Shaker-related Kv1 channels contain four channel-forming alpha subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 alpha subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels' properties. Kv1.1 and 1.2 alpha genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K(+) currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2-1.1-1.2-1.1). Pore-blocking petidergic toxins, alpha dendrotoxin, agitoxin-1, tityustoxin-Kalpha, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of alpha subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity.

After removal of the fast N-type inactivation gate, voltage-sensitive Shaker (Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches--the so called C inactivation--is a constriction of the external mouth of the channel pore that prevents K(+) ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a two-pulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was -90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a significant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814-826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a "foot in the door" mechanism, implying a significant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics.

Two-pore domain K⁺ channels (K₂P) display a characteristic extracellular cap structure formed by two M1-P1 linkers, the functional role of which is poorly understood. It has been proposed that the presence of the cap explains the insensitivity of K₂P channels to several K⁺ channel blockers including tetraethylammonium (TEA). We have explored this hypothesis using mutagenesis and functional analysis, followed by molecular simulations. Our results show that the deletion of the cap structure of TASK-3 (TWIK-related acid-sensitive K⁺ channel) generates a TEA-sensitive channel with an IC50 of 11.8 ± 0.4 mM. The enhanced sensitivity to TEA displayed by the cap-less channel is also explained by the presence of an extra tyrosine residue at position 99. These results were corroborated by molecular simulation analysis, which shows an increased stability in the binding of TEA to the cap-less channel when a ring of four tyrosine is present at the external entrance of the permeation pathway. Consistently, Y99A or Y205A single-residue mutants generated in a cap-less channel backbone resulted in TASK-3 channels with low affinity to external TEA.

Identifying the mechanisms that drive suprachiasmatic nucleus (SCN) neurons to fire action potentials with a higher frequency during the day than during the night is an important goal of circadian neurobiology. Selective chemical tools with defined mechanisms of action on specific ion channels are required for successful completion of these studies. The transient K(+) current (I(A)) plays an active role in neuronal action potential firing and may contribute to modulating the circadian firing frequency. Tetraethylammonium (TEA) is frequently used to inhibit delayed rectifier K(+) currents (I(DR)) during electrophysiological recordings of I(A). Depolarizing voltage-clamped hamster SCN neurons from a hyperpolarized holding potential activated both I(A) and I(DR). Holding the membrane potential at depolarized values inactivated I(A) and only the I(DR) was activated during a voltage step. The identity of I(A) was confirmed by applying 4-aminopyridine (5 mM), which significantly inhibited I(A). Reducing the TEA concentration from 40 mM to 1 mM significantly decreased the I(A) inactivation time constant (tau(inact)) from 9.8+/-3.0 ms to 4.9+/-1.2 ms. The changes in I(A)tau(inact) were unlikely to be due to a surface charge effect. The I(A) amplitude was not affected by TEA at any concentration or membrane potential. The isosmotic replacement of NaCl with choline chloride had no effect in I(A) kinetics demonstrating that the TEA effects were not due to a reduction of extracellular NaCl. We conclude that TEA modulates, in a concentration dependent manner, tau(inact) but not I(A) amplitude in hamster SCN neurons.

Low threshold mechanoreceptors (LTMRs) are important for environmental exploration, social interaction, and tactile discrimination. Whisker hair follicles are mechanical sensory organs in non-primate mammals that are functionally equivalent to human fingertips. Several functional types of LTMRs have been identified in rodent whisker hair follicles, including rapidly adapting (RA), slow adapting type 1 (SA1), and slowly adapting type 2 (SA2) LTMRs. Properties of these LTMRs have not been fully characterized. In the present study, we have used pressure-clamped single-fiber recording technique to record impulses of RA, SA1, and SA2 LTMRs in mouse whisker hair follicles, and tested effects of 5-HT, Cd2+, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Ba2+ on the LTMR impulses. We show that 5-HT at 2 mM suppresses SA1 impulses but has no effects on RA and SA2 impulses. Cd2+ at 100 μM suppresses both SA1 and SA2 impulses but has no effects on RA impulses. TEA at 10 mM has no effects on RA and SA1 impulses but increased SA2 impulses. However, TEA at 1 mM and 200 μM decreases SA2 impulses. 4-AP at 1 mM suppresses both SA1 and SA2 impulses but has no effects on RA impulses. Ba2+ at 5 mM increases both RA and SA1 impulses but suppresses SA2 impulses. Collectively, RA, SA1, and SA2 LTMRs show distinct pharmacological properties, suggesting that these LTMRs may use different mechanisms to tune their mechanical signaling.

