expression via E7-mediated COX-2 stimulation. However, E7 and COX-2 were down-regulated in the IL-32γ over-expressing cells and recovered by IL-32 small interfering RNA, indicating that E7 and COX-2 were feedback-inhibited by IL-32γ in cervical cancer cells.     

The afterhyperpolarization conductance exerts the same control over the gain and variability of motoneurone firing in anaesthetized cats

Does the afterhyperpolarization current control the gain and discharge variability of motoneurones according to the same law? We investigated this issue in lumbar motoneurones of anaesthetized cats. Using dynamic clamp, we measured the conductance, time constant and driving force of the AHP current in a sample of motoneurones and studied how the gain was correlated to these quantities. To study the action of the AHP on the discharge variability and to compare it to its action on the gain, we injected an artificial AHP-like current in motoneurones. This increased the natural AHP. In three motoneurones, we abolished most of the natural AHP with the calcium chelator BAPTA to investigate the condition where the discharge was essentially controlled by the artificial AHP. Our results demonstrate that both the gain and the coefficient of variation of the firing rate are inversely proportional to the magnitude and to the time constant of the artificial AHP conductance. This indicates that the AHP exerts the same control over the gain and the variability. This mechanism ensures that the variability of the discharge is modulated with the gain. This guarantees a great regularity of the discharge when the motoneurone is in a low excitability state and hence good control of the force produced.     

Sp1过表达载体的构建以及过表达Sp1对IPS诱导效率的影响

目的诱导多能干细胞(i PSCs)是通过基因转染技术诱发体细胞重编程为具有胚胎干细胞特性和功能的多能干细胞,在再生医学领域具有广阔的应用前景。然而,细胞重编程的效率非常低,探索其他安全、有效地增加i PSCs生成效率的方法显得尤为重要。基本转录因子Sp1在多种组织中广泛表达,参与几乎所有细胞功能,包括细胞增殖、凋亡、分化和新生物的转化。利用Gateway技术构建Sp1过表达载体,在细胞重编程过程中过表达Sp1蛋白,观察过表达Sp1对hiPSCs诱导效率的影响。方法通过Gateway技术,构建出表达Sp1基因的载体质粒pFinal/PGK-puro-EF1α-Sp1-IRESEGFP,将其与包装质粒共转染293FT细胞产生慢病毒,将该病毒加入到细胞重编程过程中过表达Sp1,通过碱性磷酸酶染色观察其对细胞重编程效率的影响。结果表达载体包装出的病毒感染后的人成纤维细胞中Sp1蛋白水平提高。在重编程过程中,增加Sp1表达令hiPSCs的克隆形成率略有提高(P0.05)。结论成功构建表达Sp1基因的载体质粒p Final/PGK-puro-EF1α-Sp1-IRES-EGFP。过表达Sp1能提高hiPSCs克隆形成率,但其增加效果并不显著。     

Mutation in the nerve-specific 5'non-coding region of Cx32 gene and absence of specific mRNA in a CMTX1 Italian family. Mutations in brief no. 195. Online

Charcot-Marie-Tooth type I demyelinating neuropathies are genetically heterogeneous disorders (chrmosome 17,1,X). There are at least three genes on X chromosome, the more frequently involved being Cx32 in Xq13.1. Cx32 encodes for connexin-32, a gap junction protein of 283 aminoacids. We report the results of molecular studies in a CMTX1 Italian family, in which the mutation, found in the 5'-UTR, resulted in an abnormal mRNA connexin-32 expression. Mutations in PMP22 and P0 genes were also excluded in this family. Cx32 gene analysis carried out by PCR-SSCP on family members genomic DNAs, running a 321 bp fragment spanning the TATA box, the trasciptional start site, and the non coding exon 1b, revealed a shift correlated with a transition from C to T at position 40 of exon 1b of the 12 affected members, while was not found in the controls. Then the RT PCR-SSCP on cDNA from two peripheral nerve biopsies of two heterozygous females of the family were sequenced showing only the wild-type alleles and suggesting that mutated mRNAs were too unstable to be detected. The result also suggests a regulating role of the 5'-UTR of Cx32 mRNA.     

