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Large-conductance Ca2+-dependent K+ (BKCa) channels are activated by intracellular Ca2+ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BKCa-type K+ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BKL. K+ selectivity, high conductance, activation by Mg2+ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BKCa channels. However, unlike conventional BKCa channels, BKL channels activated in the absence of free cytosolic Ca2+ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BKCa channels. Half-maximal Ca2+-dependent activation was observed at 0.4 μ M for BKL (at −20 mV) and at 4.1 μ M for BKCa channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BKL conductance. Expression of hSlo-β 1 in LNCaP cells shifted voltage-dependent activation to values between that of BKL and BKCa channels and reduced the slope of the P open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BKCa subunit, which is responsible for the BKL phenotype in a dominant manner. BKL-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BKL in LNCaP cells is regulated by serum-derived factors, however not by androgens. [ABSTRACT FROM AUTHOR]

One of the major sources of despair for the investigator who hopes to explore properties of the Thematic Apperception Test (TAT) is the deadly business of collecting this kind of data. Clinician and research worker alike find this feature of the test a barrier to learning more about the instrument and thus eventually about human behavior. To some extent the time involved in administering the test may be seen as a matter of inconvenience for the sake of a valued end since the potential power of the instrument is certainly related to the richness of the material it elicits, and this in turn is linked to its awkwardness as a data-collection procedure. However, if the process of data collection can be streamlined without seriously impairing the test's sensitivity, this will clearly represent a valuable contribution. The study reported here addresses itself to the problem of finding out to what extent findings secured from the TAT under conditions of group administration may be considered equivalent to those secured with individual administration. Related to this is the question of whether there is any striking superiority of one method of administration over the other.

Phosphoinositid 3-Kinase γ (PI3Kγ), the only known member of class Ib PI3K's, is activated by G-Protein coupled receptors (GPCRs), producing the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3). In addition, PI3Kγ possesses an intrinsic protein kinase activity and might express a scaffold function in the signaling network of different cells. PI3Kγ is expressed in hematopoietic cells acting as a main mediator of proliferation, differentiation, migration and survival of leukocytes. Recent studies expand these well-established cell specific functions of PI3Kγ to other cell types like cardiomyocytes. In heart PI3Kγ exhibits a negative regulatory function on the contractility by controlling the intracellular camp-level in concert with Phosphodiesterase 3B (PDE3B). In the present study we describe for the first time PI3Kγ expression and functional pattern in cell types of neuronal origin. PI3Kγ-protein is expressed in small diameter neurons of dorsal root ganglia (DRG), which form widely unmyelinated C-fibers into the body's periphery. These primary sensory neurons exhibit nociceptive functions and can lead to a slow, dull and long lasting pain perception. These neurons are heavily involved in the processes of neuropathic and inflammatory pain accompanied by molecular modifications in the interplay of extra- and intracellular milieus -- the playground of PI3Kγ. Now we take advantage of the PI3Kγ-knockout mice to evaluate the impact of PI3Kγ in behavioral studies and primary dorsal root ganglia cultures. Analysis of nociceptive behaviors and studies on DRG-cultures suggest an involvement of the signaling protein in the μ-opioid-receptor path. An important regulation of the opioid-receptor system is the sensitization and desensitization of the receptors. Effects of different μ-receptor-agonists point to a differential involvement of PI3Kγ in antinociceptive signaling in primary sensory neurons. [ABSTRACT FROM AUTHOR]

