Gating Charge Research Articles

Not so optimal: The evolution of mutual information in potassium voltage-gated channels.

Potassium voltage-gated (Kv) channels need to detect and respond to rapidly changing ionic concentrations in their environment. With an essential role in regulating electric signaling, they would be expected to be optimal sensors that evolved to predict the ionic concentrations. To explore these assumptions, we use statistical mechanics in conjunction with information theory to model how animal Kv channels respond to changes in potassium concentrations in their environment. By measuring mutual information in representative Kv channel types across a variety of environments, we find two things. First, under weak conditions, there is a gating charge that maximizes mutual information with the environment. Second, as Kv channels evolved, they have moved towards decreasing mutual information with the environment. This either suggests that Kv channels do not need to act as sensors of their environment or that Kv channels have other functionalities that interfere with their role as sensors of their environment.

Elucidating the differential mutational effects of gating charges in voltage-gated sodium channels.

Recent advances in genetics and pathophysiology have identified a large number of mutations in voltage-gated sodium (NaV) channels that cause various heart, muscle, and brain excitability disorders. A significant feature of mutation distribution is that the disease-associated mutations are enriched in gating charges in voltage-sensing domains (VSDs). Intriguingly, the same mutation at the equivalent positions in different VSDs brings distinctive phenotypes.To address this mechanistic mystery, we studied the mutational effects on structural transitions of two gating charges (R2 and R3) in both VSD1 and VSD2 using molecular dynamics simulation and conformational free energy calculation. The application of an external electric field allows us to simulate the VSD transitions between “up” and “down” conformations at a microsecond scale. Our computational study shows that the structural impact of the same mutation (R to Q substitution) not only alters between different gating charges within the same VSD but also differs at the equivalent position between VSD1 and VSD2. The differential impacts are determined by a salt-bridge network. This network formed by gating charges and countercharges differs from one VSD to another. Our salt-bridge analysis reveals that the perturbations induced by mutations on this VSD-specific network bring unique impacts to the formation and stability of the intermediate state. The salt bridges in the intermediate play an unexpected role in determining the transition rate of VSDs, which elucidates a general principle behind the diverse mutational effects and will help us to predict the effects of more disease-associated mutations in voltage-gated ion channels.

Effects of electric-field reshaping on voltage sensing in voltage-gated sodium channels.

Voltage-gated sodium channels (Navs) are responsible for the depolarizing phase of the action potential with essential roles in fast electrical signalling. Membrane depolarization triggers opening of Nav channels via activation of their voltage-sensing domains (VSD). The electric field across the membrane acts on a series of positively charged residues and induces their transmembrane relocation, which in turn triggers conformational changes that eventually lead to the opening of the sodium-conducting pore. Voltage dependence of the activation process can be described in terms of the gating charge that links energetics of VSD activation to the transmembrane voltage. Notably, the gating charge is defined by coupling of charged residues to the external electric field, whose shape is therefore crucial for Nav activation. Here, we employed molecular dynamics simulations of cardiac Nav1.5 and bacterial NavAb to gain atomic-level insights into the voltage-sensing mechanisms of Navs. Using our recently developed tool g_elpot to quantify VSD electrostatics with high spatial resolution, we found that, in contrast to earlier low-resolution studies, the electric field within VSDs of Nav channels has a complex isoform- and domain-specific shape, which prominently depends on the activation state of a VSD. Due to this field reshaping, not only translocated basic but also relatively static acidic residues contribute significantly to the gating charge. In the case of NavAb, we found that the transition between the resolved activated- and resting-state structures results in the gating charge of 8e, which is noticeably lower than experimental values of 12-16e. Our analysis of VSD electrostatics thus indicates that the resting-state structure represents an intermediate state of channel activation. In conclusion, our results provide an atomic-level description of the gating charge in Nav channels, and reveal the importance of electric-field reshaping for the energetics of voltage gating.

Ionic interactions between gating charges and countercharges in voltage-sensing domain I independently regulate kinetics and voltage-dependence of Cav1.1 gating.

