Increase In Current Amplitude Research Articles

Divergent Effects of AF- or LQTS-Associated HERG Mutations on Endogenous IKr

Mutations in HERG not only reduce IKr to cause QT syndrome (LQTS) but have also been associated with atrial fibrillation (AF). The mechanisms in AF are unknown. To identify genetic defects conferring AF susceptibility, we screened HERG in 375 patients with typical and lone AF, and identified three probands with rare, non-synonymous HERG variants absent in control populations (284). The first was a C-terminal HERG variant (R1047L), previously reported in LQTS, in 2 probands. One proband was part of a kindred that included 2 other family members with AF or palpitations, and all 3 were mutation carriers; no family was available in the 2nd proband. A second variant (R954C) located only six residues from a previously identified LQTS variant (S960N) was also identified in a lone AF proband. In mutation carriers, QT intervals during sinus rhythm were normal. These variants are particularly interesting because AF and LQTS mutations are likely to be located in close structural proximity. We compared the functional effects of these mutations and WT in two heterologous cell systems: HEK cells stably expressing endogenous HERG (HERG-HEK) or ‘empty’ HEK cells. R1047L caused a 1.4 fold increase in current amplitude in HERG-HEK. In empty HEK cells, there was no difference between R1047L and WT. R954C generated currents that were similar to WT in both HERG-HEK and empty HEK cells, although the nearby S960N variant reduced current 1.6 fold in HERG-HEK and 2 fold in empty HEK cells. These results suggest that relative expression levels of normal and mutant alleles determine net effect on ionic current and action potential controls. Variability in these mechanisms, across or within chambers, may contribute to phenotypes that manifest in only one chamber.

Differential effects of the transient outward K + current activator NS5806 in the canine left ventricle

To examine the electrophysiological and molecular properties of the transient outward current ( I to) in canine left ventricle using a novel I to activator, NS5806, I to was measured in isolated epicardial (Epi), midmyocardial (Mid) and endocardial (Endo) cells using whole-cell patch-clamp techniques. NS5806 activation of K v4.3 current was also studied in CHO-K1 cells and Xenopus laevis oocytes. In CHO-K1 cells co-transfected with K v4.3 and KChIP2, NS5806 (10 μM) caused a 35% increase in current amplitude and a marked slowing of current decay with τ increasing from 7.0 ± 0.4 to 10.2 ± 0.3 ms. In the absence of KChIP2, current decay was unaffected by NS5806. In ventricular myocytes, NS5806 increased I to density by 80%, 82%, and 16% in Epi, Mid, and Endo myocytes, respectively (at + 40 mV) and shifted steady-state inactivation to negative potentials. NS5806 also significantly slowed decay of I to, increasing total charge to 227%, 192% and 83% of control in Epi, Mid and Endo cells, respectively (+ 40 mV, p < 0.05). Quantification of K v4.3 and KChIP2 mRNA in the 3 ventricular cell types revealed that levels of K v4.3 message was uniform but those of KChIP2 were significantly greater in Epi and Mid cells. The KChIP2 gradient was confirmed at the protein level by Western blot. Our results suggest that NS5806 augments I to by increasing current density and slowing decay and that both depend on the presence of KChIP2. I to and its augmentation by NS5806 are greatest in Epi and Mid cells because KChIP2 levels are highest in these cell types.

Ca2+- and Thromboxane-Dependent Distribution of Functional MaxiK Channels in Cultured Astrocytes

Large-conductance, voltage- and Ca2+-activated K+ channels (MaxiK, BK) are broadly expressed ion channels typically observed as a plasma membrane protein in various cell types. In murine astrocyte primary cultures, which are more indicative of in-vivo reactive astrocytes rather than resting astrocytes, our previous results using high-resolution confocal microscopy have revealed the novel finding that MaxiK pore-forming α subunit (MaxiKα) is distributed intracellularly, colocalized along the microtubule network. This MaxiKα association with microtubules was further confirmed by in vitro His-tag pulldown assays, co-immunoprecipitation assays from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine (20mM) or thapsigargin (1μM), resulted in increased MaxiKα labeling at the plasma membrane. More notably U46619, a stable analog of thromboxane A2 (TXA2) which triggers Ca2+-release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiKα surface labeling. We now show using whole-cell patch clamp recordings that U46619 stimulated cells develop a ∼3-fold increase in current amplitude. This data indicates that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. These changes in MaxiKα plasma membrane distribution are effectively blocked by preincubating astrocytes with a cell permeable Ca2+-chelator, BAPTA-AM, or by microtubule disruption prior to stimulation. While microtubules are largely absent in mature astrocytes, our immunohistochemistry results in brain slices show that cortical astrocytes in the developing newborn mouse brain (P1) have a robust expression of microtubules that significantly colocalize with MaxiKα. The results of this study provide the novel insight that suggests Ca2+ released from intracellular stores, may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiKα stores to the plasma membrane of reactive astrocytes. Supported by NIH.