Abstract Voltage-activated, Ca2+ -sensitive K+ channels (BK or maxi K,Ca channels) play a major role in the control of neuronal excitability. We have cloned pSlo, the BK channel α subunit of the cockroach Periplaneta americana . The amino acid sequence of pSlo shows 88% identity to dSlo from Drosophila . There are five alternatively spliced positions in pSlo showing differential expression in various tissues. A pSlo-specific antibody prominently stained the octopaminergic dorsal unpaired median (DUM) neurons and peptidergic midline neurons in Periplaneta abdominal ganglia. HEK293 cells expressing pSlo exhibit K+ channels of 170 pS conductance. They have a tendency for brief closures, exhibit subconductance states and show slight inward rectification. Activation kinetics and voltage dependence are controlled by cytoplasmic [Ca2+ ]. In contrast to dSlo, pSlo channels are sensitive to charybdotoxin and iberiotoxin. Mutagenesis at two positions (E254 and Q285) changed blocking efficacy of charybdotoxin. In contrast to pSlo expressed in HEK293 cells, native IbTx-sensitive K,Ca currents in DUM and in peptidergic neurons, exhibited rapid, partial inactivation. The fast component of the K,Ca current partly accounts for the repolarization and the early after-hyperpolarization of the action potential. By means of Ca2+ -induced repolarization, BK channels may reduce the risk of Ca2+ overload in cockroach neurons. Interestingly, the neurons expressing pSlo were also found to express taurine, a messenger that is likely to limit overexcitation by an autocrine mechanism in mammalian central neurons. [ABSTRACT FROM AUTHOR]

Voltage- and Ca(2+)-activated K(+) channels of big conductance (BK channels) are abundantly found in various organs and their relevance for smooth muscle tone and neuronal signaling is well documented. Dysfunction of BK channels is implicated in an array of human diseases involving many organs including the nervous, pulmonary, cardiovascular, renal, and urinary systems. In humans a single gene (KCNMA1) encodes the pore-forming α subunit (Slo1) of BK channels, but the channel properties are variable because of alternative splicing, tissue- and subcellular-specific auxiliary subunits (β, γ), posttranslational modifications, and a multitude of endogenous signaling molecules directly affecting the channel function. Initiatives to develop drugs capable of activating BK channels (channel openers) therefore need to consider the tissue-specific variability of BK channel structure and the potential interference with endogenously produced regulatory factors. The atomic structural basis of BK channel function is only beginning to be revealed. However, building on detailed knowledge of BK channel function, including its single-channel characteristics, voltage- and Ca(2+) dependence of channel gating, and modulation by diffusible messengers, a multi-tier allosteric model of BK channel gating (Horrigan and Aldrich (HA) model) has become a valuable tool in studying modulation of the channel. Using the conceptual framework of the HA model, we here review the functional impact of endogenous modulatory factors and select small synthetic compounds that regulate BK channel activity. Furthermore, we devise experimental approaches for studying BK channel-drug interactions with the aim to classify BK-modulating substances according to their molecular mode of action., (© 2016 Elsevier Inc. All rights reserved.)

G protein-coupled opioid receptors undergo desensitization after prolonged agonist exposure. Recent in vitro studies of mu-opioid receptor (MOR) signaling revealed an involvement of phosphoinositide 3-kinases (PI3K) in agonist-induced MOR desensitization. Here we document a specific role of the G protein-coupled class IB isoform PI3Kgamma in MOR desensitization in mice and isolated sensory neurons. The tail-withdrawal nociception assay evidenced a compromised morphine-induced tolerance of PI3Kgamma-deficient mice compared to wild-type animals. Consistent with a role of PI3Kgamma in MOR signaling, PI3Kgamma was expressed in a subgroup of small-diameter dorsal root ganglia (DRG) along with MOR and the transient receptor potential vanilloid type 1 (TRPV1) receptor. In isolated DRG acute stimulation of MOR blocked voltage-gated calcium currents (VGCC) in both wild-type and PI3Kgamma-deficient DRG neurons. By contrast, following long-term opioid administration the attenuating effect of MOR was strongly compromised in wild-type DRG but not in PI3Kgamma-deficient DRG. Our results uncover PI3Kgamma as an essential modulator of long-term MOR desensitization and tolerance development induced by chronic opioid treatment in sensory neurons., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Large-conductance Ca2+-dependent K+ (BK(Ca)) channels are activated by intracellular Ca2+ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK(Ca)-type K+ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK(L). K+ selectivity, high conductance, activation by Mg2+ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK(Ca) channels. However, unlike conventional BK(Ca) channels, BK(L) channels activated in the absence of free cytosolic Ca2+ at physiological membrane potentials; the half-maximal activation voltage was shifted by about -100 mV compared with BK(Ca) channels. Half-maximal Ca2+-dependent activation was observed at 0.4 microM: for BK(L) (at -20 mV) and at 4.1 microM: for BK(Ca) channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK(L) conductance. Expression of hSlo-beta1 in LNCaP cells shifted voltage-dependent activation to values between that of BK(L) and BK(Ca) channels and reduced the slope of the P (open) (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK(Ca) subunit, which is responsible for the BK(L) phenotype in a dominant manner. BK(L)-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK(L) in LNCaP cells is regulated by serum-derived factors, however not by androgens.