Voltage-gated calcium channels control a variety of processes in excitable cells. However, the exact mechanisms regulating kinetics and voltage-dependence of channel activation are not fully understood. Voltage-gated activation is determined by four distinct voltage-sensing domains (VSD I-IV) coupled to a common pore. Each VSD consists of four transmembrane helices (S1-S4) with S4 containing four to five gating charges. Upon membrane depolarization, consecutive interactions of these gating charges with negatively charged countercharges are believed to facilitate an upward movement of the S4 helices, leading to the opening of the pore and activation of the channel. Previous studies linked naturally occurring mutations of the innermost gating charge R4 (R174W) and its negative countercharge (E100) in CaV1.1 to muscle disease. To investigate the contribution of VSD I and the roles of its gating- and countercharges in channel gating, we combined structure-guided site-directed mutagenesis with patch-clamp analysis in dysgenic myotubes (CaV1.1-null). As E100 in helix S2 together with D126 in helix S3 form the highly conserved charge-transfer center of VSDs, we included this second negative countercharge (D126) in our analysis. While mutation of R174A resulted in a strong right-shift of voltage dependence, charge neutralizing mutations of the countercharges E100 and D126 both lead to a left-shift of voltage-dependence and to a slowing of channel activation. The double mutant of both countercharges resulted in an additive effect on activation kinetics, but left the voltage dependence of activation unaffected compared to the single mutants. Our findings indicate that VSD I substantially contributes to the regulation of both kinetics and voltage-dependence of activation; that the same ionic interactions are critical for both properties; but that the molecular mechanisms governing the two gating properties are functionally separate.

Structural basis for severe pain caused by mutations in voltage gated sodium channels Nav1.7.

Gain-of-function mutations in voltage-gated sodium channel NaV1.7 cause severe inherited pain syndromes, including Inherited Erythromelalgia (IEM). We introduced IEM mutations into the bacterial sodium channel NaVAb and confirmed the negative shift in the voltage dependence of activation that recapitulates the gain-of-function of these mutants. Unexpectedly, our structures of NaVAb IEM mutations reveal two complementary pathogenic mechanisms at the atomic level. Mutations in the voltage sensor domain facilitate the outward movement of S4 gating charges by widening the pathway for gating charge movement and modifying hydrophobic interactions with surrounding amino acid side chains or membrane phospholipids. In contrast, IEM mutations in the S4-S5 linker stabilize the activated state by forming new hydrogen bonds with residues in the pore domain, including a conserved asparagine near the activation gate. These results provide key structural insights into the dual mechanisms by which these IEM mutations cause hyperexcitability and severe pain in this debilitating disease.

Voltage-dependent transitions between resting and activated states of the four Cav1.1 voltage sensors observed in MD simulation.

The sliding helix hypothesis describes how, upon depolarization and repolarization of the membrane, the positive gating charges of the S4 helices of voltage-sensing domains (VSDs) one by one move across the membrane electrical field and thereby effect the opening and closing of ion channels. Voltage-gated calcium channels contain four VSDs, which together regulate gating of a common pore. Although these four VSDs are highly homologous, they differ from each other, e.g. in the number of gating charges and in the number and position of their countercharges. Recent atomic-resolution structures of voltage-gated calcium and sodium channels delineate the endpoints of the VSD movement upon depolarization, showing the S4 helix in the activated up-state. While the first structures of VSDs in resting states have been solved for homotetrameric (prokaryotic) channels, resting state structures of the (pseudo-)heterotetrameric eukaryotic sodium and calcium channels are still lacking. Accordingly, structural models showing the transitions from the resting to the activated state and back are still elusive. Here we addressed this problem using enhanced sampling molecular dynamics simulations of the CaV1.1 calcium channel exposed to a strong membrane potential. Indeed, starting from the known up-state, the S4 helices of the four VSDs moved down to positions closely matching the experimentally determined resting state of the bacterial NaVAb channel. Importantly, the trajectories, the transiently formed ionic interactions between gating- and countercharges, and the speed of the S4 transitions differed substantially between the four VSDs. This is consistent with data from recent mutagenesis and voltage-clamp fluorometry studies on CaV1.1 and allows relating the individual VSD movements with their putative roles in the regulation of distinct channel gating properties, as well as their role in the activation of excitation-contraction coupling in skeletal muscle.