In the Kv2.1 potassium channel, binding of K(+) to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na(+) current, K(+) interacts with the TEA modulation site at concentrations

Aquaporin-1 (AQP1) channels are constitutively active water channels that allow rapid transmembrane osmotic water flux, and also serve as cyclic-GMP-gated ion channels. Tetraethylammonium chloride (TEA; 0.05 to 10 mM) was shown previously to inhibit the osmotic water permeability of human AQP1 channels expressed in Xenopus oocytes. The purpose of the present study was to determine if TEA blocks osmotic water flux of native AQP1 channels in kidney, and recombinant AQP1 channels expressed in a kidney derived MDCK cell line. We also demonstrate that TEA does not inhibit the cGMP-dependent ionic conductance of AQP1 expressed in oocytes, supporting the idea that water and ion fluxes involve pharmacologically distinct pathways in the AQP1 tetrameric complex.

1. [Ca2+] was recorded in voltage-clamped gastric myocytes from Bufo marinus. Repolarization to -110 mV following a 300 ms depolarization to +10 mV led to triphasic [Ca2+]i decay, with a fast-slow-fast pattern. After a conditioning train of repetitive depolarizations the duration of the second, slow phase of decay was shortened, while the rate of decay during the third, faster phase was increased by 34 +/- 6% (mean +/- S.E.M., n = 21) when compared with unconditioned transients. 2. [Ca2+]i decay was biphasic in cells injected with the calmodulin-binding peptide RS20, with a prolonged period of fast decay followed by a slow phase. There was no subsequent increase in decay rate during individual transients and no acceleration of decay following the conditioning train (n = 8). Decline of [Ca2+]i in cells injected with the control peptide NRS20 was triphasic and the decay rate during the third phase was increased by 50 +/- 19% in conditioned transients (n = 6). 3. Cell injection with CK3AA, a pseudo-substrate inhibitor of calmodulin-dependent protein kinase II, prevented the increase in the final rate of decay following the conditioning train (n = 6). In cells injected with an inactive peptide similar to CK3AA, however, there was a 45 +/- 17% increase after the train (n = 5). 4. Inhibition of Ca2+ uptake by the sarcoplasmic reticulum with cyclopiazonic acid or thapsigargin did not prevent acceleration of decay. 5. These results demonstrate that [Ca2+]i decay is accelerated by Ca(2+)-calmodulin and calmodulin-dependent protein kinase II. This does not depend on Ca2+ uptake by the sarcoplasmic reticulum but may reflect upregulation of mitochondrial Ca2+ removal.

The diagonal band of Broca (DBB) is involved in a wide array of physiological functions which are, in part, mediated by activation of GABAA receptors. DBB is enriched in GABA and protein tyrosine kinase (PTK) immunoreactivity. Whole-cell patch-clamp recording were performed from acutely dissociated DBB neurons to investigate the involvement of PTK in GABAA receptor function. The activation of GABAA receptor by the selective agonist, muscimol (5 microM) was dependent on the presence of intracellular ATP. Omission of ATP in the intracellular medium resulted in a fast decrement of the response whereas inclusion of sodium orthovanadate (100 microM), a non-specific phosphatase inhibitor, augmented the response and inhibited 'run down' of the response. Genistein (100 microM) and tyrphostin B-44 (-), specific inhibitors of PTK, attenuated the response to muscimol. The muscimol response was not affected by daidzein (100 microM); an inactive analogue of genistein) nor by tetraethylammonium bromide (1 mM). These observations suggest that phosphorylation is important for the activation and long term maintenance of GABAA receptor function. PTK phosphorylation, which has been previously identified as an important event in signal transduction, may modulate GABA mediated neurotransmission in the forebrain.