Human IGR1 cells are a model for malignant melanoma. Since progression through the cell cycle is accompanied by transient cell hyperpolarization, we studied the properties of potassium and chloride ion channels and their impact on cell growth. The major potassium current components were mediated by outward rectifying ether à go-go (hEAG) channels and Ca2+-activated channels (KCa) of the IK/SK type. The major chloride channel component was activated by osmotic cell swelling (Clvol). To infer about the contribution of these channels to proliferation, specific inhibitors are required. Since there is no specific blocker for hEAG available, we used the tricyclic antidepressant imipramine, which blocked all channels mentioned, in combination with blockers for KCa (charybdotoxin) and Clvol (DIDS and pamoic acid). Incubation of IGR1 cells for 48 hr in 10-15 mM imipramine reduced DNA synthesis and metabolism without significant effects on apoptosis. hEAG channels were most sensitive to imipramine (IC50: 3.4 microM at +50 mV), followed by KCa (13.8 microM at +50 mV) and Clvol (12 microM at -100 mV), indicating that hEAG expression may be of importance for proliferation of melanoma cells. The contribution of KCa channels could be excluded, as 500 nM charybdotoxin, which completely blocked KCa, had no effect on proliferation. The impact of Clvol also seems to be minor, because 500 microM pamoic acid, which completely blocked Clvol, did not affect proliferation either.

P/Q-type Ca(2+) channels, which are postulated to play major roles in synaptic transmission, are regulated in a variety of ways. Ca(2+) currents through P/Q-type Ca(2+) channels (Ca(v)2.1/beta(1a)/alpha(2)delta) heterologously expressed in mammalian cells were recorded using the whole-cell patch clamp method. The oxidant H(2)O(2) increased the current amplitude and the effect was reversed by the reducing agent dithiothreitol (DTT). The stimulatory effect of H(2)O(2) on the Ca(2+) current was mimicked by the NO donors, SNAP, and diethylamine NONOate, and reversed by the reducing agent DTT. The presence of a soluble guanylate cyclase inhibitor did not abolish the ability of SNAP to increase the Ca(2+) current. Adenovirus-mediated overexpression of nitric oxide synthase in combination with application of the Ca(2+) ionophore A23187 also increased the Ca(2+) current amplitude and the effect was again reversed by DTT. The NOS inhibitor L-NAME abolished the stimulatory effect of A23187, and A23187 did not change the Ca(2+) currents in the cells treated with control adenovirus particles. The time course of the decline of the Ca(2+) current, but not of the Ba(2+) current, in response to repeated depolarization was markedly slowed by adenovirus-mediated overexpression of nitric oxide synthase. The results demonstrate that nitric oxide enhances the channel activity by promoting oxidation and suggest that Ca(2+), nitric oxide synthase, and nitric oxide could constitute a positive feedback loop for regulation of voltage-gated P/Q-type Ca(2+) channels.

