Increase In Membrane Depolarization Research Articles

Investigating postsynaptic effects of a Drosophila neuropeptide on muscle contraction.

The Drosophila neuropeptide, DPKQDFMRFamide, was previously shown to enhance excitatory junctional potentials (EJPs) and muscle contraction by both presynaptic and postsynaptic actions. Since the peptide acts on both sides of the synaptic cleft, it has been difficult to examine postsynaptic modulatory mechanisms, particularly when contractions are elicited by nerve stimulation. Here, postsynaptic actions are examined in 3rd instar larvae by applying peptide and the excitatory neurotransmitter, l-glutamate, in the bathing solution to elicit contractions after silencing motor output by removing the central nervous system (CNS). DPKQDFMRFamide enhanced glutamate-evoked contractions at low concentrations (EC50 1.3 nM), consistent with its role as a neurohormone, and the combined effect of both substances was supra-additive. Glutamate-evoked contractions were also enhanced when transmitter release was blocked in temperature-sensitive (Shibire) mutants, confirming the peptide's postsynaptic action. The peptide increased membrane depolarization in muscle when co-applied with glutamate, and its effects were blocked by nifedipine, an L-type channel blocker, indicating effects at the plasma membrane involving calcium influx. DPKQDFMRFamide also enhanced contractions induced by caffeine in the absence of extracellular calcium, suggesting increased calcium release from the sarcoplasmic reticulum (SR) or effects downstream of calcium release from the SR. The peptide's effects do not appear to involve calcium/calmodulin-dependent protein kinase II (CaMKII), previously shown to mediate presynaptic effects. The approach used here might be useful for examining postsynaptic effects of neurohormones and cotransmitters in other systems.NEW & NOTEWORTHY Distinguishing presynaptic and postsynaptic effects of neurohormones is a long-standing challenge in many model organisms. Here, postsynaptic actions of DPKQDFMRFamide are demonstrated by assessing its ability to potentiate contractions elicited by direct application of the neurotransmitter, glutamate, when axons are silent and when transmitter release is blocked. The peptide acts at multiple sites to increase contraction, increasing glutamate-induced depolarization at the cell membrane, acting on L-type channels, and acting downstream of calcium release from the sarcoplasmic reticulum.

In Vivo Efficacy, Mechanistic Study and Synergistic Interaction of Precocene II with Norfloxacin against Methicillin‐Resistant Staphylococcus aureus

AbstractAgeratum conyzoides L. (Asteraceae) possesses wound healing, antimicrobial, and anti‐inflammatory activities. Traditionally, this plant is used in skincare. The essential oil (EO) of A. conyzoides aerial parts contains the chromenes precocene I (1) and precocene II (2) as major constituents. This study evaluated precocene II (2) for its in vivo efficacy, mechanistic analysis, and synergistic interaction with norfloxacin against methicillin‐resistant Staphylococcus aureus (MRSA). The study showed that 2 interacted synergistically (Fractional Inhibitory Concentration Index, FICI≤0.5) with norfloxacin and oxacillin and reduced their MIC values significantly. These observations were further validated using the mice model and showed a significant reduction in bacterial load at much lower doses of 2 and norfloxacin without toxicity at 200 mg/kg body weight. Mechanistic studies revealed that 2 regulates bacterial resistance against clinical isolates of S. aureus through membrane disturbance in a dose‐ and time‐dependent manner. Further, precocene II (2) damaged the membrane of the bacteria, as observed from the increased membrane depolarization and uptake of propidium iodide. It also displayed high selectivity towards S. aureus over mammalian cell lines. The in vitro results highlighted the synergistic activity of 2 through the cell membrane damage without any detrimental effect on mammalian cells. In vivo results showed that 2 in combination with norfloxacin significantly reduced bacterial load at much lower concentrations through synergistic interaction without apparent toxicity.

