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Voltage-gated K+ channels have distinct gates that regulate ion flux: the activation gate (A-gate) formed by the bundle crossing of the S6 transmembrane helices and the slow inactivation gate in the selectivity filter. These two gates are bidirectionally coupled. If coupling involves the rearrangement of the S6 transmembrane segment, then we predict state-dependent changes in the accessibility of S6 residues from the water-filled cavity of the channel with gating. To test this, we engineered cysteines, one at a time, at S6 positions A471, L472, and P473 in a T449A Shaker-IR background and determined the accessibility of these cysteines to cysteine-modifying reagents MTSET and MTSEA applied to the cytosolic surface of inside-out patches. We found that neither reagent modified either of the cysteines in the closed or the open state of the channels. On the contrary, A471C and P473C, but not L472C, were modified by MTSEA, but not by MTSET, if applied to inactivated channels with open A-gate (OI state). Our results, combined with earlier studies reporting reduced accessibility of residues I470C and V474C in the inactivated state, strongly suggest that the coupling between the A-gate and the slow inactivation gate is mediated by rearrangements in the S6 segment. The S6 rearrangements are consistent with a rigid rod-like rotation of S6 around its longitudinal axis upon inactivation. S6 rotation and changes in its environment are concomitant events in slow inactivation of Shaker KV channels.

ShK fromStichodactyla helianthushas established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in actinarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family inActinia tenebrosaandTelmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom ofT.stephensoni. We identified ten SA8 genes in two clusters and six SA8 genes in five clusters forT. stephensoniandA. tenebrosa, respectively. Nine SA8T. stephensonigenes were found in a single cluster and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. Our results provide the first demonstration that SA8 is a unique gene family in actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom ofT.stephensoni.

The hEag1 (Kv10.1) K+ channel is normally found in the brain, but it is ectopically expressed in tumor cells, including osteosarcoma. Based on the pivotal role of ion channels in osteogenesis, we tested whether pharmacological modulation of hEag1 may affect osteogenic differentiation of osteosarcoma cell lines. Using molecular biology (RT-PCR), electrophysiology (patch-clamp) and pharmacology (astemizole sensitivity, IC50 = 0.135 μM) we demonstrated that SaOS-2 osteosarcoma cells also express hEag1 channels. SaOS-2 cells also express to KCa1.1 K+ channels as shown by mRNA expression and paxilline sensitivity of the current. The inhibition of hEag1 (2 μM astemizole) or KCa1.1 (1 mM TEA) alone did not induce Ca2+ deposition in SaOS-2 cultures, however, these inhibitors, at identical concentrations, increased Ca2+ deposition evoked by the classical or pathological (inorganic phosphate, Pi) induction pathway without causing cytotoxicity, as reported by three completer assays (LDH release, MTT assay and SRB protein assay). We observed a similar effect of astemizole on Ca2+ deposition in MG-63 osteosarcoma cultures as well. We propose that the increase in the osteogenic stimuli-induced mineral matrix formation of osteosarcoma cell lines by inhibiting hEag1 may be a useful tool to drive terminal differentiation of osteosarcoma.

The hEag1 (Kv10.1) K

The Cm28 in the venom of Centruroides margaritatus is a short peptide consisting of 27 amino acid residues with a mol wt of 2,820 D. Cm28 has 100× Kd value for KV1.3) did not inhibit KV1.5, KV11.1, KCa1.1, and KCa3.1 K+ channels; NaV1.5 and NaV1.4 Na+ channels; or the hHV1 H+ channel but blocked ∼27% of the KV1.1 current. In a biological functional assay, Cm28 strongly inhibited the expression of the activation markers interleukin-2 receptor and CD40 ligand in anti-CD3–activated human CD4+ effector memory T lymphocytes. Cm28, due to its unique structure, may serve as a template for the generation of novel peptides targeting KV1.3 in autoimmune diseases.

Voltage-dependent potassium channels (K v s) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K + -selective pore. Animal toxins targeting K v s are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K + conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of K v 1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K + channels as a novel pharmacological target and provides a rational framework for drug design.

Kv1.3 K

Many arthropod venom peptides have potential as bioinsecticides, drug leads, and pharmacological tools due to their specific neuromodulatory functions. Assassin flies (Asilidae) are a family of predaceous dipterans that produce a unique and complex peptide-rich venom for killing insect prey and deterring predators. However, very little is known about the structure and function of their venom peptides. We therefore used an E. coli periplasmic expression system to express four disulfide-rich peptides that we previously reported to exist in venom of the giant assassin fly Dolopus genitalis. After purification, each recombinant peptide eluted from a C18 column at a position closely matching its natural counterpart, strongly suggesting adoption of the native tertiary fold. Injection of purified recombinant peptides into blowflies (Lucilia cuprina) and crickets (Acheta domestica) revealed that two of the four recombinant peptides, named rDg3b and rDg12, inhibited escape behaviour in a manner that was rapid in onset (

Voltage dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified to “pore-blockers” that physically plug the ion conduction pathway and “gating modifiers” that disrupt voltage sensor movements. A third group of toxins blocks K+conduction by an unknown mechanism via binding to the channel turrets. Here we show that Cs1, a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+channels as a novel pharmacological target and provides a rational framework for drug design.