Type-I phosphoinositide 3-kinases (PI3Ks) were characterized as a group of intracellular signalling proteins expressing both protein and lipid kinase activities. Recent studies implicate PI3Ks as mediators of oocyte maturation, but the molecular mechanisms are poorly defined. Here we used the Xenopus oocyte expression system as a model to investigate a possible contribution of the gamma-isoform of PI3K (PI3Kgamma) in the different pathways leading to cell-cycle progression by monitoring the time course of germinal vesicle breakdown (GVBD). Expression of a constitutive active PI3Kgamma (PI3Kgamma-CAAX) induced GVBD and increased the levels of phosphorylated Akt/protein kinase B and mitogen-activated protein kinase (MAPK). Furthermore, PI3Kgamma-CAAX accelerated progesterone-induced GVBD, but had no effect on GVBD induced by insulin. The effects of PI3Kgamma-CAAX could be suppressed by pre-incubation of the oocytes with LY294002, PD98059 or roscovitine, inhibitors of PI3K, MEK (MAPK/extracellular-signal-regulated protein kinase kinase) and cdc2/cyclin B kinase, respectively. Mutants of PI3Kgamma-CAAX, in which either lipid kinase or both lipid and protein kinase activities were altered or eliminated, did not induce significant GVBD. Our data demonstrate that expression of PI3Kgamma in Xenopus oocytes accelerates their progesterone-induced maturation and that lipid kinase activity is required to induce this effect.

Binding of scorpion alpha-toxins to receptor site 3 on voltage-gated sodium channels inhibits sodium current inactivation and is voltage-dependent. To reveal the direct effect of depolarization, we analyzed binding kinetics of the alpha-toxin Lqh-II (from Leiurus quinquestriatus hebraeus) to rat brain synaptosomes and effects on rat brain II (rBII) channels expressed in mammalian cells. Our results indicated that the 33-fold decrease in toxin affinity for depolarized (0 mV, 90 mM [K(+)](out), K(d) = 5.85 +/- 0.5 nM) versus polarized (-55 mV, 5 mM [K(+)](out), K(d) = 0.18 +/- 0.04 nM) synaptosomes at steady state results from a 48-fold reduction in the association rate (k(on) at 5 mM [K(+)] = (12.0 +/- 4) x 10(6) M(-1) s(-1) and (0.25 +/- 0.03) x 10(6) M(-1) s(-1) at 90 mM [K(+)](out)) with nearly no change in the dissociation rate. Electrophysiological analyses of rBII channels expressed in mammalian cells revealed that approximately 75% and 40% of rBII occupied fast- and slow-inactivated states, respectively, at resting membrane potential of synaptosomes (-55 mV), and Lqh-II markedly increased the steady-state fast and slow inactivation. To mimic electrophysiological conditions we induced fast depolarization of toxin-bound synaptosomes, which generated a biphasic unbinding of Lqh-II from toxin-receptor complexes. The first fast off rate closely resembled values determined electrophysiologically for rBII in mammalian cells. The second off rate was similar to the voltage-independent steady-state value, attributed to binding to the slow-inactivated channel states. Thus, the Lqh-II voltage-dependent affinity highlights two independent mechanisms representing conformational changes of sodium channels associated with transitions among electrically visible and invisible inactivated states.

The effects of the scorpion alpha-toxins Lqh II, Lqh III, and LqhalphaIT on human cardiac sodium channels (hH1), which were expressed in human embryonic kidney (HEK) 293 cells, were investigated. The toxins removed fast inactivation with EC(50) values of <2.5 nM (Lqh III), 12 nM (Lqh II), and 33 nM (LqhalphaIT). Association and dissociation rates of Lqh III were much slower than those of Lqh II and LqhalphaIT, such that Lqh III would not dissociate from the channel during a cardiac activation potential. The voltage dependence of toxin dissociation from hH1 channels was nearly the same for all toxins tested, but it was different from that found for skeletal muscle sodium channels (muI; Chen et al. 2000). These results indicate that the voltage dependence of toxin binding is a property of the channel protein. Toxin dissociation remained voltage dependent even at high voltages where activation and fast inactivation is saturated, indicating that the voltage dependence originates from other sources. Slow inactivation of hH1 and muI channels was significantly enhanced by Lqh II and Lqh III. The half-maximal voltage of steady-state slow inactivation was shifted to negative values, the voltage dependence was increased, and, in particular for hH1, slow inactivation at high voltages became more complete. This effect exceeded an expected augmentation of slow inactivation owing to the loss of fast inactivation and, therefore, shows that slow sodium channel inactivation may be directly modulated by scorpion alpha-toxins.