Ca2+- and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: from microtubules to the plasma membrane.

Large-conductance, voltage- and Ca2+-activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming alpha-subunits (MaxiKalpha) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiKalpha is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiKalpha was visualized by three independent labeling approaches: (1) MaxiKalpha-specific antibodies, (2) expressed EGFP-labeled MaxiKalpha, and (3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiKalpha association with microtubules was further confirmed by in vitro His-tag pulldown, co-immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiKalpha labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+-release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiKalpha surface labeling. Whole-cell patch clamp recordings of U46619-stimulated cells develop a approximately 3-fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiK. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiK stores to the plasma membrane of developing murine astrocytes.

Site-specific regulation of CA V2.2 channels by protein kinase C isozymes βII and ε

Ca v2.2 high voltage-gated calcium channels are regulated by phorbol-12-myristae, 13-acetate (PMA) via Ser/Thr protein kinase C (PKC) phosphorylation sites in the I–II linker and C-terminus of the α 1 2.2 subunit. Here we show that PMA enhancement of Ca v2.2 currents expressed in Xenopus oocytes can be blocked by inhibitors of PKC βII or PKC ε isozymes, as shown previously for Ca v2.3 currents, and that microinjection of PKC βII or PKC ε isozymes in the oocytes expressing the WT Ca v2.2 channels increases the basal barium current ( I Ba). The I–V plot shows a large increase in current amplitude with PKC βII and PKC ε isozymes with only a small shift in the peak I Ba in the hyperpolarizing direction. The potentiation of Ca v2.2 currents by microinjection of PKC βII and PKC ε isozymes was not altered by the inhibition of G proteins with GDPβS. The combination of isozyme specific inhibitors with previously generated Ser/Thr to Ala mutants of α 1 2.2 subunit revealed that PKC βII or PKC ε isozymes (but not PKC α or δ) can provide full enhancement through the stimulatory site (Thr-422) in the I–II linker but that PKC ε is better at decreasing channel activity through the inhibitory site Ser-425. The enhancing effect of PKC βII or ε at Thr-422 is dominant over the inhibitory effect at Ser-425. Injected PKC βII also enhances Ca v2.2 current when any of the potential stimulatory sites (Ser-1757, Ser-2108 and Ser-2132) are available in the C-terminus. PKC ε provides lesser enhancement with C-terminal sites and only with Ser-2108 and Ser-2132. Sites Ser-1757 and Ser-2132, but not Ser-2108, are dominant over the inhibitory site Ser-425. Collectively, these results reveal a hierarchy of regulatory sites in Ca v2.2 channels. Site-specific regulation by different PKC isozymes may allow graded levels of channel activation and susceptibility or resistance to subsequent stimulatory events.

Comparison of the GABAergic currents associated with midazolam and propofol in rat hippocampal neurons.

gamma-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter, activates persistent low amplitude tonic currents in several brain regions, in addition to conventional synaptic currents. Tonic conductance is highly sensitive to low concentrations of volatile anesthetics and therefore might contribute to amnestic properties. We compared the properties of GABAergic tonic currents mediated by sedative-amnestic midazolam and anesthetic propofol in rat hippocampal neurons. Patch clamp techniques were used to characterize the GABAergic currents recorded in CA1 pyramidal neurons in rat hippocampal slices. The amplitude of the tonic currents and the decay of miniature inhibitory postsynaptic currents (mIPSCs) were measured after administration of midazolam or propofol. Both midazolam and propofol caused concentration dependent increases in the tonic currents. The enhancement of the tonic currents by midazolam concentrations of greater than 0.5 microM caused no further increase in current amplitude. Propofol continued to increase with concentrations over the range tested (0.1-10 microM). Low concentrations of midazolam 0.01 microM and propofol 0.5 microM selectively enhanced the tonic currents but failed to alter mIPSCs. Low concentrations of midazolam and propofol selectively enhanced the tonic currents but not synaptic currents of rat hippocampal pyramidal neurons. Unlike midazolam, the response to propofol did not become saturated and had a greater effect on the tonic currents.