Mechanisms of bactericidal action of Eugenol against Escherichia coli

Bioactive compounds exhibit antimicrobial activity against diverse microbial pathogens and can be an alternative to antibiotics to avoid the increasing microbial antibiotic resistance. To understand the mechanism underlying the antibacterial activity, the present study was undertaken using the bioactive compound, eugenol. A wide range of antibacterial activity was demonstrated by eugenol against the test pathogens with MIC ranging from 0.0312 to 8 μg/mL. The corresponding MBC of the compound was 2-4 fold higher. Time kill curve revealed eugenol to effectively reduce E. coli to undetectable levels within 4 h. The compound demonstrated disruptive action on the cytoplasmic membrane as measured by intracellular ATP. An increase in MIC value of the compound in the presence of divalent cations and increased membrane depolarization of the bacterium was observed. The morphological and physiological changes in E. coli were also recorded through SEM and TEM studies. The present study suggests eugenol alters membrane permeability resulting in leakage of intracellular contents. This may further allow the drug to interact with the intracellular sites causing cell damage.

Exposure to Sublethal Concentrations of Benzalkonium Chloride Induces Antimicrobial Resistance and Cellular Changes in Klebsiellae pneumoniae Clinical Isolates.

Benzalkonium chloride (BAC) is widely used as a disinfectant and preservative. This study investigated the effect on antimicrobial susceptibility and the cellular changes that occurred after exposure of Klebsiella pneumoniae clinical isolates to sublethal concentrations of BAC. Minimum inhibitory concentration and minimum bactericidal concentration of BAC were determined for the collected 50 K. pneumoniae clinical isolates by broth microdilution method, and the tested isolates were adapted to increasing sublethal concentrations of BAC. The effect of adaptation on MICs of the tested 16 antimicrobial agents, the cell ultrastructure, efflux, and membrane depolarization of the tested isolates were examined. Interestingly, most K. pneumoniae isolates that adapted to BAC showed increased antimicrobial resistance, various morphological and structural changes, increased membrane depolarization, and enhanced efflux activity. The findings of this study suggest that the extensive use of BAC at sublethal concentrations could contribute to the emergence of antibiotic resistance in K. pneumoniae clinical isolates that might complicate the therapy of infections caused by this pathogen. In conclusion, the hazard associated with the prolonged exposure to sublethal concentrations of BAC represents a public health risk and therefore it should be a focus in both hospital and community sanitation practices.

Gating charge displacement in a monomeric voltage-gated proton (Hv1) channel

The voltage-gated proton (Hv1) channel, a voltage sensor and a conductive pore contained in one structural module, plays important roles in many physiological processes. Voltage sensor movements can be directly detected by measuring gating currents, and a detailed characterization of Hv1 charge displacements during channel activation can help to understand the function of this channel. We succeeded in detecting gating currents in the monomeric form of the Ciona-Hv1 channel. To decrease proton currents and better separate gating currents from ion currents, we used the low-conducting Hv1 mutant N264R. Isolated ON-gating currents decayed at increasing rates with increasing membrane depolarization, and the amount of gating charges displaced saturates at high voltages. These are two hallmarks of currents arising from the movement of charged elements within the boundaries of the cell membrane. The kinetic analysis of gating currents revealed a complex time course of the ON-gating current characterized by two peaks and a marked Cole-Moore effect. Both features argue that the voltage sensor undergoes several voltage-dependent conformational changes during activation. However, most of the charge is displaced in a single central transition. Upon voltage sensor activation, the charge is trapped, and only a fast component that carries a small percentage of the total charge is observed in the OFF. We hypothesize that trapping is due to the presence of the arginine side chain in position 264, which acts as a blocking ion. We conclude that the movement of the voltage sensor must proceed through at least five states to account for our experimental data satisfactorily.