Margatoxin (MgTx), an alpha-KTx scorpion toxin, is considered a selective inhibitor of the Kv1.3K + channel. This peptide is widely used in ion channel research; however, a comprehensive study of its selectivity with electrophysiological methods has not been published yet. The lack of selectivity might lead to undesired side effects upon therapeutic application or may lead to incorrect conclusion regarding the role of a particular ion channel in a physiological or pathophysiological response either in vitro or in vivo. Using the patch-clamp technique we characterized the selectivity profile of MgTx using L929 cells expressing mKv1.1 channels, human peripheral lymphocytes expressing Kv1.3 channels and transiently transfected tsA201 cells expressing hKv1.1, hKv1.2, hKv1.3, hKv1.4-IR, hKv1.5, hKv1.6, hKv1.7, rKv2.1, Shaker-IR, hERG, hKCa1.1, hKCa3.1 and hNav1.5 channels. MgTx is indeed a high affinity inhibitor of Kv1.3 (Kd = 11.7 pM) but is not selective, it inhibits the Kv1.2 channel with similar affinity (Kd = 6.4 pM) and Kv1.1 in the nanomolar range (Kd = 4.2 nM). Based on our comprehensive data MgTX has to be considered a non-selective Kv1.3 inhibitor, and thus, experiments aiming at elucidating the significance of Kv1.3 in in vitro or in vivo physiological responses have to be carefully evaluated.

Immunomagnetic cell separation is a powerful tool of immunology, however the presence of beads on the cell surface might alter the electrophysiological and pharmacological parameters of the ion channels expressed in the separated cells. We addressed this issue by measuring the aforementioned properties of Kv1.3, the predominant voltage-gated K+ channel of activated T cells upon immunomagnetic separation. PBMCs from healthy donors were activated then CD4+ cells were separated with REAlease® CD4 Kit (Miltenyi Biotec Inc.). As control, we made stepwise removal of REAlease® antibody-bead complex, immunomagnetic negative separation and FACS. Purity and viability was determined by flow cytometry. Electrophysiology studies were made with patch-clamp technique in whole-cell configuration. The presence of bead-antibody complex statistically significantly increased the time constant (τact) characterizing the activation kinetics of the current as compared to negative selection (p=0.049) and antibody only (p=0.024) controls, however the increase in τact from ~0.5 ms to ~0.6 ms does not bear any biological significance. Other gating parameters of Kv1.3, such as inactivation time constant and midpoint of voltage-dependence of steady-state activation were unaffected by the beads. Moreover, the binding of a peptide (ChTx) and a small organic molecule (TEA) inhibitor to Kv1.3 was oblivious to the presence of beads. In addition, the electrical capacitance of the cell membrane was not altered when beads were attached to the cells. Based on our results we conclude that the application of immunomagnetic cell separation is completely suitable to electrophysiology studies of immune cells.

Voltage-gated K+ (Kv) channel activation depends on interactions between voltage sensors and an intracellular activation gate that controls access to a central pore cavity. Here, we hypothesize that this gate is additionally responsible for closed-state inactivation (CSI) in Kv4.x channels. These Kv channels undergo CSI by a mechanism that is still poorly understood. To test the hypothesis, we deduced the state of the Kv4.1 channel intracellular gate by exploiting the trap-door paradigm of pore blockade by internally applied quaternary ammonium (QA) ions exhibiting slow blocking kinetics and high-affinity for a blocking site. We found that inactivation gating seemingly traps benzyl-tributylammonium (bTBuA) when it enters the central pore cavity in the open state. However, bTBuA fails to block inactivated Kv4.1 channels, suggesting gated access involving an internal gate. In contrast, bTBuA blockade of a Shaker Kv channel that undergoes open-state P/C-type inactivation exhibits fast onset and recovery inconsistent with bTBuA trapping. Furthermore, the inactivated Shaker Kv channel is readily blocked by bTBuA. We conclude that Kv4.1 closed-state inactivation modulates pore blockade by QA ions in a manner that depends on the state of the internal activation gate.