Desensitization of Chemical Activation by Auxiliary Subunits: CONVERGENCE OF MOLECULAR DETERMINANTS CRITICAL FOR AUGMENTING KCNQ1 POTASSIUM CHANNELS

Chemical openers for KCNQ potassium channels are useful probes both for understanding channel gating and for developing therapeutics. The five KCNQ isoforms (KCNQ1 to KCNQ5, or Kv7.1 to Kv7.5) are differentially localized. Therefore, the molecular specificity of chemical openers is an important subject of investigation. Native KCNQ1 normally exists in complex with auxiliary subunits known as KCNE. In cardiac myocytes, the KCNQ1-KCNE1 (IsK or minK) channel is thought to underlie the I(Ks) current, a component critical for membrane repolarization during cardiac action potential. Hence, the molecular and pharmacological differences between KCNQ1 and KCNQ1-KCNE1 channels have been important topics. Zinc pyrithione (ZnPy) is a newly identified KCNQ channel opener, which potently activates KCNQ2, KCNQ4, and KCNQ5. However, the ZnPy effects on cardiac KCNQ1 potassium channels remain largely unknown. Here we show that ZnPy effectively augments the KCNQ1 current, exhibiting an increase in current amplitude, reduction of inactivation, and slowing of both activation and deactivation. Some of these are reminiscent of effects by KCNE1. In addition, neither the heteromultimeric KCNQ1-KCNE1 channels nor native I(Ks) current displayed any sensitivity to ZnPy, indicating that the static occupancy by a KCNE subunit desensitizes the reversible effects by a chemical opener. Site-directed mutagenesis of KCNQ1 reveals that residues critical for the potentiation effects by either ZnPy or KCNE are clustered together in the S6 region overlapping with the critical gating determinants. Thus, the convergence of potentiation effects and molecular determinants critical for both an auxiliary subunit and a chemical opener argue for a mechanistic overlap in causing potentiation.

Molecular, pharmacological and functional properties of GABAA receptors in anterior pituitary cells

Anterior pituitary cells express gamma-aminobutyric acid (GABA)-A receptor-channels, but their structure, distribution within the secretory cell types, and nature of action have not been clarified. Here we addressed these questions using cultured anterior pituitary cells from postpubertal female rats and immortalized alphaT3-1 and GH(3) cells. Our results show that mRNAs for all GABA(A) receptor subunits are expressed in pituitary cells and that alpha1/beta1 subunit proteins are present in all secretory cells. In voltage-clamped gramicidin-perforated cells, GABA induced dose-dependent increases in current amplitude that were inhibited by bicuculline and picrotoxin and facilitated by diazepam and zolpidem in a concentration-dependent manner. In intact cells, GABA and the GABA(A) receptor agonist muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABA(B) receptor agonist baclofen was ineffective, suggesting that chloride-mediated depolarization activates voltage-gated calcium channels. Consistent with this finding, RT-PCR analysis indicated high expression of NKCC1, but not KCC2 cation/chloride transporter mRNAs in pituitary cells. Furthermore, the GABA(A) channel reversal potential for chloride ions was positive to the baseline membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells and modulation of spontaneous electrical activity. These results indicate that secretory pituitary cells express functional GABA(A) receptor-channels that are depolarizing.