Hotspots detection and distribution pattern of disease-associated mutations in voltage-gated sodium channels.

More than 3000 mutations have been identified in voltage-gated sodium (Nav) channels that cause various inherited cardiac arrhythmias, neurological diseases, pain disorders, and periodic paralysis. Within the Nav family, the recurrence of mutations at the same position in different members is a strong indicator of the structural and functional importance of these amino acids. By mapping the most recent annotated disease-associated mutations of nine human Nav channels from the UniProt database to equivalent positions in multiple sequence alignment, we detected numerous clusters of mutation hotspots that are located in the voltage-sensing domain (VSD), the pore domain (PD), and the fast-inactivation segment. A significant feature is that the hotspots are enriched in positively charged residues, which are either gating charges in VSDs or arginines behind the selectivity filter in PD. Furthermore, 22 different sodium channelopathies relevant to different channels were categorized into two groups based on their documented gain of function (GoF) or loss of function (LoF) effect. A detailed comparison reveals that the GoF or LoF effect is relatively consistent for recurrent mutations at the equivalent positions. Some mutations can lead to both GoF and LoF effects, a phenomenon referred to as “overlapping syndrome” in Nav1.5. A number of similar overlapping syndrome mutations are also identified in other Nav channels. Intriguingly, we found that these GoF/LoF overlap mutations frequently appear in the S4-S5 linkers close to the fast inactivation segment. The GoF/LoF overlap effects of these mutations could be explained through two separate mechanisms: perturbing the VSD-PD coupling to affect activation and disrupting the binding of the fast-inactivation segment to alter the inactivation rate. This structural mapping and comparative analysis provide a novel approach to understanding the structure, function, and pathophysiology of channelopathies at the atomic level.

Control over Molecular Orbital Gating and Marcus Inverted Charge Transport in Molecular Junctions with Conjugated Molecular Wires (Adv. Electron. Mater. 2/2023)

Marcus Inverted Region In article number 2200637, Christian A. Nijhuis, Damien Thompson, Enrique Del Barco, and co-workers demonstrate that the mechanism of charge transport can be deliberately switched between the normal and Marcus Inverted regions by controlling the molecule–electrode coupling strength in a series of molecules. The cover shows two almost identical junctions. The only difference is one CH2 which lowers molecule-electrode coupling strength pushing the junctions from direct coherent tunnelling to hopping in the Marcus Inverted region. This ability can improve the energy efficiency of molecular devices. Image credit: Niels van der Velde.

Tuning the temperature dependence of Shaker potassium channel with mutations at S4-S5 linker.

We investigated the effects of temperature on mutations that uncouple the voltage sensor (VSD) from the pore (PD) in the Shaker-IR potassium channel using temperature step (Tstep). We utilized a setup that achieves fast temperature changes (millisecond) and allows the recording of the cell membrane temperature with sub-millisecond resolution (https://doi.org/10.1101/2022.07.11.499616). We used the mutants V369I, I372L, S376T (ILT), in the N-terminus of the S4-S5, that produces a split in the components of the gating charge movement, and I384N, near the S4-S5 C-terminus, that uncouples the VSD charge movement from the PD. From a bath temperature of 17 ºC, we used a Tstep of around ∼9 ºC with rising phase of 1.5 ms. In ILT, applying a Tstep, after the gating currents have subsided, promotes a right shift in the first component of gating charge and a left shift of the second component (the last concerted step associated with PD opening). Consistently, Tstep applied after the onset of a depolarizing voltage pulse produces a ∼5 mV left shift in the conductance-voltage curve (G-V). The entropy, calculated from G-V, changes from 24 for WT to −42 J/K for ILT. In I384N, applying a Tstep reduces the ionic current indicating the closure of the pore. We hypothesize that the temperature dependence of the ILT G-V arises from the changes in the last step of the charge movement. On the other hand, since the VSD and PD are loosely coupled in I384N, the increase in temperature is likely to further decrease the VSD-PD coupling, thus decreasing the measured conductance. (Support: NIH: GM030376, Fondecyt: 1190203)

A gating model for the mechanical activation of sensory TRP channels.

