Total Charge Movement per Channel : The Relation between Gating Charge Displacement and the Voltage Sensitivity of Activation

One measure of the voltage dependence of ion channel conductance is the amount of gating charge that moves during activation and vice versa. The limiting slope method, introduced by Almers (Almers, W. 1978. Rev. Physiol. Biochem. Pharmacol. 82:96–190), exploits the relationship of charge movement and voltage sensitivity, yielding a lower limit to the range of single channel gating charge displacement. In practice, the technique is plagued by low experimental resolution due to the requirement that the logarithmic voltage sensitivity of activation be measured at very low probabilities of opening. In addition, the linear sequential models to which the original theory was restricted needed to be expanded to accommodate the complexity of mechanisms available for the activation of channels. In this communication, we refine the theory by developing a relationship between the mean activation charge displacement (a measure of the voltage sensitivity of activation) and the gating charge displacement (the integral of gating current). We demonstrate that recording the equilibrium gating charge displacement as an adjunct to the limiting slope technique greatly improves accuracy under conditions where the plots of mean activation charge displacement and gross gating charge displacement versus voltage can be superimposed. We explore this relationship for a wide variety of channel models, which include those having a continuous density of states, nonsequential activation pathways, and subconductance states. We introduce new criteria for the appropriate use of the limiting slope procedure and provide a practical example of the theory applied to low resolution simulation data.     

Transfer of Rolf S3-S4 Linker to hERG Eliminates Activation Gating but Spares Inactivation

Studies in Shaker, a voltage-dependent potassium channel, suggest a coupling between activation and inactivation. This coupling is controversial in hERG, a fast-inactivating voltage-dependent potassium channel. To address this question, we transferred to hERG the S3-S4 linker of the voltage-independent channel, rolf, to selectively disrupt the activation process. This chimera shows an intact voltage-dependent inactivation process consistent with a weak coupling, if any, between both processes. Kinetic models suggest that the chimera presents only an open and an inactivated states, with identical transition rates as in hERG. The lower sensitivity of the chimera to BeKm-1, a hERG preferential closed-state inhibitor, also suggests that the chimera presents mainly open and inactivated conformations. This chimera allows determining the mechanism of action of hERG blockers, as exemplified by the test on ketoconazole.     

Gating charge movement precedes ionic current activation in hERG channels

We recently reported gating currents recorded from hERG channels expressed in mammalian TSA cells and assessed the kinetics at different voltages. We detected 2 distinct components of charge movement with the bulk of the charge being carried by a slower component. Here we compare our findings in TSA cells with recordings made from oocytes using the Cut Open Vaseline Gap clamp (COVG) and go on to directly compare activation of gating charge and ionic currents at 0 and +60 mV. The data show that gating charge saturates and moves more rapidly than ionic current activates suggesting a transition downstream from the movement of the bulk of gating charge is rate limiting for channel opening.     

Gating charge movement precedes ionic current activation in hERG channels

We recently reported gating currents recorded from hERG channels expressed in mammalian TSA cells and assessed the kinetics at different voltages. We detected 2 distinct components of charge movement with the bulk of the charge being carried by a slower component. Here we compare our findings in TSA cells with recordings made from oocytes using the Cut Open Vaseline Gap clamp (COVG) and go on to directly compare activation of gating charge and ionic currents at 0 and +60 mV. The data show that gating charge saturates and moves more rapidly than ionic current activates suggesting a transition downstream from the movement of the bulk of gating charge is rate limiting for channel opening.     

Differences Between Ion Binding to eag and HERG voltage sensors Contribute to Differential Regulation of Activation and Deactivation Gating

HERG (KCNH2) and ether-à-go-go (eag) (KCNH1) are members of the same subfamily of voltage-gated K+ channels. In eag, voltage-dependent activation is significantly slowed by extracellular divalent cations. To exert this effect, ions bind to a site located between transmembrane segments S2 and S3 in the voltage sensor domain where they interact with acidic residues that are conserved only among members of the eag subfamily. In HERG channels, extracellular divalent ions significantly accelerate deactivation. To investigate the ion-binding site in HERG, acidic residues in S2 and S3 were neutralized singly or in pairs to alanine, and the functional effects of extracellular Mg2+ were characterized in Xenopus oocytes. To modulate deactivation kinetics in HERG, divalent cations interact with eag subfamily-specific acidic residues (D460 and D509) and also with an acidic residue in S2 (D456) that is widely conserved in the voltage-gated channel superfamily. In contrast, the analogous widely-conserved residue does not contribute to the ion-binding site that modulates activation kinetics in eag. We propose that structural differences between the ion-binding sites in the eag and HERG voltage sensors contribute to the differential regulation of activation and deactivation gating in these channels. A previously proposed model for S4 conformational changes during voltage-dependent activation can account for the differential regulation of gating seen in eag and HERG.     

