Channel Activation Process Research Articles

How can we learn what attention is? Response gating via multiple direct routes kept in check by inhibitory control processes

AbstractTo explore the time course of space- and object-based attentional selection processes I analysed the shapes of the response time (RT) and accuracy distributions of left/right arrow identification responses in the two-rectangle paradigm. After cueing one of the four ends of two horizontally or vertically oriented rectangles the arrow typically appears at the cued location (valid), or sometimes at an uncued location in the same (invalid-same) or other rectangle (invalid-different). The data point to a multiple-route model in which (a) an informative cue generates response channel activation before arrow signals emerge, (b) the task-irrelevant arrow location is represented in multiple egocentric and allocentric reference frames around 150 ms after target onset, with the former including a reference frame centered on the currently attended location, (c) the task-irrelevant spatial codes activate premature response tendencies that are actively inhibited to allow gating of arrow direction signals, (d) after an invalid cue the onset of the arrow triggers an “attention shift” – acting between 150 and 240 ms after target onset – that strongly interferes with task performance in certain conditions (invalid-same cueing with horizontal rectangles, and invalid-different cueing with vertical rectangles), and (e) participants differ in which task-irrelevant codes they preferentially inhibit. These results pave the way for future confirmatory studies to temporally characterize and disentangle the contributions of different types of response channel activation processes, from those of reactive cognitive control processes including active and selective response suppression.

Proton Transport Mechanism of M2 Proton Channel Studied by Laser-Induced pH Jump.

The M2 proton transport channel of the influenza virus A is an important model system because it conducts protons with high selectivity and unidirectionally when activated at low pH, despite the relative simplicity of its structure. Although it has been studied extensively, the molecular details of the pH-dependent gating and proton conductance mechanisms are incompletely understood. We report direct observation of the M2 proton channel activation process using a laser-induced pH jump coupled with tryptophan fluorescence as a probe. Biphasic kinetics is observed, with the fast phase corresponding to the His37 protonation, and the slow phase associated with the subsequent conformation change. Unusually fast His37 protonation was observed (2.0 × 1010 M-1 s-1), implying the existence of proton collecting antennae for expedited proton transport. The conformation change (4 × 103 s-1) was about 2 orders of magnitude slower than protonation at endosomal pH, suggesting that a transporter model is likely not feasible.

Gating Mechanisms of Store-Operated CRAC Channels

In many animal cells, stimulation of cell surface receptors coupled to G proteins or tyrosine kinases mobilizes Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. The ensuing Ca2+ entry regulates a wide variety of effector cell responses including transcription, motility, and proliferation. The physiological importance of CRAC channels for human health is underscored by studies indicating that mutations in CRAC channel genes produce a spectrum of devastating diseases including chronic inflammation, muscle weakness, and a severe combined immunodeficiency syndrome. Moreover, from a basic science perspective, CRAC channels exhibit a unique biophysical fingerprint characterized by exquisite Ca2+-selectivity, store-operated gating, and distinct pore properties and therefore serve as fascinating ion channels for understanding the biophysical mechanisms of ion permeation and gating. Studies in the last decade have revealed the cellular and molecular choreography of the CRAC channel activation process, and it is now established that opening of CRAC channels is governed through direct interactions between the pore-forming Orai proteins, and the ER Ca2+ sensors, STIM1 and STIM2. Here, I will discuss recent work in our laboratory that seeks to understand the molecular rearrangements in the CRAC channel protein underlying the opening of the pore.

High glucose enhances store-operated calcium entry by upregulating ORAI/STIM via calcineurin-NFAT signalling.