Branches of the B cell antigen receptor pathway are directed by protein conduits Bam32 and Carma1

Adaptor proteins act as conduits to channel upstream signals into downstream effector branches. Two B cell-associated adaptors, Bam32 and Carma1, regulate the ERK, JNK, and NF-kappaB branches of the BCR signaling pathway. Recent studies of Bam32-/- and Carma1-/- mice suggest that each adaptor controls a distinct conduit regulating either only proliferation (Bam32) or both the proliferation and survival of B cells (Carma1).     

Activation of Ca(2+)-dependent K+ channel and Cl- conductance in canine pancreatic acinar cells through a cyclic AMP pathway.

Effects of adenosine 3',5'-cyclic monophosphate (cAMP) on Ca(2+)-dependent K+ channel and Cl- conductance in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp methods. In whole-cell current recordings on isolated cells dialyzed with K(+)-rich solution containing 0.5 mM EGTA, addition of 0.5 mM dibutyryl cAMP (dbcAMP), or 50 microM forskolin to the bath increased outward K+ and inward Cl- currents associated with depolarizing and hyperpolarizing voltage jumps, respectively. In intact cells (cell-attached configurations), addition of 0.5 mM dbcAMP or 50 microM forskolin to the bath increased the opening of single K+ channel. In Ca(2+)-free external solution (bath and pipette) 50 microM forskolin or 0.5 mM dbcAMP application evoked an increase in the opening of single K+ channel in intact cells. Addition of 0.5 mM dbcAMP to the bath solution containing 10 mM EGTA without Ca2+ increased the currents of whole-cell dialyzed with K(+)-rich solution containing 10 mM EGTA. When cell was dialyzed with 20 mM EGTA, dbcAMP, or forskolin application did not increase the whole-cell currents. In excised inside-out patches, addition of the catalytic subunit of cAMP-dependent protein kinase (16 U/ml) in the presence of 0.3 mM ATP to the cytoplasmic face of membrane activated the K+ channel, but 0.1 mM cAMP did not. These results suggest that cAMP-dependent phosphorylation can activate Ca(2+)-dependent K+ channels without increase in intracellular free Ca2+ and cAMP-dependent mechanism can activate Ca(2+)-dependent Cl- conductances without the increase in Ca2+ in canine pancreatic acinar cells.     

Ubiquitin-fusion degradation pathway plays an indispensable role in naked DNA vaccination with a chimeric gene encoding a syngeneic cytotoxic T lymphocyte epitope of melanocyte and green fluorescent protein

Antitumour immunity against murine melanoma B16 was achieved by genetic immunization with a naked chimeric DNA encoding a fusion protein linking green fluorescent protein (GFP) to the N-terminus of a major CD8(+) cytotoxic T lymphocyte (CTL) epitope of tyrosinase-related protein 2 (TRP-2(181-188)) of murine melanoma, designated as pGFP-TRP-2. Tumour growth was profoundly suppressed in C57BL/6 mice immunized with pGFP-TRP-2, while mice vaccinated with pTRP-2 showed rapid tumour growth and died within 40 days after tumour challenge. Splenocytes of mice immunized with pGFP-TRP-2 showed high CTL activity specific for TRP-2(181-188). GFP-TRP-2 expressed in COS-7 cells was rapidly degradated in vitro and the degradation was almost completely prevented by adding a proteasome inhibitor, MG-132, in the culture. Furthermore, the antimelanoma immunity induced by genetic immunization with pGFP-TRP-2 was completely cancelled in mice deficient in proteasome activator PA28alpha/beta. Taken together, GFP-TRP-2 processed by cytosolic proteasome played a central role in breaking peripheral tolerance to a melanoma/melanocyte antigen, TRP-2(181-188), by activating CD8(+) CTL specific for TRP-2(181-188). TRP-2(181-188) fused to GFP may be readily cut off from GFP by the ubiquitin-fusion degradation (UFD) pathway and efficiently presented to major histocompatibility complex class I molecules, resulting in effective induction of CD8(+) T cells specific for the CTL epitope. Furthermore, CD4(+) T cells specific for GFP were shown to play a crucial role in the antimelanoma immunity, probably potentiating activity of TRP-2-specific CTL and/or the "ubiquitin-proteasome pathway". It is noteworthy to document that genetic immunization with pGFP plus pTRP-2(181-188) failed to exert the antitumour immunity.     