Changes in Negative Charge at the Luminal Mouth of the Pore Alter Ion Handling and Gating in the Cardiac Ryanodine-Receptor

We have tested the hypothesis that a high density of negative charge at the luminal mouth of the RyR2 pore plays a pivotal role in the high cation conductance and limited selectivity observed in this channel by introducing into each monomer a double point mutation to neutralize acidic residues in this region of the mouse RyR2 channel. The resultant channel, ED4832AA, is capable of functioning as a calcium-release channel in situ. Consistent with our hypothesis, the ED4832AA mutation altered the ion handling characteristics of single RyR2 channels. The mutant channel retains the ability to discriminate between cations and anions but cation conductance is altered significantly. Unitary K⁺ conductance is reduced at low levels of activity but increases dramatically as activity is raised and shows little sign of saturation. ED4832AA no longer discriminates between divalent and monovalent cations. In addition, the gating characteristics of single RyR2 channels are altered markedly by residue neutralization. Open probability in the ED4832AA channel is substantially higher than that of the wild-type channel. Moreover, at holding potentials in excess of ±50 mV several subconductance states become apparent in ED4832AA and are more prevalent at very high holding potentials. These observations are discussed within the structural framework provided by a previously developed model of the RyR2 pore. Our data indicates that neutralization of acidic residues in the luminal mouth of the pore produces wide-ranging changes in the electric field in the pore, the interaction energies of permeant ions in the pore and the stability of the selectivity filter region of the pore, which together contribute to the observed changes ion handling and gating.     

Activation and Deactivation of H-ATPase in Intact Chloroplasts

The light activation mechanism of the latent H⁺-ATPase was investigated in intact spinach (Spinacia oleracea, Hybrid 424) chloroplasts. The following observations were made. (a) Photosystem I electron acceptors such as methyl viologen, nitrite, oxaloacetate, etc., inhibit the light activation of the enzyme. (b) The electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) fully inhibits the process. (c) Ascorbate plus diaminodurene or dithionite can restore light activation in DCMU-poisoned chloroplasts. (d) The activated state of the enzyme decays rather slowly (within a few minutes) after illumination of the intact chloroplasts. (e) The rate of dark decay is accelerated by oxidants (H₂O₂ or ferricyanide) and slowed down by dithiothreitol.

It is suggested that the physiological mechanism for regulation of the H⁺-ATPase involves oxidation and reduction reactions in a manner which resembles the regulation of the light-activated carbon cycle enzymes.

     

NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement on the Path to Activation

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V_1/2 of activation of L529I (−34 ± 4 mV) is similar to wild-type (WT) (−37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V_1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (−131 ± 4 mV for K525C and −120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.     

Charge Movement Associated with the Opening and Closing of the Activation Gates of the Na Channels

The sodium current (I_Na) that develops after step depolarization of a voltage clamped squid axon is preceded by a transient outward current that is closely associated with the opening of the activation gates of the Na pores. This "gating current" is best seen when permeant ions (Na and K) are replaced by relatively impermeant ones, and when the linear portion of capacitative current is eliminated by adding current from positive steps to that from exactly equal negative ones. During opening of the Na pores gating current is outward, and as the pores close there is an inward tail of current that decays with approximately the same time-course as I_Na recorded in Na-containing medium. Both outward and inward gating current are unaffected by tetrodotoxin (TTX). Gating current is capacitative in origin, the result of relatively slow reorientation of charged or dipolar molecules in a suddenly altered membrane field. Close association with the Na activation process is clear from the time-course of gating current, and from the fact that three procedures that reversibly block I_Na also block gating current: internal perfusion with Zn²⁺, prolonged depolarization of the membrane, and inactivation of I_Na with a short positive prepulse.     