ORAI and stromal interaction molecule (STIM) are store-operated channel molecules that play essential roles in human physiology through a coupling mechanism of internal Ca(2+) store to Ca(2+) influx. However, the roles of ORAI and STIM in vascular endothelial cells under diabetic conditions remain unknown. Here, we investigated expression and signalling pathways of ORAI and STIM regulated by high glucose or hyperglycaemia using in vitro cell models, in vivo diabetic mice and tissues from patients. We found that ORAI1-3 and STIM1-2 were ubiquitously expressed in human vasculatures. Their expression was upregulated by chronic treatment with high glucose (HG, 25mM D-glucose), which was accompanied by enhanced store-operated Ca(2+) influx in vascular endothelial cells. The increased expression was also observed in the aortae from genetically modified Akita diabetic mice (C57BL/6-Ins2(Akita)/J) and streptozocin-induced diabetic mice, and aortae from diabetic patients. HG-induced upregulation of ORAI and STIM genes was prevented by the calcineurin inhibitor cyclosporin A and NFATc3 siRNA. Additionally, in vivo treatment with the nuclear factor of activated T cells (NFAT) inhibitor A-285222 prevented the gene upregulation in Akita mice. However, HG had no direct effects on ORAI1-3 currents and the channel activation process through cytosolic STIM1 movement in the cells co-expressing STIM1-EYFP/ORAIs. We concluded that upregulation of STIM/ORAI through Ca(2+)-calcineurin-NFAT pathway is a novel mechanism causing abnormal Ca(2+) homeostasis and endothelial dysfunction under hyperglycaemia. ORAI1-3 and STIM1-2 are ubiquitously expressed in vasculatures and upregulated by high glucose. Increased expression is confirmed in Akita (Ins2(Akita)/J) and STZ diabetic mice and patients. Upregulation mechanism is mediated by Ca(2+)/calcineurin/NFATc3 signalling. High glucose has no direct effects on ORAI1-3 channel activity and channel activation process.

Stim1 Cytosolic Coiled-Coil Interactions in the Resting and Activated State

STIM1 and Orai1 are key components of the Ca2+-release activated Ca2+ (CRAC) current that plays an important role in T cell activation as well as mast cell degranulation. Activation of the CRAC channel forming subunit Orai1 occurs via a physical interaction with the ER transmembrane Ca2+ sensor protein STIM1 when ER Ca2+ stores are depleted. This CRAC channel activation process is accompanied by a conformational change of STIM1 into an extended conformation together with puncta formation. Consequently SOAR/CAD is exposed during this process and drives oligomerization, probably by interhelical rearrangements between the three cytosolic STIM1 coiled-coil (CC) domains. Here we focused on intra- and inter-molecular interactions specifically between CC2/CC3 and the three α-helices comprising CC1. In an attempt to differentially examine and map possible interactions between these three α-helices and the SOAR/CAD comprising CC2 and CC3, a system termed “FRET-based Interactions in Restricted Environments (FIRE)” was developed. Furthermore, single point mutations were introduced into these helical fragments to eliminate or strengthen their interactions. In extension of these results, we additionally inserted point mutations and deletions into full length STIM1 and the YFP-OASF-CFP-FRET sensor for further functional analysis by patch-clamp and FRET measurements. Our results revealed new insights into the mechanism linking STIM1 oligomerization to the differential interactions of specific α-helices of CC1 with CAD/SOAR and allowed us to delineate a model describing STIM1 activation following store depletion. (supported by Austrian Science Fund (FWF): P22747 to R.S., P22565 and P25172 to C.R.)

Contribution of the KCa3.1 channel-calmodulin interactions to the regulation of the KCa3.1 gating process.