Liver tumor-promoting effect of beta-naphthoflavone, a strong CYP 1A1/2 inducer, and the relationship between CYP 1A1/2 induction and Cx32 decrease in its hepatocarcinogenesis in the rat

Interrelationships among induction of cytochrome P-450 (CYP) 1A1/2, decrease in connexin 32 (Cx32), and liver tumor-promoting activity by beta-naphthoflavone (BNF) in the promotion stage were examined in a 2-stage liver carcinogenesis model. A total of 20 male Fischer 344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone. Starting 2 weeks later, they were fed a diet containing 2%, 1%, or 0% BNF for 6 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Absolute and relative liver weights were significantly increased in the DEN+BNF groups as compared to the DEN-alone group. Diffuse hepatocellular hypertrophy with cytoplasmic eosinophilia, sometimes accompanied by development of adenoma-like hepatic foci, was observed in the BNF-treated rats. Remarkable induction of cytochrome CYP 1A1/2 and significant increase in CYP 2E1 were noted in the DEN+BNF groups, and positive immunohistochemical staining for both was observed diffusely. The areas of Cx32-positive spots per hepatocyte in the centrilobular areas of livers of the BNF-treated rats were significantly decreased, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form were increased in the BNF-treated groups. These results suggest that BNF is a liver tumor promoter that, unlike phenobarbital, does not induce CYP 2B1/2 isozymes, and there seems to be no direct relationship between CYP 1A1/2 induction and Cx32 reduction in BNF hepatocarcinogenesis.     

Duplication of chromosome arms 9q and 11q: evidence for a novel, 14q32 translocation-independent pathogenetic pathway in multiple myeloma

14q32 translocations [t(14q)] represent critical but not universal events in multiple myeloma (MM). Gains of chromosome arms 1q, 9q, and 11q (+1q, +9q, and +11q) have recently been identified as frequent aberrations in this disease, but their pathogenetic significance remains unclear. We studied a series of 108 MM patients using fluorescence in situ hybridization and DNA probes mapping to chromosome bands 1q21, 9q34, 11q25, 13q14, and 14q32. Three subsets of tumors were defined: (1) MM+/+ (detection of +9q and +11q; 43.5% of cases), (2) MM+/- (+9q or +11q; 21.3%), and (3) MM-/- (neither +9q nor +11q; 35.2%). The incidence of t(14q) was significantly different in these subgroups: 23% in MM+/+, 56% in MM+/-, and 89% in MM-/-. Deletion of 13q (13q-) also was significantly less frequent in MM+/+ (23%) than in MM+/- and MM-/- (36% and 63%, respectively). The nonrandom distribution of chromosomal aberrations in the present series of MM tumors points to a novel, 14q32 translocation-independent pathogenetic pathway in plasma cell neoplasms.     

Tetrodotoxin binding to normal depolarized frog muscle and the conductance of a single sodium channel.

1. We have examined the binding of tritium-labelled and unlabelled tetrodotoxin to frog twitch muscle. Bio-assay as well as radioisotope experiments show a saturable component of tetrodotoxin binding with a binding capacity of about 22 p-mole/g wet wt., and a dissociation constant of about 5 nM. 2. If the observed uptake of tetrodotoxin by muscles represents one-to-one binding of the drug to sodium channels, the channel density is about 380 channels/mum2 of a muscle fibre's surface membrane. On the basis of this result and electrical measurements of sodium conductance in frog muscle, we calculate that the conductance of a single sodium channel is of the order of 10(-12) reciprocal ohms. This is one to two orders of magnitude less than previous estimates. 3. We have looked for an effect of membrane depolarization on saturable tetrodotoxin binding, and have found none. This suggests that there is little molecular interaction between the "gating" portion of the sodium channel molecule, and that which binds tetrodotoxin.     