Central Charged Residues in DIIIS4 Regulate Deactivation Gating in Skeletal Muscle Sodium Channels

Summary 1. Mutations in the S4 segment of domain III in the voltage gated skeletal muscle sodium channel hNa_V1.4 were constructed to test the roles of each charged residue in deactivation gating. Mutations comprised charge reversals at K1-R6, charge neutralization, and substitution at R4 and R5. 2. Charge-reversing mutations at R4 and R5 produced the greatest alteration of activation parameters compared to hNa_V1.4. Effects included depolarization of the conductance/voltage (g/V) curve, decreased valence and slowing of kinetics. 3. Reversal of charge at R2 to R4 hyperpolarized, and reversal at R5 or R6 depolarized the h _∞ curve. Most DIIIS4 mutations slowed inactivation from the open state. R4E slowed closed state fast inactivation and R5E inhibited its completion. 4. Deactivation from the open and/or inactivated state was prolonged in mutations reversing charge at R2 to R4 but accelerated by reversal of charge at R5 or R6. Effects were most pronounced at central charges R4 and R5. 5. Charge and structure each contribute to effects of mutations at R4 and R5 on channel gating. Effects of mutations on activation and deactivation at R4 and, to a lesser extent R5, were primarily owing to charge alteration, whereas effects on fast inactivation were charge independent.     

巨噬细胞的三种活化

IFN-γ诱导产生、经典活化的巨噬细胞参与Th1型免疫应答,对细胞内感染病原体如结核杆菌有很好杀伤作用。由IL-4、IL-13等所诱导的替代性活化的巨噬细胞,具有组织修复功能,是与经典活化的巨噬细胞不同的表现型。近来研究表明,FcγRs与其配体结合所诱导活化的巨噬细胞——Ⅱ型活化的巨噬细胞与上述两种活化的巨噬细胞不同,具有抗炎症并参与,Th2型免疫应答的作用。该主要讨论这三种类型活化的巨噬细胞的发现、活化的信号及其生物学功能。     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

Distortion Component Analysis of Outer Hair Cell Motility-Related Gating Charge

The underlying Boltzmann characteristics of motility-related gating currents of the outer hair cell (OHC) are predicted to generate distortion components in response to sinusoidal transmembrane voltages. We studied this distortion since it reflects the mechanical activity of the cell that may contribute to peripheral auditory system distortion. Distortion components in the OHC electrical response were analyzed using the whole-cell voltage clamp technique, under conditions where ionic conductances were blocked. Single or double-sinusoidal transmembrane voltage stimulation was delivered at various holding voltages, and distortion components of the current responses were detected by Fourier analysis. Current response magnitude and phase of each distortion component as a function of membrane potential were compared with characteristics of the voltage-dependent capacitance, obtained by voltage stair-step transient analysis or dual-frequency admittance analysis. The sum distortion was most prominent among the distortion components at all holding voltages. Notches in the sum (f1+f2), difference (f2−f1) and second harmonic (2f) components occur at the voltage where peak voltage-dependent capacitance resides (V _pkCm ). Rapid phase reversals also occurred at V _pkCm , but phase remained fairly stable at more depolarized and hyperpolarized potentials. Thus, it is possible to extract Boltzmann parameters of the motility-related charge movement from these distortion components. In fact, we have developed a technique to follow changes in the voltage dependence of OHC motility and charge movement by tracking the voltage at phase reversal of the f2−f1 product. When intracellular turgor pressure was changed, V _pkCm and distortion notch voltages shifted in the same direction. These data have important implications for understanding cochlear nonlinearity, and more generally, indicate the usefulness of distortion analysis to study displacement currents. Received: 31 December 1998/Revised: 12 March 1999     

Activation and Deactivation of F0F1-ATPase in Yeast Mitochrondia

The regulation of membrane-bound proton F₀F₁ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F₁.IF1 complex into an inactive form.     

Assembly of Nsp1 Nucleoporins Provides Insight into Nuclear Pore Complex Gating

Nuclear pore complexes (NPCs) form gateways for material transfer across the nuclear envelope of eukaryotic cells. Disordered proteins, rich in phenylalanine-glycine repeat motifs (FG-nups), form the central transport channel. Understanding how nups are arranged in the interior of the NPC may explain how NPC functions as a selectivity filter for transport of large molecules and a sieve-like filter for diffusion of small molecules (< or ). We employed molecular dynamics to model the structures formed by various assemblies of one kind of nup, namely the 609-aa-long FG domain of Nsp1 (Nsp1-FG). The simulations started from different initial conformations and geometrical arrangements of Nsp1-FGs. In all cases Nsp1-FGs collectively formed brush-like structures with bristles made of bundles of 2–27 nups, however, the bundles being cross-linked through single nups leaving one bundle and joining a nearby one. The degree of cross-linking varies with different initial nup conformations and arrangements. Structural analysis reveals that FG-repeats of the nups not only involve formation of bundle structures, but are abundantly present in cross-linking regions where the epitopes of FG-repeats are highly accessible. Large molecules that are assisted by transport factors (TFs) are selectively transported through NPC apparently by binding to FG-nups through populated FG-binding pockets on the TF surface. Therefore, our finding suggests that TFs bind concertedly to multiple FGs in cross-linking regions and break-up the bundles to create wide pores for themselves and their cargoes to pass. In addition, the cross-linking between Nsp1-FG bundles, arising from simulations, is found to set a molecular size limit of < for passive diffusion of molecules. Our simulations suggest that the NPC central channel, near the periphery where tethering of nups is dominant, features brush-like moderately cross-linked bundles, but in the central region, where tethering loses its effect, features a sieve-like structure of bundles and frequent cross-links.     