Gamma Oscillations Are Generated Locally in an Attention-Related Midbrain Network

Gamma-band (25-140 Hz) oscillations are a hallmark of sensory processing in the forebrain. The optic tectum (OT), a midbrain structure implicated in sensorimotor processing and attention, also exhibits gamma oscillations. However, the origin and mechanisms of these oscillations remain unknown. We discovered that in acute slices of the avian OT, persistent (>100 ms) epochs of large amplitude gamma oscillations can be evoked that closely resemble those recorded in vivo. We found that cholinergic, glutamatergic, and GABAergic mechanisms differentially regulate the structure of the oscillations at various timescales. These persistent oscillations originate in the multisensory layers of the OT and are broadcast to visual layers via the cholinergic nucleus Ipc, providing a potential mechanism for enhancing the processing of visual information within the OT. The finding that the midbrain contains an intrinsic gamma-generating circuit suggests that the OT could use its own oscillatory code to route signals to forebrain networks.

Synaptic drive at developing synapses: Transient upregulation of kainate receptors

At the onset of a period of intense synaptic refinement initiated by synchronized eye opening (EO), rapid changes in postsynaptic NMDA receptor and AMPA receptor currents (NMDARcs and AMPARcs) occur within the superficial visual layers of the rodent superior colliculus (sSC; Lu and Constantine-Paton [2004]: Neuron 43:237-249). Subsequently, evoked non-NMDARc amplitudes increase, but by 2 weeks after EO (AEO) they decrease significantly. Here, using whole-cell patch-clamp recording, we demonstrate that small, slowly desensitizing excitatory kainate receptor currents (KARcs) are responsible for the rise and subsequent fall in non-NMDARcs. The increase in KAR transmission parallels inhibitory GABA(A) responses that plateau at 7 days AEO. By 2 weeks AEO, KARcs are gone. AMPARcs remain unchanged during the appearance and disappearance of the KARcs, despite increases in sSC neuropil activity and continued refinement of inputs to individual sSC neurons. We suggest that in the interval of heightened activity, before SC inhibition matures, many AMPARcs desensitize and are relatively ineffective at relieving the Mg(2+) block on NMDARs. This transient appearance of slowly desensitizing, long-duration KARcs may provide increased membrane depolarization necessary for NMDAR function and continuation of synaptic refinement.

Depolarization, Bacterial Membrane Composition, and the Antimicrobial Action of Ceragenins

Ceragenins are cholic acid-derived antimicrobial agents that mimic the activity of endogenous antimicrobial peptides. Ceragenins target bacterial membranes, yet the consequences of these interactions have not been fully elucidated. The role of the outer membrane in allowing access of the ceragenins to the cytoplasmic membrane of gram-negative bacteria was studied using the ML-35p mutant strain of Escherichia coli that has been engineered to allow independent monitoring of small-molecule flux across the inner and outer membranes. The ceragenins CSA-8, CSA-13, and CSA-54 permeabilize the outer membrane of this bacterium, suggesting that the outer membrane does not play a major role in preventing the access of these agents to the cytoplasmic membrane. However, only the most potent of these ceragenins, CSA-13, was able to permeabilize the inner membrane. Interestingly, neither CSA-8 nor CSA-54 caused inner membrane permeabilization over a 30-min period, even at concentrations well above those required for bacterial toxicity. To further assess the role of membrane interactions, we measured membrane depolarization in gram-positive bacteria with different membrane lipid compositions, as well as in gram-negative bacteria. We found greatly increased membrane depolarization at the minimal bactericidal concentration of the ceragenins for bacterial species containing a high concentration of phosphatidylethanolamine or uncharged lipids in their cytoplasmic membranes. Although membrane lipid composition affected bactericidal efficiency, membrane depolarization was sufficient to cause lethality, providing that agents could access the cytoplasmic membrane. Consequently, we propose that in targeting bacterial cytoplasmic membranes, focus be placed on membrane depolarization as an indicator of potency.