Generation and characterization of the humoral immune response to DNA immunization with a chimeric beta-amyloid-interleukin-4 minigene

Active immunization with fibrillar beta-amyloid peptide (Abeta(42)) as well as passive transfer of anti-Abeta antibodies significantly reduces Abeta plaque deposition, neuritic dystrophy, and astrogliosis in the brain of mutant amyloid precursor protein (APP)-transgenic mice. Although the mechanism(s) of clearance of Abeta from the brain following active or passive immunization remains to be determined, it is clear that anti-Abeta antibodies are critical for clearance. DNA immunization provides an attractive alternative to direct peptide and adjuvant approaches for inducing a humoral response to Abeta. We constructed a DNA minigene with Abeta fused to mouse interleukin-4 (pAbeta(42)-IL-4) as a molecular adjuvant to generate anti-Abeta antibodies and enhance the Th2-type of immune responses. Gene gun immunizations induced primarily IgG1 and IgG2b anti-Abeta antibodies. Fine epitope analysis with overlapping peptides of the Abeta(42) sequence identified the 1-15 region as a dominant B cell epitope. The DNA minigene-induced anti-Abeta antibodies bound to Abeta plaques in brain tissue from an Alzheimer's disease patient demonstrating functional activity of the antibodies and the potential for therapeutic efficacy.     

A single M protein mutation affects the acid inactivation threshold and growth kinetics of a chimeric flavivirus

Numerous viruses of the Flaviviridae family, including dengue, yellow fever, Japanese encephalitis, and West Nile, cause significant disease in humans and animals. The structure and function of the molecular components of the flavivirus envelope are therefore of significant interest. To our knowledge, a membrane (M) protein mutation which affects the pH at which flavivirus particles are inactivated in vitro has never been reported. Here we show that substitution of proline for glutamine at residue M5 (MQ5P) of a Japanese encephalitis-yellow fever chimera (ChimeriVax-JE) increases its acid sensitivity in vitro by 0.3 pH units (i.e., increases the pH at which virus titer is reduced by 50% from 6.08 to 6.38). In addition, growth kinetics of this mutant virus are accelerated in Vero cells, while neurovirulence and neuroinvasiveness measured in a mouse model are unaffected. A possible interpretation of these observations is that M can modulate the envelope (E) protein function during cell infection.     

Sleep-related neural activity in a premotor and a basal-ganglia pathway of the songbird

During singing, neurons in premotor nucleus RA (robust nucleus of the arcopallium) of the zebra finch produce complex temporal sequences of bursts that are recapitulated during sleep. RA receives input from nucleus HVC via the premotor pathway, and also from the lateral magnocellular nucleus of the anterior nidopallium (LMAN), part of a basal ganglia-related circuit essential for vocal learning. We explore the propagation of sleep-related spike patterns in these two pathways and their influences on RA activity. We promote sleep in head-fixed birds by injections of melatonin and make single-neuron recordings from the three major classes of neurons in HVC: RA-projecting neurons, Area X-projecting neurons, and interneurons. We also record LMAN neurons that project to RA. In paired recordings, spike trains from identified HVC neuron types are strongly coherent with spike trains in RA neurons, whereas LMAN projection neurons on average exhibit only a weak coherency with neurons in HVC and RA. We further examine the relative roles of HVC and LMAN in generating RA burst sequences with reversible inactivation. Lidocaine inactivation of HVC completely abolishes bursting in RA, whereas inactivation of LMAN has no effect on burst rates in RA. In combination, our data suggest that in adult birds, RA burst sequences in sleep are driven via the premotor pathway from HVC. We present a simple generative model of spike trains in HVC, RA, and LMAN neurons that is able to qualitatively reproduce observed coherency functions. We propose that commonly observed coherency peaks at positive and negative time lags are caused by sequentially correlated HVC activity.