The Ca2+-activated potassium channel of intermediate conductance, KCa3.1, is now emerging as a therapeutic target for a large variety of health disorders. The Ca2+ sensitivity of KCa3.1 is conferred by the Ca2+-binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C terminus. It was proposed on the basis of the crystal structure obtained for the C-terminal region of the rat KCa2.2 channel (rSK2) with CaM that the binding of Ca2+ to the CaM N-lobe results in CaM interlocking the C-terminal regions of two adjacent KCa3.1 subunits, leading to the formation of a dimeric structure. A study was thus undertaken to identify residues of the CaM N-lobe–KCa3.1 complex that either contribute to the channel activation process or control the channel open probability at saturating Ca2+ (Pomax). A structural homology model of the KCa3.1–CaM complex was first generated using as template the crystal structure of the C-terminal region of the rat KCa2.2 channel with CaM. This model was confirmed by cross-bridging residues R362 of KCa3.1 and K75 of CaM. Patch-clamp experiments were next performed, demonstrating that the solvation energy of the residue at position 367 in KCa3.1 is a key determinant to the channel Pomax and deactivation time toff. Mutations of residues M368 and Q364 predicted to form anchoring points for CaM binding to KCa3.1 had little impact on either toff or Pomax. Finally, our results show that channel activation depends on electrostatic interactions involving the charged residues R362 and E363, added to a nonpolar energy contribution coming from M368. We conclude that electrostatic interactions involving residues R362 and E363 and hydrophobic effects at M368 play a prominent role in KCa3.1 activation, whereas hydrophobic interactions at S367 are determinant to the stability of the CaM–KCa3.1 complex throughout gating.

Lubiprostone Improves Not Only Spontaneous Bowel Movement but Also Quality of Life in Patients With Chronic Idiopathic Constipation: Phase III Randomized, Double-Blind, and Placebo-Controlled Study and Long-Term Treatment Study in Japan

Aim: Lubiprostone is a prostone analogue with a novel type-2 chloride channel activation process. We previously reported the efficacy of lubiprostone for chronic idiopathic constipation (CIC) with or without irritable bowel syndrome (IBS) as phase II trial in Japan (Gastroenterology 138: S226, 2010). However, report of phase a III randomized controlled trial and long-term efficacy of lubiprostone in Asian population was lacking. We tested the following hypotheses; (1) Lubiprostone is effective on spontaneous bowel movements (SBMs) in CIC patients in Japan. (2) Treatment with lubiprostone for long (but not short) term improves quality of life (QOL) in CIC patients.Methods: Essential feature of this studywas randomized, double-blind, placebo-controlled, phase III trial of lubiprostone. Patients with CIC (n = 124) were randomly allocated for treatments using a placebo (n = 62) or 24 μg twice daily dose of lubiprostone (n = 62) for 4 weeks. The primary efficacy endpoint was the change from baseline in the weekly average number of SBMs at week 1. The secondary efficacy endpoints included the change from baseline in the weekly average number of SBMs at week 2-4, gastrointestinal symptoms, SF-36, and IBS-QOL. In the long-term treatment study, 209 patients with CICwere enrolled. Twice daily dose of lubiprostone 24 μgwas administered for 48 weeks. Efficacy and safety were checked at visits. Results: Administration of 48 μg of lubiprostone induced significantly more average number of SBMs at week 1 (3.7 ± 2.8) than that of placebo (1.3 ± 1.8, p < 0.001). SBMs at week 2, 3, and 4 in lubiprostone 48 μg/day arm were significantly superior to those in placebo arm. Stool form at week 1, 2, 3, and 4 in lubiprostone arm was also significantly better than in placebo arm. There was significantly more global improvement during the treatment period with lubiprostone than placebo. However, changes in SF-36 or IBS-QOL in lubiprostone arm did not differ from those in placebo arm. In the long-term treatment study, SBMs during administration of lubiprostone were significantly more than those at baseline (p < 0.001). Lubiprostone significantly maintained better stool form, straining, incomplete evacuation, abdominal discomfort, abdominal distention, and global improvement during treatment period than baseline. Lubiprostone for long-term therapy induced significantly better SF-36 and IBS-QOL than baseline. Conclusion: Our results confirmed effect of lubiprostone on constipation regardless of different culture. This study also suggests that improvement of QOL may follow better bowel movements for certain duration in patients with CIC.