Histamine potentiates acid-induced responses mediating transient receptor potential V1 in mouse primary sensory neurons

In inflamed tissues, extracellular pH decreases and acidosis is an important source of pain. Histamine is released from mast cells under inflammatory conditions and evokes the pain sensation in vivo, but the cellular mechanism of histamine-induced pain has not been well understood. In the present study, we examined the effects of histamine on [Ca 2+] i and membrane potential responses to acid in isolated mouse dorsal root ganglion (DRG) neurons. In capsaicin-sensitive DRG neurons from wild-type mice, acid (>pH 5.0) evoked [Ca 2+] i increases, but not in DRG neurons from transient receptor potential V1 (TRPV1) (−/−) mice. Regardless of isolectin GS-IB4 (IB4)-staining, histamine potentiated [Ca 2+] i responses to acid (≥pH 6.0) that were mediated by TRPV1 activation. Histamine increased membrane depolarization induced by acid and evoked spike discharges. RT-PCR indicated the expression of all four histamine receptors (H1R, H2R, H3R, H4R) in mouse DRG. The potentiating effect of histamine was mimicked by an H1R agonist, but not H2R-H4R agonists and was inhibited only by an H1R antagonist. Histamine failed to potentiate the [Ca 2+] i response to acid in the presence of inhibitors for phospholipase C (PLC) and protein kinase C (PKC). A lipoxygenase inhibitor and protein kinase A inhibitor did not affect the potentiating effects of histamine. Carrageenan and complete Freund's adjuvant produced inflammatory hyperalgesia, but these inflammatory conditions did not change the potentiating effects of histamine in DRG neurons. The present results suggest that histamine sensitizes acid-induced responses through TRPV1 activation via H1R coupled with PLC/PKC pathways, the action of which may be involved in the generation of inflammatory pain.

Reduction of Hspa9 Expression, a Del(5q) Candidate Gene, in a Murine Bone Marrow Transplantation Model Recapitulates Key Feature of Ineffective Hematopoiesis Observed in MDS.