Anterior pituitary cells excited by GABA

The pituitary, sometimes called the master gland due to its governance over other glands, consists of three distinct anatomical and functional parts: posterior, intermediate (pars intermedia) and anterior lobes (Fig. 1). While the posterior pituitary consists of axon terminals originating from neurons with somata in the hypothalamus, cells in the pars intermedia release peptidergic hormones under the control of neurotransmitters released into the synaptic cleft from the innervating neurons. On the other hand, the secretory activity of anterior pituitary cells, which control important bodily functions including growth, development, reproduction and responses to stress, release hormones in response to blood-borne hypothalamic factors. Following their interaction with specific surface membrane receptors and subsequent activation of intracellular signalling mechanisms they control exocytosis and thus the secretory output from anterior pituitary cells. Figure 1 Diagram depicting hypothalamus pituitary gland and an anterior pituitary cell with the GABAA receptor, Na+-K−-2Cl− co-transporter (NKCCi), the K+-Cl− co-transporter (KCC2), voltage-dependent calcium channel (VDCC). The paper by Zemkova et al. (2008) in this issue of The Journal of Physiology, brings a new insight into the regulation of anterior pituitary cells. It seems that a novel regulating factor, γ-aminobutyric acid (GABA), will have to be firmly positioned into the list of factors stimulating hormone release from these cells. Although it has been known previously that anterior pituitary cells express ionotropic and metabotropic GABA receptors which mediate the secretory hormonal output from these cells (Anderson & Mitchell, 1986; Nakayama et al. 2006), it was unclear how the ionotropic GABA receptors, which gate chloride-specific channels, function in the stimulus–secretion coupling. Specifically, it was controversial whether GABA was stimulatory or inhibitory for the secretory output of anterior pituitary cells. To address these issues Zemkova et al. (2008) first used molecular biology techniques to study the expression of molecular subunits of GABAA receptor channels in cultured anterior pituitary cells from postpubertal female rats and immortalized αT3-1 and GH3 clonal pituitary cells. Interestingly, mRNAs for all GABAA receptor subunits were found to be expressed in pituitary cells and α1/β1 subunit proteins are present in all secretory cells. Second, by using electrophysiology to voltage clamp gramicidin-perforated cells to monitor GABA-induced currents, they have shown that GABA-induced dose-dependent increases in current amplitude were inhibited by bicuculline and picrotoxin, inhibitors of GABAA receptor channels, and facilitated by diazepam and zolpidem in a concentration-dependent manner. Third, they used fluorescence methods to test whether the application of GABA and the GABAA receptor agonist muscimol affected cytosolic calcium activity, an important cytosolic stimulus of anterior pituitary cell secretory activity. Since both GABA and muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABAB receptor agonist baclofen was ineffective, their data are consistent with the view that chloride-mediated depolarization activates voltage-gated calcium channels. Furthermore, the GABAA channel reversal potential for chloride ions was positive to the resting membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells. This prompted Zemkova et al. (2008) to consider the mechanism of chloride homeostasis, which in most brain cells is controlled by NKCC1 and KCC2, two electrically neutral cation/chloride cotransporters. The ubiquitously expressed NKCC1 derives energy from the electrochemical gradient for Na+ to take up Cl−, promoting cytosolic accumulation of Cl−, whereas KCC2 facilitates Cl− extrusion coupled to the K+ gradient (Fiumelli & Woodin, 2007). The experiments have shown that the mRNA expression of NKCC1 greatly exceeded that of KCC2, which is consistent with the relatively positive reversal potential for chloride currents. While the results of Zemkova et al. (2008) establish the excitatory role of GABA in anterior pituitary cells, a number of questions have now been opened. For example, we will have to consider that GABA is now listed as a regulatory factor of anterior pituitary cells. However, it is unclear if it is delivered from the hypothalamus via the portal vascular system. While GABA is the major inhibitory neurotransmitter in the central nervous system and is released from nerve terminals innervating pars intermedia cells (Schneggenburger & Lopez-Barneo, 1992), the mode of delivery of GABA to the cells in the anterior pituitary is probably different. Is the source of GABA the portal vasculature capturing GABA released from neurons in the median eminence? It is known that radioactive GABA readily accumulates in the pituitary (Kuroda et al. 2000; Duvilanski et al. 2000). Is it possible that GABA is released from anterior pituitary cells and thus can be considered an autocrine regulator? These questions will have to be addressed in the future.