S1309 Efficacy of Lubiprostone in Patients With Constipation With or Without Irritable Bowel Syndrome in Japan: Randomized, Placebo-Controlled and Dose-Finding Study

Aim: Ethnic factors may contribute to drug efficacy in functional bowel disorders (Gastroenterology 2006; 130: 1435-46). Lubiprostone is an innovative prostone analogue with a novel type-2 chloride channel activation process. The efficacy of lubiprostone for chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C) was unknown in Japanese population. This study was performed to test the hypothesis that lubiprostone is effective on CIC including IBS-C in Japan and to find an optimal dose.Methods: Essential feature of this study was randomized, double-blind, placebo-controlled, and dose-finding phase 2 trial of lubiprostone. A total of 170 patients with CIC, 75.3% without IBS-C and 24.7% with IBS-C, were registered and were randomly allocated for treatments using a placebo (n = 42) or one of the three daily doses of lubiprostone: 16 mcg (n = 41), 32 mcg (n = 43), or 48 mcg (n = 44) for 2 weeks. Each took one capsule twice a day. The primary efficacy endpoint was the change from baseline in the weekly average number of spontaneous bowel movements (SBMs) at Week 1. The secondary efficacy endpoints included the change from baseline in the weekly average number of SBMs at Week 2, the percentages of patients who experienced bowel movements within 24 hours and/or 48 hours windows after the initial dose, the degree of incomplete evacuation, straining, stool form, and abdominal symptoms, global assessment of constipation severity, global assessment of treatment effectiveness, SF-36, IBS-QOL, and satisfactory relief. Results: There was a statistically significant and dose-dependent increase in change from baseline in the weekly average number of SBMs at Week 1 (ANOVA: p < 0.0001). There were statistically significant differences in the favor of lubiprostone 48 mcg/day and 32 mcg/day over placebo (P < 0.0001, P = 0.0017, respectively). The primary endpoint results were also significant by the stratified analyses when CIC patients were limited to those without IBS-C (P < 0.0001). Despite the small sample size, the primary endpoint in patients with IBS-C treated with 48 mcg of lubiprostone was significantly better than those with placebo (P = 0.0086). Dose-response effects were also seen for the secondary efficacy endpoints. No serious side effects were produced by lubiprostone. Conclusion: Our results suggest that lubiprostone shows a steady and effective improvement in the symptoms of CIC including IBS-C in a dose dependent manner and that lubiprostone 48 mcg/day is the optimal dose. These data support continued development of lubiprostone for Japanese patients with constipation.

Voltage-gated sodium channels are targets for toxins from the venom of the spider Heriaeus melloteei

Three novel peptides were isolated from the venom of the spider Heriaeus melloteei (Thomisidae) and characterized. The peptides named Hm-1, 2 and 3 blocked voltage-gated Na+ channels at concentrations in the order of 100 nM. Activity of the purified peptides was investigated in Na+ channel isoforms of mammals and insects. Hm-1 and 2 appeared to act as pore blockers, whereas Hm-3 modulated the channel activation process. The toxins described exhibit minor similarity with other known peptides and may therefore constitute new groups of Na+ channel ligands.

Aromatic Residues ∈Trp-55 and δTrp-57 and the Activation of Acetylcholine Receptor Channels

The two transmitter binding sites of the neuromuscular acetylcholine (ACh) receptor channel contain several aromatic residues, including a tryptophan located on the complementary, negative face of each binding pocket. These two residues, Trp-55 in the epsilon subunit and Trp-57 in the delta subunit, were mutated (AEFHILRVY), and for most constructs the rate constants for acetylcholine binding and channel gating were estimated by using single channel kinetic analyses. The rate constants for unliganded channel opening and closing were also estimated for some mutants. From these measurements we calculated all of the equilibrium constants of the "allosteric" cycle as follows: diliganded gating, unliganded gating, dissociation from the C(losed) conformation, and dissociation from the O(pen) conformation. The results indicate the following. (i) These aromatic side chains play a relatively minor role in ACh receptor channel activation. (ii) The main consequence of mutations is to reduce the affinity of the O conformation of the binding site for ACh, with the effect being greater at the epsilon subunit. (iii) In epsilon (but not delta) the aromatic nature of the side chain is important in determining affinity, to a slightly greater degree in the O conformation. Phi value analyses (of both tryptophan residues) show Phi approximately 1 for both the ACh binding and diliganded gating reactions. (iv) This suggests that the structural boundaries of the dynamic elements of the gating conformational change may not be subunit-delimited, and (v) the mutated tryptophan residues experience energy changes that occur relatively early in both the ligand-binding and channel-gating reactions.