Abstract 946Deletions spanning chromosome 5q31.2 are among the most common karyotypic abnormalities in MDS, and evidence suggests that haploinsufficiency of del(5q) genes maybe an early genetic event. To test whether haploinsufficiency of del(5q31.2) genes contribute to ineffective hematopoiesis observed in MDS (i.e. abnormal proliferation, apoptosis, and differentiation), we reduced the expression of HSPA9 (Hspa9 for the murine orhologue), one of the 28 genes, in primary human hematopoietic cells and a murine transplantation model using lentiviral mediated gene silencing.We purified human CD34+ hematopoietic progenitors from cord blood samples (>90% purity) and infected them individually with 5 unique lentiviral shRNAs targeting HSPA9 (each achieving 30% to >90% knockdown) and 2 control shRNAs. HSPA9 knockdown cells expanded an average of 6 fold less than control cells from days 4-7 in erythroid unileage differentiation culture conditions (SCF, IL-6, IL-3, and EPO) (p≤0.007). The reduced cell numbers observed in HSPA9 knockdown cultures was associated with an increase in apoptosis and a decrease in the number of cells in S-phase compared to control shRNA expressing cells (p≤0.008) (increased membrane depolarization and active Caspase-3 expression were also observed with HSPA9 knockdown). In addition, after 7 days, there was a significant reduction in maturing erythroid precursors (p≤0.004), but not myeloid or megakaryocytic precursors, in HSPA9 knockdown cultures vs. control cultures. These results suggest that reduced expression of HSPA9 in primary hematopoietic cells recapitulates key features of ineffective erythropoiesis observed in MDS.We extended these studies using a bone marrow lentiviral transduction/transplantation mouse model. Bone marrow progenitors were infected with a shRNA expressing lentivirus with yellow fluorescence protein (YFP) as a reporter. Loss of Hspa9 shRNA transduced peripheral blood cells occurred over 4-months, necessitating evaluation of mice from 3-8 weeks post-transplant. Two independent transplantation cohorts were performed and analyzed (control shRNA, n=8 mice; Hspa9 shRNA, n=9 mice). The percent of YFP donor leukocytes was significantly reduced in bone marrow (36.3% vs. 27.9%, p≤0.02), spleen (41.6% vs. 19.5%, p≤0.001), and peripheral blood (39.5% vs. 29.5%, p≤0.007) in mice receiving Hspa9 knockdown cells, consistent with in vitro results. The absolute number of YFP+, Ter119High/CD71+ erythroid cells was reduced by 2.5 fold in the bone marrow (2.21×106 vs. 8.97×105, p≤0.001), 8.9 fold in the spleen (3.46×106 vs. 3.87×105, p≤0.002), and 4.4 fold in the peripheral blood (20.9×103/μl vs. 4.80×103/μl, p≤0.01), whereas the total number of myeloid cells (CD11b+/Gr-1− monocytes and CD11b+/Gr-1+ neutrophils) in the three compartments was no different in mice receiving control vs. Hspa9 knockdown cells, respectively. Furthermore, the absolute number of reticulocytes, polychromatic normoblasts, and basophillic normoblasts (defined by flow cytometry) in Hspa9 knockdown recipients were significantly reduced in the bone marrow, spleen, and peripheral blood vs. control (p≤0.01 for all comparisons). The absolute number of B-lymphocytes (B220+), but not T-lymphocytes (CD3e+), were reduced by 2.4 fold in the bone marrow (1.16×107 vs. 4.81×106, p≤0.001), 3.4 fold in the spleen (4.26×108 vs. 1.24×108, p≤0.001), and 3.5 fold in the peripheral blood (1.06×103/μl vs. 3.05×102/μl p≤0.004). These results suggest that erythroid and B-cell progenitors are preferentially affected by Hspa9 knockdown, both findings that are also observed in patients with MDS. Following a 24-hour BrdU labeling in vivo, the percent of YFP+ cells in S-phase was significantly reduced in Hspa9 knockdown cells vs. control cells (21.3% vs. 27.2%, respectively, p≤0.02) in the bone marrow. Furthermore, the absolute number of YFP+ progenitor cells (lineage−/Sca-1+/C-kit+) in the bone marrow was reduced by 3.9 fold (2.59×104 vs. 6.59×103, p≤0.03), indicating that Hspa9 knockdown alters the early progenitor pool.Collectively, these results suggest that haploinsufficiency of HSPA9 alone contributes to ineffective erythropoiesis, but that other cooperating genes are necessary for clonal dominance to occur in MDS. Because HSPA9 has been shown to physically bind and sequester p53 in the cytoplasm, we are testing whether the apoptosis and cell cycle abnormalities are p53 dependent using p53-/- donor murine bone marrow cells in a transduction/transplantation model. Disclosures:No relevant conflicts of interest to declare.

Flow cytometric assessment of distinct physiological stages withinCryptosporidium parvumsporozoites post-excystation

Cryptosporidium parvum are protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring within Cryptosporidium sporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37 degrees C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.

RNA suppression of ERK2 leads to collapse of mitochondrial membrane potential with acute oxidative stress in human lens epithelial cells

17beta-Estradiol (E(2)) reduces oxidative stress-induced depolarization of mitochondrial membrane potential (MMP) in cultured human lens epithelial cells (HLE-B3). The mechanism by which the nongenomic effects of E(2) contributed to the protection against mitochondrial membrane depolarization was investigated. Mitochondrial membrane integrity is regulated by phosphorylation of BAD, and it is known that phosphorylation of Ser(112) inactivates BAD and prevents its participation in the mitochondrial death pathway. We found that E(2) rapidly increased both the phosphorylation of ERK2 and Ser(112) in BAD. Ser(112) is phosphorylated by p90 ribosomal S6 kinase (RSK), a Ser/Thr kinase, which is a downstream effector of ERK1/2. Inhibition of RSK by the RSK-specific inhibitor SL0101 did not reduce the level of E(2)-induced phosphorylation of Ser(112). Silencing BAD using small interfering RNA did not alter mitochondrial membrane depolarization elicited by peroxide insult. However, under the same conditions, silencing ERK2 dramatically increased membrane depolarization compared with the control small interfering RNA. Therefore, ERK2, functioning through a BAD-independent mechanism regulates MMP in humans lens epithelial cells. We propose that estrogen-induced activation of ERK2 acts to protect cells from acute oxidative stress. Moreover, despite the fact that ERK2 plays a regulatory role in mitochondrial membrane potential, estrogen was found to block mitochondrial membrane depolarization via an ERK-independent mechanism.