Second coiled‐coil domain of KCNQ channel controls current expression and subfamily specific heteromultimerization by salt bridge networks

KCNQ channels carry the slowly activating, voltage-dependent M-current in excitable cells such as neurons. Although the KCNQ2 homomultimer can form a functional voltage-gated K(+) channel, heteromultimerization with KCNQ3 produces a > 10-fold increase in current amplitude. All KCNQ channels contain double coiled-coil domains (TCC1 and TCC2, or A-domain Head and Tail), of which TCC2 (A-domain Tail) is thought to be important for subunit recognition, channel assembly and surface expression. The mechanism by which TCC2 recognizes and associates with its partner is not fully understood, however. Our aim in the present study was to elucidate the recognition mechanism by examining the phenotypes of TCC2-deletion mutants, TCC2-swapped chimeras and point mutants. Electrophysiological analysis using Xenopus oocytes under two-electrode voltage clamp revealed that homotetrameric KCNQ3 TCC2 is a negative regulator of current expression in the absence of KCNQ2 TCC2. Recent structural analysis of KCNQ4 TCC2 revealed the presence of intercoil salt bridge networks. We therefore swapped the sign of the charged residues reportedly involved in the salt bridge formation and functionally confirmed that the intercoil salt bridge network is responsible for the subunit recognition between KCNQ2 and KCNQ3. Finally, we constructed TCC2-swapped KCNQ2/KCNQ3 mutants with KCNQ1 TCC2 or GCN4-pLI, a coiled-coil domain from an unrelated protein, and found that TCC2 is substitutable and even GCN4-pLI can work as a substitute for TCC2. Our present data provide some new insights into the role played by TCC2 during current expression, and also provide functional evidence of the importance of the intercoil salt bridge network for subunit recognition and coiled-coil formation, as is suggested by recent crystallographic data.

ISA Channel Complexes Include Four Subunits Each of DPP6 and Kv4.2

Kv4 potassium channels produce rapidly inactivating currents that regulate excitability of muscles and nerves. To reconstitute the neuronal A-type current I(SA), Kv4 subunits assemble with DPP6, a single transmembrane domain accessory subunit. DPP6 alters function-accelerating activation, inactivation, and recovery from inactivation-and increases surface expression. We sought here to determine the stoichiometry of Kv4 and DPP6 in complexes using functional and biochemical methods. First, wild type channels formed from subunit monomers were compared with channels carrying subunits linked in tandem to enforce 4:4 and 4:2 assemblies (Kv4.2-DPP6 and Kv4.2-Kv4.2-DPP6). Next, channels were overexpressed and purified so that the molar ratio of subunits in complexes could be assessed by direct amino acid analysis. Both biophysical and biochemical methods indicate that I(SA) channels carry four subunits each of Kv4.2 and DPP6.

Probing the Binding Sites and Mechanisms of Action of Two HumanEther-a-go-go-Related Gene Channel Activators, 1,3-bis-(2-Hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) and 2-[2-(3,4-Dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid (PD307243)

We studied the mechanisms and sites of activator actions of 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro-1H-isoindol-5-ylamino]-nicotinic acid [PD307243 (PD)] and 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea [NS1643 (NS)] on the human ether-a-go-go-related gene (hERG) channel expressed in oocytes and COS-7 cells. PD and NS affected hERG in a concentration-dependent manner, reaching a maximal increase in current amplitude by 100% and > or = 300% (1-s test pulse to 0 mV), with apparent K(d) values of 3 and 20 microM, respectively. Both drugs slowed hERG inactivation. NS additionally shifted the activation curve in the negative direction, accelerated activation, and slowed deactivation. Kinetic model simulations suggested that the activator effects of PD and NS could be largely accounted for by their effects on the hERG gating kinetics. Both drugs worked from outside the cell membrane but their binding sites seemed to be distinctly different. Perturbing the conformation of outer vestibule/external pore entrance (by cysteine substitution at high-impact positions or cysteine side chain modification at intermediate-impact positions) prevented the activator effect of NS but not that of PD. Furthermore, the peptide toxin BeKm-1, which bound to the outer mouth of the hERG channel, suppressed NS effect but potentiated PD effect. We propose that NS is a "gating-modifier": it binds to the outer vestibule/pore entrance of hERG and increases current amplitudes by promoting channel activation while retarding inactivation. The activator effect of PD was prevented by external quaternary ammonium cations or dofetilide, which approached the hERG selectivity filter from opposite sides of the membrane and depleted K(+) ions in the selectivity filter. We suggest that PD may work as a "pore-modifier" of the hERG channel.