It has been reported earlier that the slow (C-type) inactivated conformation in Kv channels is stabilized by a multipoint hydrogen-bond network behind the selectivity filter. Furthermore, MD simulations revealed that structural water molecules are also involved in the formation of this network locking the selectivity filter in its inactive conformation. We found that the application of an extracellular, but not intracellular, solution based on heavy water (D2O) dramatically slowed entry into the slow inactivated state in Shaker-IR mutants (T449A, T449A/I470A, and T449K/I470C, displaying a wide range of inactivation kinetics), consistent with the proposed effect of the dynamics of structural water molecules on the conformational stability of the selectivity filter. Alternative hypotheses capable of explaining the observed effects of D2O were examined. Increased viscosity of the external solution mimicked by the addition of glycerol had a negligible effect on the rate of inactivation. In addition, the inactivation time constants of K+ currents in the outward and the inward directions in asymmetric solutions were not affected by a H2O/D2O exchange, negating an indirect effect of D2O on the rate of K+ rehydration. The elimination of the nonspecific effects of D2O on our macroscopic current measurements supports the hypothesis that the rate of structural water exchange at the region behind the selectivity filter determines the rate of slow inactivation, as proposed by molecular modeling.

Slow inactivation of Shaker-related voltage-gated potassium channels is caused by closure of the inactivation gate in the selectivity filter while the intracellular activation gate (A-gate), that controls ion access to the central pore cavity, is open ([OI] gate configuration). Using internally applied benzyl-tributylammonium (bTBuA) ions and the trap-door paradigm we suggested earlier that the A-gate in Kv4.1 channels might play a novel role as the inactivation gate and being responsible for closed-state inactivation. The current study investigated whether bTBuA shows the characteristics of QA block in slow-inactivated Shaker-IR having open A-gate ([OI]), and therefore, validate the conclusions drawn for Kv4.1. Currents were recorded in excised inside-out patches expressing T449K Shaker-IR channels that displays relatively fast slow inactivation (τi=15 ms). A train of 5.0-s-long depolarizing pulses to +50 mV was applied and the peak currents were determined upon exposure of the intracellular surface of open or open-inactivated channels to 100 µM bTBuA. We found that bTBuA shows a classical open-channel block when applied intracellularly to Shaker channels having both the A-gate and the P-gate open, similarly to when applied to Kv4.1. Next bTBuA was applied to fully inactivated Shaker channels having [OI] configuration (at +50 mV). We found that the compound becomes trapped behind the activation gate upon repolarization: although excess bTBuA was washed out between two pulses, the peak current of the second pulse was greatly reduced indicating trapping of the blocker. On the contrary, bTBuA fails to block inactivated Kv4.1 channels. Our results therefore validate the use of bTBuA as a tool to prove the hypothesis that the A-gate in fully inactivated Kv4.x channels is closed. Futhermore, bTBuA can be locked into Shaker in the absence of the I470C previously shown to be required for trapping of TEA. Support: KTIA_NAP_13-2-2015-0009.

In the absence of N-type inactivation Shaker potassium channels display slow (C-type) inactivation. Functional and structural studies indicated that channel opening precedes slow inactivation in voltage-gated and KcsA K+ channels, whereas others argued for slow inactivation from the closed sate of the channel as well. None of the previous studies correlated the opening of the of the activation gate (A-gate), formed by the bundle crossing of the S6 segments, with the development of inactivation, which is associated with the structural rearrangement in the selectivity filter. These two gates are coupled and thus, the current experiments addressed the hypothesis that opening of the activation gate must precede slow inactivation.To address this hypothesis we compared the voltage dependence of A-gate opening and that of the development of inactivation in T449A/V474C Shaker-IR channels. Opening of the A-gate was monitored by the accessibility of 474C to Cd2+ from the intracellular side. The membrane potential was changed repeatedly from a holding potential of −120 mV to test potentials ranging from −110 mV to −60 mV in the presence or absence of Cd2+.Our results show that the function describing the voltage dependence of Cd2+ block is shifted toward the negative potentials compared to the voltage dependence of steady-state inactivation curve. This indicates that A-gate opening already occurs at such negative potentials where no inactivation can be detected. Furthermore, the curve representing Cd2+ block is also negative-shifted compared to the voltage dependence of steady-state activation (G-V) curve. This suggests that even at fairly negative holding potentials, at which no macroscopic current can be detected, rare channel openings occur yielding access to the channel cavity. Based on these results we suggest that A-gate opening always precedes structural changes associated with slow inactivation.Supported by OTKA K75904