Na +/Ca 2+ exchanger 2 is neuroprotective by exporting Ca 2+ during a transient focal cerebral ischemia in the mouse

Na +/Ca 2+ exchanger (NCX), by mediating Na + and Ca 2+ fluxes bi-directionally, assumes a role in controlling the Ca 2+ homeostasis in the ischemic brain. It has been suggested that the three isoforms of NCX (NCX1, 2 and 3) may be differentially involved in permanent cerebral ischemia. However, the role of NCX2 has not been defined in ischemic reperfusion injury after a transient focal cerebral ischemia. Furthermore, it is not known whether NCX2 imports or exports intracellular Ca 2+ ([Ca 2+] i) following ischemia and reperfusion. To define the role of NCX2 in ischemia and reperfusion, we examined mice lacking NCX2, in vivo and in vitro. After an in vitro ischemia, a significantly slower recovery in population spike amplitudes, a sustained elevation of [Ca 2+] i and an increased membrane depolarization were developed in the NCX2-deficient hippocampus. Moreover, a transient focal cerebral ischemia in vivo produced a larger infarction and more cell death in the NCX2-deficient mouse brain. In particular, in the wild type brain, NCX2-expressing neurons were largely spared from cell death after ischemia. Our results suggest that NCX2 exports Ca 2+ in ischemia and thus protects neuronal cells from death by reducing [Ca 2+] i in the adult mouse brain.

Calcium and Small-Conductance Calcium-Activated Potassium Channels in Gonadotropin-Releasing Hormone Neurons before, during, and after Puberty

The pubertal increase in GnRH secretion resulting in sexual maturation and reproductive competence is a complex process involving kisspeptin stimulation of GnRH neurons and requiring Ca(2+) and possibly other intracellular messengers. To determine whether the expression of Ca(2+) channels, or small-conductance Ca(2+)-activated K(+) (SK) channels, whose activity reflects cytoplasmic free Ca(2+) concentration, changes at puberty in GnRH neurons, Ca(2+) and SK currents in GnRH neurons were recorded in brain slices of juvenile [postnatal day (P) 10-21], pubertal (P28-P42), and adult (> or =P56) male GnRH-green fluorescent protein transgenic mice using perforated-patch and whole-cell techniques. Ca(2+) currents were inhibited by the Ca(2+) channel blocker Cd(2+) and showed marked heterogeneity but were on average similar in juvenile, pubertal, and adult GnRH neurons. SK currents, which were inhibited by the SK channel blocker apamin and enhanced by the SK and intermediate-conductance Ca(2+)-activated K(+) channel activator 1-ethyl-2-benzimidazolinone, were also on average similar in juvenile, pubertal, and adult GnRH neurons. These findings suggest that whereas Ca(2+) and SK channels may participate in the pubertal increase in GnRH secretion and there may be changes in Ca(2+) or SK channel subtypes, overall Ca(2+) and SK channel expression in GnRH neurons remains relatively constant across pubertal development. Hence, the expected increase in GnRH neuron cytoplasmic free Ca(2+) concentration required for increased GnRH secretion at puberty appears to be due to mechanisms other than altered Ca(2+) or SK channel expression, e.g. increased membrane depolarization and subsequent activation of preexisting Ca(2+) channels after increased excitatory synaptic input.