Activation of Oncogenic Pathways in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause. The most recent hypotheses on IPF pathogenesis suggest a central role of epithelial cell damage, followed by a dysregulated molecular cross talk between epithelial cells and fibroblasts. Thus, IPF progression has often been assimilated to that of cancer, and several signaling patterns appear to be disrupted in both diseases. Here, we analyze the expression in an IPF series of a panel of molecules, which are known to play a role in tumorigenic process. Our findings, although preliminary, reveal that IPF landscape is enriched in neoplastic potential expressed in a context of complex genomic polyclonality and cellular heterogeneity. These results provide a rationale for further investigations aimed to exploit—in a similar fashion to cancer—targeted therapies for a “precision medicine” approach to IPF.     

Coordinated Movement of Cytoplasmic and Transmembrane Domains of RyR1 upon Gating

Ryanodine receptor type 1 (RyR1) produces spatially and temporally defined Ca²⁺ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 Å resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic “inner branches” and the transmembrane “inner helices”). Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 Å diameter increase of the ion gate. Whereas the inner helices of the K⁺ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K⁺ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right-handed bundle structures along a common 4-fold axis.     

Charge Movement of a Voltage-Sensitive Fluorescent Protein

The N-terminus of Ciona intestinalis (Ci-VSP) is a voltage-sensing domain (VSD) controlling the activity of a phosphatase domain on the C terminus. By replacing the phosphatase domain with a tandem of fluorescent proteins, CFP and YFP, a family of fluorescence resonance energy transfer-based, genetically encoded voltage-sensing fluorescent protein (VSFP) was created. VSFP2.3, one of the latest versions of this family, showed large changes in YFP emission upon changes in membrane potential with CFP excitation when expressed in Xenopus laevis oocytes. The time course of the fluorescence has two components: the fast component correlates with the time course of sensing current produced by the charge movement, while the slow component is at least one order-of-magnitude slower than the sensing current. This suggests that the tandem of fluorescent proteins reports a secondary conformational transition of the VSD which resembles the relaxation of the VSD of Ci-VSP described in detail for the Ci-VSP. This observation indicates that the relaxation of the VSD of VSFP2.3 is a global conformational change that encompasses the entire S4 segment.     

Movement of the S4 segment in the hERG potassium channel during membrane depolarization

Abstract

The hERG potassium channel is a member of the voltage gated potassium (Kv) channel family, comprising a pore domain and four voltage sensing domains (VSDs). Like other Kv channels, the VSD senses changes in membrane voltage and transmits the signal to gates located in the pore domain; the gates open at positive potentials (activation) and close at negative potentials, thereby controlling the ion flux. hERG, however, differs from other Kv channels in that it is activated slowly but inactivated rapidly – a property that is crucial for the role it plays in the repolarization of the cardiac action potential. Voltage-gating requires movement of gating charges across the membrane electric field, which is accomplished by the transmembrane movement of the fourth transmembrane segment, S4, of the VSD containing the positively charged arginine or lysine residues. Here we ask if the functional differences between hERG and other Kv channels could arise from differences in the transmembrane movement of S4. To address this, we have introduced single cysteine residues into the S4 region of the VSD, expressed the mutant channels in Xenopus oocytes and examined the effect of membrane impermeable para-chloromercuribenzene sulphonate on function by the two-electrode voltage clamp technique. Our results show that depolarization results in the accessibility of seven consecutive S4 residues, including the first two charged residues, K525 and R528, to extracellularly applied reagent. These data indicate that the extent of S4 movement in hERG is similar to other Kv channels, including the archabacterial KvAP and the Shaker channel of Drosophila.