Computing transient gating charge movement of voltage-dependent ion channels.

The opening of voltage-gated sodium, potassium, and calcium ion channels has a steep relationship with voltage. In response to changes in the transmembrane voltage, structural movements of an ion channel that precede channel opening generate a capacitative gating current. The net gating charge displacement due to membrane depolarization is an index of the voltage sensitivity of the ion channel activation process. Understanding the molecular basis of voltage-dependent gating of ion channels requires the measurement and computation of the gating charge, Q. We derive a simple and accurate semianalytic approach to computing the voltage dependence of transient gating charge movement (Q-V relationship) of discrete Markov state models of ion channels using matrix methods. This approach allows rapid computation of Q-V curves for finite and infinite length step depolarizations and is consistent with experimentally measured transient gating charge. This computational approach was applied to Shaker potassium channel gating, including the impact of inactivating particles on potassium channel gating currents.

Effects of extracellular pH on the interaction of sipatrigine and lamotrigine with high-voltage-activated (HVA) calcium channels in dissociated neurones of rat cortex

Acidic extracellular pH reduced high-voltage-activated (HVA) currents in freshly isolated cortical pyramidal neurones of adult rats, shifting activation to more positive voltages ( V 1/2=−18 mV at pH 7.4, −11 mV at pH 6.4). Sipatrigine inhibited HVA currents, with decreasing potency at acidic pH (IC 50 8 μM at pH 7.4, 19 μM at pH 6.4) but the degree of maximal inhibition was >80% in all cases (pH 6.4–8.0). Sipatrigine has two basic groups (p K A values 4.2, 7.7) and at pH 7.4 is 68% in monovalent cationic form and 32% uncharged. From simple binding theory, the pH dependence of sipatrigine inhibition indicates a protonated group with p K A 6.6. Sipatrigine (50 μM) shifted the voltage dependence of channel activation at pH 7.4 (−7.6 mV shift) but not at pH 6.4. Lamotrigine has one basic site (p K A 5.5) and inhibited 34% of the HVA current, with similar potency over the pH range 6.4–7.4 (IC 50 7.5–9 μM). These data suggest that the sipatrigine binding site on HVA calcium channels binds both cationic and neutral forms of sipatrigine, interacts with a group with p K A=6.6 and with the channel activation process, and differs from that for lamotrigine.

A patch-clamp study of the Ca2+ mobilization from internal stores in bovine aortic endothelial cells. I. Effects of caffeine on intracellular Ca2+ stores.