Transient receptor potential (TRP) channels are notorious for their proposed roles as sensors of chemical, thermal and mechanical stimuli in multiple cell types. While the roles of TRP channels as mechanotransducers in physiological conditions remain debated, it has been proposed that most of them can be activated by mechanical stimulation (Liu and Montell, BBRC, 2015; Startek et al., IJMS, 2019). The main objective of this study was to provide a theoretical framework serving to assess the determinants of TRP channel mechanosensitivity. We hypothesized that the mechanically-induced activation is boosted by the weak voltage dependence of TRP channels, as previously proposed for thermal stimuli and chemical agonists. To test this hypothesis, we examined the properties of an existing close-open gating model, which we extended to consider the influence of an external mechanical stimulus, in addition to the known contribution of temperature and the transmembrane membrane potential. The model predicts that the thermal and mechanical sensitivities are determined by the balance between an energy component associated to the protein volume and another associated to the protein-membrane surface. The model supports the hypothesis linking the mechanical- and voltage-dependent gating by predicting inverse relationships between the mechanical sensitivity and the gating valence. In addition, it explains how TRP channels can act as secondary mechanosensors by being stimulated by second messengers generated upon mechanical activation of signaling pathways. We speculate that, natural selection of ionic channels with low gating change yielded better sensors of thermal, chemical and mechanical stimuli. On the other hand, it may be possible that a TRP channel functioning primarily as thermo- or chemo-sensor is also found to the mechanosensitive in special experimental conditions, by the reason of having a low gating charge, without necessarily having a physiological role as mechanotransducer.

Voltage-sensor trapping in Hv1 channel (CiHv1).

Previous studies on monomeric N264R showed that the OFF-gating current is completely trapped and that, from a kinetic point of view, this charge displacement takes a long time to return. Currently, the molecular mechanism governing this entrapment of charge remains unknown. In order to understand the OFF-gating charge trapping mechanism, we performed patch-clamp in its inside-out configuration using Xenopus laevis oocytes as a heterologous expression system, combined with molecular dynamics and kinetic modeling. Our results shows that the ΔNΔC D160N mutant does not have a trapped charge at the OFF-gating charge; but, when the N264R mutation is added to this mutant, the OFF-gating charge trapping appears in a diminished state. This is possibly because the residue in position 160, which is also the selectivity filter of the channel, interacts with the arginines of the voltage sensor. In addition, the 2GBI blocker possesses a guanidinium group which also presents an arginine in its side chain. This inhibitor generates an effect similar to ΔNΔC N264R OFF-gating charge displacement. This suggests that arginine is required for charge trapping. By performing mutations at position 160 it was found that hydrophobicity near that position obstructs the movement of the voltage sensor exerting an effect similar to that of the aromatic rings of 2GBI. Furthermore, when ΔpH is increased and especially when acidity is increased on the intracellular side, the OFF-gating charge is also trapped. We performed molecular dynamics simulations on ΔNΔC N264R, and our results suggest that the positive charge of arginine “push-up” the arginines 258 and 261 and form a stable salt-bridge over time with D160. Finally, our results confirm that for the gating charge to be trapped it is necessary to change the electrostatic configuration on the intracellular side near position 264.

High-level expression of voltage-gated sodium channels in thymidine-arrested human embryonic kidney cells.