Given the anatomical and physiological similarities to the human heart, canine in vivo heart models may facilitate the analysis of molecular mechanisms underlying cardiac repolarization abnormalities. The development of such models depends, however, on information about canine K+ channels responsible for the establishment of IK currents. In this context, we isolated and sequenced the reverse transcript of the canine ether à go-go-related gene (cERG). The complementary deoxyribonucleic acid (cDNA-derived cERG polypeptide consists of 1,158 amino acids, the sequence of which shows striking homology to human, rat and mouse ERG subunits (97%, 94% and 95% identity respectively). In highly conserved peptide domains like the PAS domain, the membrane-spanning segments S1, S3-S6 and the pore-forming region, there was 100% identity. Analysis of cERG transcription revealed abundant expression of cERG messenger ribonucleic acid (mRNA) in heart and brain and low expression in liver, spleen and kidney. Membrane currents recorded in Xenopus oocytes expressing cERG channels showed functional properties very similar to the human K+ channel hERG, which encodes the alpha-subunit of the cardiac rapidly activating, delayed rectifier (IKr) channel.

Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca(2+)-activated K(+) channels (BK(Ca) or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca(2+), the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K(+) channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism.

Scorpion alpha-toxins from Leiurus quinquestriatus hebraeus, LqhII and LqhIII, are similarly toxic to mice when administered by a subcutaneous route, but in mouse brain LqhII is 25-fold more toxic. Examination of the two toxins effects in central nervous system (CNS), peripheral preparations and expressed sodium channels revealed the basis for their differential toxicity. In rat brain synaptosomes, LqhII binds with high affinity, whereas LqhIII competes only at high concentration for LqhII-binding sites in a voltage-dependent manner. LqhII strongly inhibits sodium current inactivation of brain rBII subtype expressed in HEK293 cells, whereas LqhIII is weakly active at 2 microM, suggesting that LqhIII affects sodium channel subtypes other than rBII in the brain. In the periphery, both toxins inhibit tetrodotoxin-sensitive sodium current inactivation in dorsal root ganglion neurons, and are strongly active directly on the muscle and on expressed muI channels. Only LqhII, however, induced repetitive end-plate potentials in mouse phrenic nerve-hemidiaphragm muscle preparation by direct effect on the motor nerve. Thus, rBII and sodium channel subtypes expressed in peripheral nervous system (PNS) serve as the main targets for LqhII but are mostly not sensitive to LqhIII. Toxicity of both toxins in periphery may be attributed to the direct effect on muscle. Our data elucidate, for the first time, how different toxins affect mammalian central and peripheral excitable cells, and reveal unexpected subtype specificity of toxins that interact with receptor site 3.

Intracellular Ca(2+) inhibits voltage-gated potassium channels of the ether à go-go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in Xenopus oocytes. The channels lost Ca(2+) sensitivity when measured in cell-free membrane patches. However, Ca(2+) sensitivity could be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca(2+). Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrated direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673-770). Point mutations within this section revealed a novel CaM-binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca(2+)-activated potassium channels, Ca(2+) dependent, with an apparent K(D) of 480 nM. Co-expression experiments of wild-type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition.

Time constants of slow inactivation were investigated in NH(2)-terminal deleted Shaker potassium channels using macro-patch recordings from Xenopus oocytes. Slow inactivation is voltage insensitive in physiological solutions or in simple experimental solutions such as K(+)(o)//K(+)(i) or Na(+)(o)//K(+)(i). However, when [Na(+)](i) is increased while [K(+)](i) is reduced, voltage sensitivity appears in the slow inactivation rates at positive potentials. In such solutions, the I-V curves show a region of negative slope conductance between approximately 0 and +60 mV, with strongly increased outward current at more positive voltages, yielding an N-shaped curvature. These changes in peak outward currents are associated with marked changes in the dominant slow inactivation time constant from approximately 1.5 s at potentials less than approximately +60 mV to approximately 30 ms at more than +150 mV. Since slow inactivation in Shaker channels is extremely sensitive to the concentrations and species of permeant ions, more rapid entry into slow inactivated state(s) might indicate decreased K(+) permeation and increased Na(+) permeation at positive potentials. However, the N-shaped I-V curve becomes fully developed before the onset of significant slow inactivation, indicating that this N-shaped I-V does not arise from permeability changes associated with entry into slow inactivated states. Thus, changes in the relative contributions of K(+) and Na(+) ions to outward currents could arise either: (a) from depletions of [K(+)](i) sufficient to permit increased Na(+) permeation, or (b) from voltage-dependent changes in K(+) and Na(+) permeabilities. Our results rule out the first of these mechanisms. Furthermore, effects of changing [K(+)](i) and [K(+)](o) on ramp I-V waveforms suggest that applied potential directly affects relative permeation by K(+) and Na(+) ions. Therefore, we conclude that the voltage sensitivity of slow inactivation rates arises indirectly as a result of voltage-dependent changes in the ion occupancy of these channels, and demonstrate that simple barrier models can predict such voltage-dependent changes in relative permeabilities.