Cumulative Activation of Voltage-Dependent KVS-1 Potassium Channels

In this study, we reveal the existence of a novel use-dependent phenomenon in potassium channels, which we refer to as cumulative activation (CA). CA consists of an increase in current amplitude in response to repetitive depolarizing step pulses to the same potential. CA persists for up to 20 s and is similar to a phenomenon called "voltage-dependent facilitation" observed in some calcium channels. The KVS-1 K+ channel, which exhibits CA, is a rapidly activating and inactivating voltage-dependent potassium channel expressed in chemosensory and other neurons of Caenorhabditis elegans. It is unusual in being most closely related to the Shab (Kv2) family of potassium channels, which typically behave like delayed rectifier K+ channels in other species. The magnitude of CA depends on the frequency, voltage, and duration of the depolarizing step pulse. CA also radically changes the activation and inactivation kinetics of the channel, suggesting that the channel may undergo a physical modification in a use-dependent manner; thus, a model that closely simulates the behavior of the channel postulates the existence of two populations of channels, unmodified and modified. Use-dependent changes in the behavior of potassium channels, such as CA observed in KVS-1, could be involved in functional mechanisms of cellular plasticity such as synaptic depression that represent the cellular basis of learning and memory.

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Potentiation of acid-sensing ion channels by sulfhydryl compounds

The acid-sensing ion channels (ASICs) are voltage-independent ion channels activated by acidic extracellular pH. ASICs play a role in sensory transduction, behavior, and acidotoxic neuronal death, which occurs during stroke and ischemia. During these conditions, the extracellular concentration of sulfhydryl reducing agents increases. We used perforated patch-clamp technique to analyze the impact of sulfhydryls on H(+)-gated currents from Chinese hamster ovary (CHO) cells expressing human ASIC1a (hASIC1a). We found that hASIC1a currents activated by pH 6.5 were increased almost twofold by the sulfhydryl-containing reducing agents dithiothreitol (DTT) and glutathione. DTT shifted the pH-dose response of hASIC1a toward a more neutral pH (pH(0.5) from 6.54 to 6.69) and slowed channel desensitization. The effect of reducing agents on native mouse hippocampal neurons and transfected mouse ASIC1a was similar. We found that the effect of DTT on hASIC1a was mimicked by the metal chelator TPEN, and mutant hASIC1a channels with reduced TPEN potentiation showed reduced DTT potentiation. Furthermore, the addition of DTT in the presence of TPEN did not result in further increases in current amplitude. These results suggest that the effect of DTT on hASIC1a is due to relief of tonic inhibition by transition metal ions. We found that all ASICs examined remained potentiated following the removal of DTT. This effect was reversed by the oxidizing agent DTNB in hASIC1a, supporting the hypothesis that DTT also impacts ASICs via a redox-sensitive site. Thus sulfhydryl compounds potentiate H(+)-gated currents via two mechanisms, metal chelation and redox modulation of target amino acids.

G Protein-coupled Receptor Kinase 2-mediated Phosphorylation of Downstream Regulatory Element Antagonist Modulator Regulates Membrane Trafficking of Kv4.2 Potassium Channel

Downstream regulatory element antagonist modulator (DREAM)/potassium channel interacting protein (KChIP3) is a multifunctional protein of the neuronal calcium sensor subfamily of Ca2+-binding proteins with specific roles in different cell compartments. In the nucleus, DREAM acts as a Ca2+-dependent transcriptional repressor, and outside the nucleus DREAM interacts with Kv4 potassium channels, regulating their trafficking to the cell membrane and their gating properties. In this study we characterized the interaction of DREAM with GRK6 and GRK2, members of the G protein-coupled receptor kinase family of proteins, and their phosphorylation of DREAM. Ser-95 was identified as the site phosphorylated by GRK2. This phosphorylation did not modify the repressor activity of DREAM. Mutation of Ser-95 to aspartic acid, however, blocked DREAM-mediated membrane expression of the Kv4.2 potassium channel without affecting channel tetramerization. Treatment with the calcineurin inhibitors FK506 and cyclosporin A also blocked DREAM-mediated Kv4.2 channel trafficking and calcineurin de-phosphorylated GRK2-phosphorylated DREAM in vitro. Our results indicate that these two Ca2+-dependent posttranslational events regulate the activity of DREAM on Kv4.2 channel function.