Characterization of single L-type Ca2+ channels in myocytes isolated from the cricket lateral oviduct

The single Ca2+ channel activity was obtained from cell-attached patch recordings with the use of pipettes filled with 100 mM Ba2+ as the charge carrier in myocytes isolated from the lateral oviduct of cricket Gryllus bimaculatus. The following results were obtained. (1) The channel had a unitary conductance of 18 pS. (2) The open time histogram of the channel could be fitted with a single exponential while the closed time histogram could be fitted with the sum of two exponentials, suggesting that there are at least one open state and two closed states for this channel. (3) The open probability of the channel increased with increasing membrane depolarization. (4) The mean current reconstructed by averaging individual current trace responses inactivated slowly and the current-voltage relationship for the peak mean current showed a bell-shaped relation. (5) The dihydropyridine (DHP) Ca2+ antagonist, nifedipine, reduced the mean current by increasing the proportion of "blank" sweeps. On the other hand, the DHP Ca2+ agonist, Bay K 8644, increased the mean current by increasing the mean open-times of the channel. These results confirm a presence of DHP-sensitive L-type Ca2+ channel in myocytes isolated from the lateral oviduct of cricket G. bimaculatus.

Molecular Determinants of Voltage-dependent Human Ether-a-Go-Go Related Gene (HERG) K+ Channel Block

The structural determinants for the voltage-dependent block of ion channels are poorly understood. Here we investigate the voltage-dependent block of wild-type and mutant human ether-a-go-go related gene (HERG) K(+) channels by the antimalarial compound chloroquine. The block of wild-type HERG channels expressed in Xenopus oocytes was enhanced as the membrane potential was progressively depolarized. The IC(50) was 8.4 +/- 0.9 microm when assessed during 4-s voltage clamp pulses to 0 mV. Chloroquine also slowed the apparent rate of HERG deactivation, reflecting the inability of drug-bound channels to close. Mutation to alanine of aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of HERG greatly reduced the potency of channel block by chloroquine (IC(50) > 1 mm at 0 mV). However, mutation of Tyr-652 also altered the voltage dependence of the block. In contrast to wild-type HERG, block of Y652A HERG channels was diminished by progressive membrane depolarization, and complete relief from block was observed at +40 mV. HERG channel block was voltage-independent when the hydroxyl group of Tyr-652 was removed by mutating the residue to Phe. Together these findings indicate a critical role for Tyr-652 in voltage-dependent block of HERG channels. Molecular modeling was used to define energy-minimized dockings of chloroquine to the central cavity of HERG. Our experimental findings and modeling suggest that chloroquine preferentially blocks open HERG channels by cation-pi and pi-stacking interactions with Tyr-652 and Phe-656 of multiple subunits.

Effect of Mentha × piperita essential oil and monoterpenes on cucumber root membrane potential

Peppermint ( Mentha piperita L.) essential oil and its main components were assessed for their ability to interfere with plant plasma membrane potentials. Tests were conducted on root segments isolated from etiolated seedlings of cucumber ( Cucumis sativus L.). Increasing the concentration of peppermint essential oil from 5 to 50 ppm caused a decrease in membrane potential ( V m) hyperpolarization of 10–3 mV, whereas concentrations from 100 up to 900 ppm caused an increasing depolarization of V m (from 5 to 110 mV). When tested at 300 ppm, (+)-menthyl acetate, (−)-limonene and 1,8-cineole did not exert any significant effect on V m, whereas (+)-menthofuran (73 mV), (+)-pulegone (85 mV), (+)-neomenthol (96 mV), (−)-menthol (105 mV) and (−)-menthone (111 mV) showed increased ability to depolarize V m. A plot of log of octanol–water partition coefficient ( K ow) against their depolarizing effect showed a significant negative correlation, suggesting that among all monoterpenoids increased membrane depolarization depends on lower K ow. However, among monoterpene ketones, alcohols and furans, increased membrane depolarization is associated with a decline in water solubility. The possible effect of monoterpenoids on membrane ion fluxes is also discussed, since changes in the bioelectric potential of cells imply changes in the flux of ions across the plasma membrane