The scorpion alpha-toxins Lqh II, Lqh III, and Lqh alphaIT from Leiurus quinquestriatus hebraeus are representatives of typical alpha-toxins, specific for either mammals (Lqh II) or insects (Lqh alphaIT), and alpha-like toxins (Lqh III) which act on both mammals and insects. For a comparative study of the effects of these toxins on mammalian sodium channels we stably expressed rat skeletal muscle sodium channel alpha subunits (microI) in HEK 293 cells and measured Na+ currents in the whole-cell patch-clamp mode. The alpha- and alpha-like toxins strongly slowed down channel inactivation with a half-maximal effect at 1.4 nM (Lqh II), 5.4 nM (Lqh III), and 0.5 nM (Lqh alphaIT). The recovery from fast inactivation was accelerated by all toxins with the potency sequence: Lqh II>Lqh alphaIT>Lqh III. The voltage dependence of inactivation and recovery from inactivation were reduced while the threshold for activation was only slightly shifted by approximately equal to 10 mV without altering the slope factors, suggesting uncoupling of the impaired inactivation from the activation. The toxins induced an increase in peak inward current, which was accounted for by an increased maximal open-channel probability. Although all three toxins induced similar modifications of the channel properties, their kinetics of association and dissociation were very different. Between -140 and -80 mV toxin association was not voltage dependent. In 100 nM toxin the association time constants were: 1.3 s (Lqh II), 20 s (Lqh III), and 3.8 s (Lqh alphaIT). At positive voltages the toxin dissociated from the channel; at +100 mV the dissociation time constants were 30, 321, and 135 ms, respectively. In contrast to the association, dissociation was voltage dependent with a similar slope of about 12 mV per e-fold change for all three toxins. The strong differences in the association and dissociation kinetics of these toxins may identify them as members of different scorpion alpha-toxin subgroups.

Reactive oxygen species (ROS) and nitric oxide (NO) are important participants in signal transduction that could provide the cellular basis for activity-dependent regulation of neuronal excitability. In young rat cortical brain slices and undifferentiated PC12 cells, paired application of depolarization/agonist stimulation and oxidation induces long-lasting potentiation of subsequent Ca(2+) signaling that is reversed by hypoxia. This potentiation critically depends on NO production and involves cellular ROS utilization. The ability to develop the Ca(2+) signal potentiation is regulated by the developmental stage of nerve tissue, decreasing markedly in adult rat cortical neurons and differentiated PC12 cells.

Oxidation of amino acid residues causes noticeable changes in gating of many ion channels. We found that P/C-type inactivation of Shaker potassium channels expressed in Xenopus oocytes is irreversibly accelerated by patch excision and that this effect was mimicked by application of the oxidant H(2)O(2), which is normally produced in cells by the dismutase action on the superoxide anion. The inactivation time course was also accelerated by high concentration of O(2). Substitution of a methionine residue located in the P-segment of the channel with a leucine largely eliminated the channel's sensitivity to patch excision, H(2)O(2), and high O(2). The results demonstrate that oxidation of methionine is an important regulator of P/C-type inactivation and that it may play a role in mediating the cellular responses to hypoxia/hyperoxia.