Crosstalk between the activation and slow inactivation gates in Shaker potassium channels is now well-established. The activation gate perceives the conformation of the inactivation gate (Panyi and Deutsch, 2006, 2007). Closure of the inactivation gate speeds opening and slows closing of the activation gate, i.e., stabilizing the gate in the open configuration. If this coupling involves movement of the S6 transmembrane segment, then we predict state-dependent changes in accessibility of residues lining the channel cavity. We engineered cysteines, one at a time, at positions 470, 471, 472, 473, and 474 in a T449A Shaker-IR background and determined modification rates for the cysteine modifying reagents, MTSET and MTSEA, in the open, closed, and inactivated state of the channel. Neither reagent, applied from the intracellular side, modifies cysteines at 470-474 in the closed state. Both 470C and 474C are rapidly modified in the open state and at approximately one-tenth this rate in the inactivated state. In contrast, 471C is not modified in the open state but can be modified by MTSEA but not MTSET in the inactivated state. Residue 472C cannot be modified in any of the three states. Mutant 473C did not express current. Our findings are consistent with a rotation of S6 in the inactivated state, which increases the accessibility of residue 471 while simultaneously decreasing accessibility of residues 470 and 474. Any model of C-type inactivation in the Shaker Kv channel must conform to these experimental observations.[Supported by NIH grant GM 069837 (CD) and OTKA K 75904 (GP)].

Low-frequency, high-amplitude pH-oscillations observed experimentally in the H2O2-HSO3(-)-S2O3(-) flow reaction system at 21.0 degrees C undergo period-doubling cascades to chemical chaos upon decreasing the temperature to 19.0 degrees C in small steps. Period-4 oscillations are observed at 20.0 degrees C and can be calculated on the basis of a simple model. A reverse transition from chaos to high-frequency limit cycle oscillations is also observable in the reaction system upon decreasing further the temperature step by step to 15.0 degrees C. Period-2 oscillations are measured at 18.0 degrees C. Such a temperature-change-induced transition between periodic and chaotic oscillatory states can be understood by taking into account the different effects of temperature on the rates of composite reactions in the oscillatory system. Small differences in the activation energies of the composite reactions are responsible for the observed transitions. Temperature-change-induced period doubling is suggested as a simple tool for determining whether an experimentally observed random behavior in chemical systems is of deterministic origin or due to experimental noise.

Large-amplitude pH oscillations have been measured during the oxidation of sulfur (IV) species by the bromate ion in aqueous solution in a continuous-flow stirred tank reactor in the absence of any additional oxidizing or reducing reagent. The source of the oscillation in this simple chemical reaction is a two-way oxidation of sulfur (IV) by the bromate ion: (1) the hydrogen-ion-producing self-accelerating oxidation to sulfur (VI) (SO4(2-)), and (2) a hydrogen-ion-consuming oxidation to sulfur (V) (S2O6(2-)). In such a way, both the H+-producing and H+-consuming composite processes required for a pH oscillator take place in parallel in a reaction between two reagents in this system. A simple reaction scheme, consisting of the protonation equilibria of SO3(2-) and HSO3-, the oxidation of HSO3- and H2SO3 by BrO3- to SO4(2-), and the oxidation of H2SO3 to S2O6(2-) has successfully been used to simulate the observed dynamical behavior. Simulation with this simple scheme shows that oscillations can be calculated even if only about 1% of sulfur (IV) is oxidized to S2O6(2-) along with the main product SO4(2-). Agreement between calculated and measured dynamical behavior is found to be quite good. Increasing temperature decreases both the period length of oscillations in a CSTR and the Landolt time measured in a closed reactor. No temperature compensation of the oscillatory frequency is found in this reaction.

It has been reported earlier that the activation and inactivation gates of Shaker channels are coupled, and this coupling might be mediated by a rotational motion of the S6 helix. We hypothese that either the electrostatics or the volume/hydrophobicity of the side chains pointing into the cavity can affect this coupling. Accordingly, we have introduced side-chains at position 470 in a T449A Shaker-IR background with different hydrophobicities according to the Wimley-White hydrophobicity scale (Ala, Cys, or Trp) and charges (Glu, Asp, Arg, Lys) and measured the inactivation kinetics. Mutant channels were transiently expressed in tsA_201 cells. All ionic current experiments were performed with excised inside-out patches. The introduction of an ionizable cysteine and the small but neutral alanine dramatically slowed entry into the inactivated state (the inactivation time constants for Shaker mutant T449A/I470C, T449A/I470A and T449A were 1.4 s, 250 ms and 50 ms, respectively). We could not detect any current on mutant T449A/I470X (X = Asp, Glu, Trp) but the transfection with a mixture of mutant and wild-type expression plasmids expressed currents. These heterotetramers had slower inactivation kinetics with respect to the T449A channels. Almost all of the channels had a midpoint voltage for activation in the range −50 mV to −20 mV. The only exception was the I470C mutant which had a large shift in voltage dependence (midpoint ∼5 mV). Mutant channels containing Arg or Lys (I470R, I470K) did not give functional expression either as homotetramers or as heterotetramers. Our results show that not the size but the physicochemical properties of the side chains (hydrophobicity and charge) at position 470 determine the inactivation kinetics of voltage-gated K+ channels, which may reflect interaction of the side chains with permeant ions.