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH3) cells, using the whole cell configuration with Cs+ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of -50 mV activated the voltage-dependent L-type Ca2+ current (ICa,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba2+ replacement of external Ca2+ as the charge carrier through Ca2+ channels, removal of Ca2+ from the bath solution, or buffering intracellular Ca2+ with EGTA (10 mM). The reversal potential of inward tail current was approximately -25 mV. When intracellular Cl- was changed, the reversal potential of the Ca2+-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate ICa,L and allow Ca2+ influx, and is referred to as Ca2+-activated nonselective cationic current (ICAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca2+ spike was also observed with a hybrid current-voltage protocol. Thus, the present studies clearly indicate that Ca2+-activated nonselective cationic channels are expressed in GH3 cells, and can be elicited by the depolarizing stimuli that lead to the activation of ICa,L.

Solvent selection is a pressing challenge in developing efficient and selective liquid phase catalytic processes, as predictive understanding of the solvent effect remains lacking. In this work, an attenuated total reflection infrared spectroscopy technique is developed to quantitatively measure adsorption isotherms on porous materials in solvent and decouple the thermodynamic contributions of van der Waals interactions within zeolite pore walls from those of pore-phase proton transfer. While both the pore diameter and the solvent identity dramatically impact the confinement (adsorption) step, the solvent identity plays a dominant role in proton-transfer. Combined computational and experimental investigations show increasingly favorable pore-phase proton transfer to pyridine in the order: water < acetonitrile < 1,4 - dioxane. Equilibrium methods unaffected by mass transfer limitations are outlined for quantitatively estimating fundamental thermodynamic values using statistical thermodynamics.

Since peroxide-based explosives (PBEs) lack reactive functional groups, they cannot be determined directly by most detection methods and are often detected indirectly by converting them to H2O2. However, H2O2 may originate from many sources, causing false positives in PBE detection. Here, we developed a novel electrochemical sensor for the direct sensitive and selective determination of PBEs such as triacetone triperoxide (TATP) and hexamethylene triperoxide diamine (HMTD) using electrochemical modification of the glassy carbon (GC) electrode with PBE-memory polycarbazole (PCz) films decorated with gold nanoparticles (AuNPs) by cyclic voltammetry (CV). The prepared electrodes were named TATP-memory-GC/PCz/AuNPs (used for TATP determination) and HMTD-memory-GC/PCz/AuNPs (used for HMTD detection). The calibration lines of TATP and HMTD were found in the concentration range of 0.1-1.0 mg L-1 using the net current intensities of differential pulse voltammetry (DPV) versus analyte concentrations. The limit of detection (LOD) commonly found was 15 μg L-1 for TATP and HMTD. The sensor electrodes could separately determine intact TATP and HMTD in the presence of nitro-aromatic, nitramine, and nitrate ester energetic materials. The proposed electrochemical sensing method was not interfered by electroactive substances such as paracetamol, caffeine, acetylsalicylic acid, aspartame, d-glucose, and detergent (containing perborate and percarbonate) used as camouflage materials for PBEs. This is the first molecularly imprinted polymeric electrode for PBEs accomplishing such low LODs, and the DPV method was statistically validated in contaminated clay soil samples against the GC-MS method for TATP and a spectrophotometric method for HMTD using t- and F-tests.

The synthesis, spectral properties, thermal analysis, structural characterization and in silico prediction of pharmacokinetic parameters of tetramethylammonium (compound 1) and tetraethylammonium (compound 2) salt of nimesulide were described in this article. Both compounds crystallize in the monoclinic P21/n space group, with one tetraalkylammonium cation and one nimesulide anion in the asymmetric unit and their crystal structures are stabilized by C-H···O hydrogen bonds between ions. Additionally, structures of title compounds are stabilized by π-π interactions (compound 1), or C-H···π interactions (compound 2) between nimesulide anions. The TG and DSC measurements show that compound 1 melts at a temperature higher than nimesulide, whereas the compound 2 melts at a temperature lower than nimesulide. The MALDI-TOF, 1H NMR, 13C NMR and ATR-FTIR analyses confirm the SCXRD study, that in compounds 1 and 2 nimesulide exists in an ionized form. Studies performed by SWISS ADME and ProTOX II tools, predict to be oral bioavailability of both salts obtained, and one of them (compound 1) is predicted to be well-absorbed by digestive system, while both compounds obtained are classified into toxicity class 4.