Auto-modulation of neuroactive steroids on GABA A receptors: A novel pharmacological effect

GABA A receptor function is modulated by various important drugs including neuroactive steroids that act on allosteric modulatory sites and can directly activate GABA A receptor channels at high concentrations. We used whole cell patch-clamp recordings and rapid applications of the neuroactive steroid alphaxalone to investigate repetitive steroid effects. Alphaxalone potentiation of submaximal GABA-evoked currents was enhanced significantly by repetitive coapplications at all investigated recombinant isoforms (α1β3δ, α1β3γ2L, α6β3δ, α6β3γ2L) and at GABA A receptors of differentiated human NT2 neurons. A similar increase of current amplitudes was induced by repetitive applications of a high steroid concentration without GABA. We refer to these reversible effects as auto-modulation because repeated interactions of steroids enhanced their own pharmacological impact at the receptor sites in a time and concentration dependent manner without affecting GABA controls. Pronounced auto-modulatory actions were also measured using the neurosteroid 5α-THDOC in contrast to indiplon, THIP, and pentobarbital indicating a steroid specificity. Protein kinase A inhibition significantly reduced alphaxalone auto-modulation at α1β3γ2L, α6β3γ2L, and α6β3δ subtypes while it enhanced potentiation at α1β3δ isoforms suggesting a crucial influence of receptor subunit composition and phosphorylation for steroid actions. Especially at extrasynaptic GABA A receptor sites containing the δ subunit steroid auto-modulation may have a critical role in enhancing potentiation of GABA-induced currents.

Oxidative modification of M-type K+ channels as a mechanism of cytoprotective neuronal silencing

Voltage-gated K(+) channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2-S3 linker is critical for this effect. Oxidation-induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia-induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M-channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress.

Two PEST‐like motifs regulate Ca2+/calpain‐mediated cleavage of the CaVβ3 subunit and provide important determinants for neuronal Ca2+ channel activity

An increase in intracellular Ca2+ due to voltage-gated Ca2+ (CaV) channel opening represents an important trigger for a number of second-messenger-mediated effects ranging from neurotransmitter release to gene activation. Ca2+ entry occurs through the principal pore-forming protein but several ancillary subunits are known to more precisely tune ion influx. Among them, the CaVbeta subunits are perhaps the most important, given that they largely influence the biophysical and pharmacological properties of the channel. Notably, several functional features may be associated with specific structural regions of the CaVbeta subunits emphasizing the relevance of intramolecular domains in the physiology of these proteins. In the current report, we show that CaVbeta3 contains two PEST motifs and undergoes Ca2+ -dependent degradation which can be prevented by the specific calpain inhibitor calpeptin. Using mutant constructs lacking the PEST motifs, we present evidence that they are necessary for the cleavage of CaVbeta3 by calpain. Furthermore, the deletion of the PEST sequences did not affect the binding of CaVbeta3 to the ion-conducting CaV2.2 subunit and, when expressed in human embryonic kidney-293 cells, the PEST motif-deleted CaVbeta3 significantly increased whole-cell current density and retarded channel inactivation. Consistent with this observation, calpeptin treatment of human embryonic kidney-293 cells expressing wild-type CaVbeta3 resulted in an increase in current amplitude. Together, these findings suggest that calpain-mediated CaVbeta3 proteolysis may be an essential process for Ca2+ channel functional regulation.

Thermal diffusion of indium in perylenetetracarboxylic dianhydride

Current-voltage (I-V) characteristic measurements of Ag/3,4,9,10-Perylenetetracarboxylic Dianhydride/In/Si(001) heterostructures demonstrate that when the structure temperature approaches the In melting point the I-V characteristic changes from rectifying to Ohmic and the current amplitude increases by several orders of magnitude. The synchrotron radiation photoemission investigation of the 3,4,9,10-Perylenetetracarboxylic Dianhydride (PTCDA) layers of the same thickness grown on In in the same temperature range show strong In diffusion throughout the PTCDA layers as thick as 1 μm. The In 4 PTCDA coordination compound was not observed on the PTCDA surface and it appears to be limited to the region near the In/PTCDA interface [1].