The benefit of using heterologous mammalian cell lines such as human embryonic kidney (HEK293) cells for expressing voltage-gated ion channels includes efficient translation and processing of such large membrane proteins. Still, the biophysical assessment of these channels is hindered by their low expression and reduced localization on the surface of a cell. Having a higher expression level of ion channels is crucial for investigating mutations that cause a decrease in the current amplitude as well as for measuring tiny currents such as gating currents and gating pore currents that are in the range of 0.1 to 1% of the peak current. We aimed at improving the efficiency of ion channel expression and enhancing their localization in cell membranes by modifying the protocol of cell cycle arrest at the G1/S boundary using thymidine to direct the cells into increasing the expression machinery during viral transduction of mammalian voltage-gated sodium channels. The combination of cell arrest at the G1/S boundary and mammalian baculovirus system (BacMam) resulted in a 5-fold increase in the current density of different mammalian voltage-gated sodium channels in HEK293 cells compared to the current literature. By applying this modified protocol for expression, we succeeded to measure a pathogenic gating pore current in HEK293 cells of ∼0.5% of the central pore current induced by an autism-related mutation in the R2 gating charge in Domain II (R853Q) of Nav1.2, which is blocked in a voltage-dependent manner when the voltage sensors activate. These results show that our protocol is a universal one that can be applied on different voltage-gated ion channels and possibly other membrane proteins. This ultimately will pave the way to decipher the structure and function of ion channels and their association with ion channelopathies and other diseases.

The voltage-dependent structural transitions governing the opening of the human t-type Cav3.1 channel: Insights from voltage-clamp fluorometry.

Low-voltage-activated CaV3.1 (T-type) Ca2+ channels contribute to neuronal burst-mode firing and heart pacemaker activity. Similar to other voltage-gated channels, the four Voltage-Sensing Domains (VSDs) are expected to activate before the pore opens. However, previous studies attempting to characterize the CaV3.1 voltage-sensing apparatus in channels blocked by La3+ found that most of the gating charge moves after the channel has opened. To shed light on the molecular mechanisms underlying the unique voltage-dependent gating of CaV3.1, we expressed the human CaV3.1 pore-forming subunit α1G in Xenopus oocytes and used voltage-clamp fluorometry (VCF) to optically track the structural rearrangements of the four VSDs. In conducting channels under physiologically-relevant conditions (2mM [Ca2+]out) we found that the activation curves, F(V), of all VSDs preceded the activation of the ionic conductance, G(V): the half-activation potentials of the four F(V) curves ranged between −61±2 mV and −42±1 mV, while G(V) was V1/2(1) = −36±2 mV; V1/2(2) = 6±3 mV. However, in channels blocked by 200 µM La3+ (as in gating current measurements), the voltage dependence of VSD-I, -III and -IV shifted towards depolarized potentials, to the right of the G(V) curve. Only VSD-II activation was not perturbed by La3+. These results suggest that VSD-I, -III and -IV are energetically coupled with the pore and likely responsible for the voltage-dependent activation. Accordingly, the ataxia-causing mutation R1715H (VSD-IV S4) caused a +20 mV shift in VSD-IV activation and a concomitant +10 mV shift in the G(V). Our findings demonstrate that under conducting conditions all VSDs of Cav3.1 operate in a canonical way: they activate before pore opening. We propose that the impaired activation of CaV3.1 carrying the ataxia-causing mutation is caused by the impaired activation of VSD-IV.

Charge-voltage curves of Shaker potassium channel are not hysteretic at steady state.