Interzeolite transformation has been used to produce a novel family of hierarchical catalysts featuring excellent textural properties, strong acidity, and superior catalytic performance for the Friedel-Crafts alkylation of indole with benzhydrol, the Claisen-Schmidt condensation of benzaldehyde and hydroxyacetophenone, and the cracking of polystyrene. Intermediate solids of the FAU interzeolite transformation into BEA display both increased accessibility─due to the development of mesoporosity─and strong acidity─caused by the presence of ultrasmall crystals or zeolitic fragments in their structure. The use of surfactants allows for the development of the hierarchical catalysts with very narrow pore size distribution. The properties of interzeolite transformation intermediates (ITIs) can be fine-tuned simply by stopping the interconversion at different times.

Zeolite-based catalysts are globally employed in many industrial processes, such as crude-oil refining and bulk chemical production. In this work, the cracking of low-density polyethylene (LDPE) was thoroughly followed in a FTIR operando study to examine the catalytic efficiency of purely microporous zeolites of various textural characteristics. To provide complementary and valuable information on the catalytic activity of the zeolite studied, the thermogravimetric analysis results were compared with yields of the products generated under operating conditions. The reaction products were analyzed via GC-MS to determine the hydrocarbon chain distribution in terms of paraffin, olefins, and aromatics. The individual impact of textural and acidic parameters on catalytic parameters was assessed. The accumulation of bridging hydroxyls of high strength in the zeolite benefited the decrease in polymer decomposition temperature. Through a strategic comparison of purely microporous zeolites, we showed that the catalytic cracking of LDPE is dominated by the acidic feature inherent to the microporous environment.

Unmyelinated nociceptive fibres are a key element in the human nociceptive system, however, it is very difficult to investigate such fibres in vivo in more detail. An alternate approach are studies on isolated human nerves. Here we describe that confocal Ca2+ imaging reveals new information about the physiology of human nociceptive C fibres. Confocal images at two emission wavelengths were collected from regions with unmyelinated nerve fibres within segments of biopsied human sural nerves stained with the Ca2+-sensitive fluorescent dyes Calcium Green-1 and Fura Red. Short trains of supramaximal electrical stimuli applied to one end of the nerve as well as bath application of capsaicin resulted in an increase in the free intracellular Ca2+ concentration. Intracellular Ca2+ transients were seen at action potential frequencies above 1 Hz. They were absent in Ca2+-free bathing solution and reduced during bath application of cadmium. This indicates an extracellular source of the activity-dependent rise in [Ca2+]i. Furthermore, Ca2+ transients were also observed during elevation of the extracellular K+ concentration or during short trains of calcium action potentials. Such 'Ca2+ spikes' were elicited by a combination of tetrodotoxin and potassium channel blockers. These data suggest the presence of voltage-dependent Ca2+ channels in the membrane of nociceptive human nerve fibres.

Single-walled carbon nanotubes (SWCNTs) have been modified with ester groups using typical organic radical chemistry. Consequently, traps for mobile excitons have been created, which enhanced the optical properties of the material. The proposed methodology combines the benefits of mainstream approaches to create luminescent defects in SWCNTs while it simultaneously avoids their limitations. A step change was achieved when the aqueous medium was abandoned. The selection of an appropriate organic solvent enabled much more facile modification of SWCNTs. The presented technique is quick and versatile as it can engage numerous reactants to tune the light emission capabilities of SWCNTs. Importantly, it can also utilize SWCNTs sorted by chirality using conjugated polymers to enhance their light emission capabilities. Such differentiation is conducted in organic solvents, so monochiral SWCNT can be directly functionalized using the demonstrated concept in the same medium without the need to redisperse the material in water.