The effects of agents known to interfere with Ca2+ release processes of endoplasmic reticulum were investigated in bradykinin (BK)-stimulated bovine aortic endothelial cells (BAE cells), via the activation of Ca(2+)-activated potassium channels [K(Ca2+) channels]. In cell-attached patch experiments, the external application of caffeine (1 mM) caused a brief activation of K(Ca2+) channels in Ca(2+)-free and Ca(2+)-containing external solutions. The application of BK (10 nM) during cell stimulation by caffeine (1-20 mM) invariably led to a drastic channel activation which was maintained during a recording period longer than that observed in caffeine-free conditions. In addition, the cell exposure to caffeine (20 mM) during the BK stimulation enhanced systematically the channel activation process. Since a rapid inhibition of BK-evoked channel activity was also produced by removing caffeine from the bath medium, it is proposed that the sustained single-channel response recorded in the concomitant presence of both agents was due to their synergic action on internal stores and/or the external Ca2+ entry pathway resulting in an increased [Ca2+]i. In addition, the local anesthetic, procaine, depressed the initial BK-induced K(Ca2+) channel activity and completely blocked the secondary phase of the channel activation process related to the external Ca2+ influx into stimulated cells. In contrast, this blocking effect of procaine was not observed on the initial caffeine-elicited channel activity and could not suppress the external Ca(2+)-dependent phase of this channel activation process. Our results confirm the existence of at least two pharmacologically distinct types of Ca(2+)-release from internal stores in BAE cells: an inositol 1,4,5-triphosphate (InsP3)-dependent and a caffeine-induced Ca(2+)-release process.

Modulation by extracellular pH of bradykinin-evoked activation of Ca(2+)-activated K+ channels in endothelial cells

We used the patch-clamp technique to investigate, via the activation of Ca(2+)-activated potassium channels [K(Ca2+)channels], the effects of extracellular pH (pHo) on the bradykinin (BK)-stimulated rise in cytosolic Ca2+ concentration in bovine aortic endothelial cells (BAE). In cell-attached experiments, the external application of BK caused a transient activation of the K(Ca2+) channels. Increasing pHo from 7.3 to 9 maintained the channel activity, which was not inhibited by withdrawing the agonist. The channel-activation process could be blocked either by removing external Ca2+ or by depolarizing the cells with a high-K+ external solution. These results indicate that the Ca2+ influx triggered by BK contributes in maintaining the agonist-evoked response in high pHo. Changes in pHo produced a slight increase in the intracellular pH (pHi) measured fluorimetrically with the H+ indicator dye 2',7-bis(carboxyethyl)5(6')-carboxyfluorescein. However, increasing pHi by the external application of NH4Cl at physiological pHo caused a rapid decline and not an increase in the K(Ca2+) channel activity triggered by BK. In fura-2-loaded cells, alkaline pHo had no effect on the time course of the Ca2+ response to BK in external Ca(2+)-free conditions, suggesting that the Ca2+ extrusion process is not affected by pHo. Our results suggest that the BK-evoked Ca2+ influx, which is required to reload internal Ca2+ stores, is controlled by a mechanism depending on extracellular H+.

External ATP triggers a biphasic activation process of a calcium-dependent K+ channel in cultured bovine aortic endothelial cells.

We have used the patch-clamp method in order to investigate the single-channel events underlying the effect of external ATP on the potassium permeability of bovine aortic endothelial cells (BAE). The results obtained from cell-attached and inside-out experiments led first to conclude that BAE cells possess an inward rectifying potassium channel activated by internal calcium at micromolar concentrations. The channel conductance for inward currents was estimated at 40 pS in symmetrical 200 mM KCl and the open-channel probability was found to be voltage insensitive within the membrane voltage range -50 to -100 mV. Based on results obtained in the cell-attached configuration, it could next be established that external ATP and ADP at micromolar concentrations could trigger, via the stimulation of P2 purinergic receptors, a time variable activation process of the observed calcium-dependent potassium channel. This activation process was found to occur in a biphasic manner with an initial phase independent of the presence of calcium in the cell bathing medium. The second phase which could be blocked by calcium channel blockers such as Co2+ or La3+ required, however, the presence of external calcium and could be abolished by depolarizing the cells using high K+ external solutions. Another important aspect related to this phenomenon was the observation that removing ATP from the external medium during the second phase led to a complete abolition of the associated calcium-dependent potassium channel activation process. It is suggested from these results that the action of ATP on the potassium permeability of BAE cells is related to a second messenger mediated release of calcium from internal calcium stores coupled to an ATP-dependent calcium influx abolished at depolarizing voltages.