Triazolyl–benzimidazolones and triazolyl–benzotriazoles: new potential potassium channel activators. II

This paper reports the synthesis and pharmacological evaluation of a series of 5-substituted-triazolyl–benzotriazoles ( 2a–f) and the corresponding series of 5-substituted-triazolyl–benzimidazolones ( 6a–f), as potential activators of the big-conductance calcium-activated potassium channels (BK Ca). The synthesis and structure demonstration of the stock compounds of the two series have been described in our previous works, as well as the common starting compounds 4-carboxamido-5-(4-substituted-2-amino-anilino)-1,2,3-triazoles ( 1a–f). The triazolyl–benzotriazoles were obtained by diazotization, while the triazolyl–benzimidazolones were obtained by thermal intramolecular cyclization of ethoxycarbonylamino derivatives or directly with phosgene. Benzimidazolone compounds generally showed little effect whilst the compounds with a benzotriazole ring showed full efficacy, with vasorelaxing properties and potency parameters a little lower than that of the reference compound NS 1619. These effects were significantly reduced by an increased membrane depolarization. This depolarization-sensitive response is in agreement with the pharmacodynamic hypothesis of activation of potassium channels.

Activity of respiratory laryngeal motoneurons during fictive coughing and swallowing.

Membrane potential changes and discharges from 28 laryngeal motoneurons were recorded intracellularly in the caudal nucleus ambiguus of decerebrate, paralyzed and ventilated cats. Electrical activities were recorded from 17 expiratory laryngeal motoneurons (ELMs) with maximal depolarizing membrane potential in early expiration, and from 11 inspiratory laryngeal motoneurons (ILMs) with maximal depolarizing membrane potential in inspiration. Activities during breathing were compared with those observed during fictive coughing and swallowing evoked by electrical stimulation of the superior laryngeal nerves. These non-respiratory behaviors were evidenced in paralyzed animals by characteristic discharge patterns of the phrenic, abdominal nerves and pharyngeal branch of the vagus nerve. We recorded the activity of 11 ELMs and 5 ILMs during coughing in which ELMs, but not ILMs, exhibited increased membrane depolarization and discharge frequencies. Membrane depolarization and discharge frequencies of all ELMs were also significantly increased during swallowing. In addition, membrane depolarization of most ELMs (15/17) was preceded by a short-lasting hyperpolarization due to chloride-dependent inhibitory mechanisms occurring at the onset of swallowing. Out of 10 ILMs tested during swallowing, 7 exhibited membrane depolarization, preceded in 5 cases by a short-lasting hyperpolarization. Discharge frequencies of ILMs were significantly reduced during swallowing. The same pattern of phasic activities of ILMs and ELMs was observed during coughing and breathing, suggesting the involvement of similar excitatory pathways in both behaviors. These results imply that the duration of activation and the discharge frequency of neurons of the central generator for breathing that drive laryngeal motoneurons are enhanced during coughing. During swallowing, in addition to central excitatory mechanisms, laryngeal motoneurons are subjected to an initial inhibition of unknown origin. This inhibition probably contributes to the temporal organization of the swallowing motor sequence.

Acid pH augments excitatory action of ATP on a dissociated mammalian sensory neuron.

ATP stimulates nociceptive neurons via an action on ligand-gated ion channels. Since tissue injury and inflammation result in both localized acidosis and release of ATP, we studied the effect of acid pH on ATP-gated ion channels in rat nodose ganglion neurons. Lowering pH dramatically increased membrane depolarization and action potential firing elicited by ATP. ATP-activated current was enhanced by acid pH and suppressed by alkaline pH. A pH of 7.2 produced the half-maximal effect. Acidification increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. The observations suggest that the localized acidosis associated with tissue injury may enhance pain perception via an action on ATP-gated ion channels on mammalian sensory neurons.