1. The cell-attached and excised inside-out configurations of the patch-clamp technique were used to demonstrate the presence of two different types of ion channels in the membrane of trout red blood cells under isotonic and normoxic conditions, in the absence of hormonal stimulation. The large majority (93%) of successful membrane seals allowed observation of at least one channel type. 2. In the cell-attached mode with Ringer solution in the bath and Ringer solution, 145 mM KCl or 145 NaCl in the pipette, a channel of intermediate conductance (15-25 pS at clamped voltage, Vp = 0 mV) was present in 85% of cells. The single channel activity reversed between 5 and 7 mV positive to the spontaneous membrane potential. A small conductance channel of 5-6 pS and +5 mV reversal potential was also present in 62% of cells. 3. After excision into the inside-out configuration (with 145 mM KCl or NaCl, pCa 8 in the bath, 145 mM KCl or NaCl, pCa 3 in the pipette) the intermediate conductance channel was present in 439 out of 452 successful seals. This channel was spontaneously active in 90% of patches and in the other 10% of patches the channel was activated by suction. The current-voltage relationship showed slight inward rectification. The channel conductance was in the range 15-20 pS between -60 and 0 mV and increased to 25-30 pS between 0 and 60 mV, with a reversal potential close to zero. Substitution of K+ for Na+ in the pipette or in the bath did not significantly change the single channel conductance. Dilution of the bathing solution KCl concentration shifted the reversal potential towards the Nernst equilibrium for cations. Substitution of N-methyl-D-glucamine (NMDG) for K+ or Na+ in the bath almost abolished the outward current whilst the divalent cation Ca2+ permeated the channel with a higher permeability than K+ and Na+. Inhibition of channel openings was obtained with flufenamic acid, quinine, gadolinium or barium. Taken together these data demonstrate that the intermediate conductance channel belongs to a class of non-selective cation (NSC) channels. 4. In excised patches, under the same control conditions, the conductance of the small conductance non-rectifying channel was 8.6 +/- 0.8 pS (n = 12) between -60 and +60 mV and the reversal potential was close to 0 mV. This channel could be blocked by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) but not by flufenamic acid, DIDS, barium or gadolinium. Selectivity and substitution experiments made it possible to identify this channel as a non-rectifying small conductance chloride (SCC) channel.

Ciguatoxins, mainly produced by benthic dinoflagellate Gambierdiscus species, are responsible for a complex human poisoning known as ciguatera. Previous pharmacological studies revealed that these toxins activate voltage-gated Na+ channels. In frog nodes of Ranvier, ciguatoxins induce spontaneous and repetitive action potentials (APs) and increase axonal volume that may explain alterations of nerve functioning in intoxicated humans. The present study aimed determining the ionic mechanisms involved in Pacific ciguatoxin-1B (P-CTX-1B)-induced membrane hyperexcitability and subsequent volume increase in frog nodes of Ranvier, using electrophysiology and confocal microscopy. The results reveal that P-CTX-1B action is not dependent on external Cl- ions since it was not affected by substituting Cl- by methylsulfate ions. In contrast, substitution of external Na+ by Li+ ions suppressed spontaneous APs and prevented nodal swelling. This suggests that P-CTX-1B-modified Na+ channels are not selective to Li+ ions and/or are blocked by these ions, and that Na+ influx through Na+ channels opened during spontaneous APs is required for axonal swelling. The fact that the K+ channel blocker tetraethylammonium modified, but did not suppress, spontaneous APs and greatly reduced nodal swelling induced by P-CTX-1B indicates that K+ efflux might also be involved. This is supported by the fact that P-CTX-1B, when tested in the presence of both tetraethylammonium and the K+ ionophore valinomycin, produced the characteristic nodal swelling. It is concluded that, during the action of P-CTX-1B, water movements responsible for axonal swelling depend on both Na+ influx and K+ efflux. These results pave the way for further studies regarding ciguatera treatment.

Fast chromatography systems especially developed for high sample throughput applications require sensitive detectors with a high repetition rate. These high throughput techniques, including various chip-based microfluidic designs, often benefit from detectors providing subsequent separation in another dimension, such as mass spectrometry or ion mobility spectrometry (IMS), giving additional information about the analytes or monitoring reaction kinetics. However, subsequent separation is required at a high repetition rate. Here, we therefore present an ultra-fast drift tube IMS operating at ambient pressure. Short drift times while maintaining high resolving power are reached by several key instrumental design features: short length of the drift tube, resistor network of the drift tube, tristate ion shutter, and improved data acquisition electronics. With these design improvements, even slow ions with a reduced mobility of just 0.94 cm2/(V s) have a drift time below 1.6 ms. Such short drift times allow for a significantly increased repetition rate of 600 Hz compared with previously reported values. To further reduce drift times and thus increase the repetition rate, helium can be used as the drift gas, which allows repetition rates of up to 2 kHz. Finally, these significant improvements enable IMS to be used as a detector following ultra-fast separation including chip-based chromatographic systems or droplet microfluidic applications requiring high repetition rates.