Charge-voltage curves of many voltage-gated ion channels exhibit hysteresis but such curves are also a direct measure of free energy of channel gating and, hence, should be path-independent. Here, we identify conditions to measure steady-state charge-voltage curves and show that these are curves are not hysteretic. Charged residues in transmembrane segments of voltage-gated ion channels (VGICs) sense and respond to changes in the electric field. The movement of these gating charges underpins voltage-dependent activation and is also a direct metric of the net free-energy of channel activation. However, for most voltage-gated ion channels, the charge-voltage (Q-V) curves appear to be dependent on initial conditions. For instance, Q-V curves of Shaker potassium channel obtained by hyperpolarizing from 0 mV is left-shifted compared to those obtained by depolarizing from a holding potential of -80 mV. This hysteresis in Q-V curves is a common feature of channels in the VGIC superfamily and raises profound questions about channel energetics because the net free-energy of channel gating is a state function and should be path independent. Due to technical limitations, conventional gating current protocols are limited to test pulse durations of <500 ms, which raises the possibility that the dependence of Q-V on initial conditions reflects a lack of equilibration. Others have suggested that the hysteresis is fundamental thermodynamic property of voltage-gated ion channels and reflects energy dissipation due to measurements under non-equilibrium conditions inherent to rapid voltage jumps (Villalba-Galea. 2017. Channels. https://doi.org/10.1080/19336950.2016.1243190). Using an improved gating current and voltage-clamp fluorometry protocols, we show that the gating hysteresis arising from different initial conditions in Shaker potassium channel is eliminated with ultra-long (18-25 s) test pulses. Our study identifies a modified gating current recording protocol to obtain steady-state Q-V curves of a voltage-gated ion channel. Above all, these findings demonstrate that the gating hysteresis in Shaker channel is a kinetic phenomenon rather than a true thermodynamic property of the channel and the charge-voltage curve is a true measure of the net-free energy of channel gating.

Control over Molecular Orbital Gating and Marcus Inverted Charge Transport in Molecular Junctions with Conjugated Molecular Wires

AbstractRecently it is discovered that molecular junctions can be pushed into the Marcus Inverted region of charge transport, but it is unclear which factors are important. This paper shows that the mechanism of charge transport across molecular wires can be switched between the normal and Marcus Inverted regions by fine‐tuning the molecule–electrode coupling strength and the tunneling distance across oligophenylene ethynylene (OPE) wire terminated with ferrocene (Fc) abbreviated as S‐OPEnFc (n = 1–3). Coherent tunneling dominates the mechanism of charge transport in junctions with short molecules (n = 1), but for n = 2 or 3 redox reactions become important. By weakening the molecule—electrode interaction by interrupted conjugation, S‐CH2‐OPEnFc, intramolecular orbital gating can occur pushing the junctions completely into the Marcus Inverted region. These results indicated that weak molecule—electrode coupling is important to push junctions into the Marcus Inverted Region.

New 'kids' on the voltage-gated proton channel block.

So far one gene for Hv1 has been detected in studied species. The work presented by Chaves etal. in The FEBS Journal reported an 'Unexpected expansion of the voltage-gated proton channel family'. They searched for proton channel candidates and found three sequences in the genome of Aplysia californica (Ac), which were named AcHv1, AcHv2 and AcHv3. Based on electrophysiological experiments, AcHv1 and AcHv2 are voltage-gated channels. While AcHv1 behaves like Hv1 in other species, that is, it is voltage and pH-dependent, it can be inhibited by zinc and conducts protons outwardly, AcHv2 conducts protons inwards at symmetrical pH. AcHv3 constantly leaks protons, and its C-terminal part contains several cytoplasmic retention motifs. Through carefully designed and carried out electrophysiological experiments, Chaves etal. determined the biophysical parameters of all three proton channels, such as the voltage and the pH dependence, the threshold-voltage, the gating charge and the time constants of activation and inactivation. Comment on: https://doi.org/10.1111/febs.16617.

Unwinding and spiral sliding of S4 and domain rotation of VSD during the electromechanical coupling in Nav1.7.

Voltage-gated sodium (Nav) channel Nav1.7 has been targeted for the development of nonaddictive pain killers. Structures of Nav1.7 in distinct functional states will offer an advanced mechanistic understanding and aid drug discovery. Here we report the cryoelectron microscopy analysis of a human Nav1.7 variant that, with 11 rationally introduced point mutations, has a markedly right-shifted activation voltage curve with V1/2 reaching 69 mV. The voltage-sensing domain in the first repeat (VSDI) in a 2.7-Å resolution structure displays a completely down (deactivated) conformation. Compared to the structure of WT Nav1.7, three gating charge (GC) residues in VSDI are transferred to the cytosolic side through a combination of helix unwinding and spiral sliding of S4I and ∼20° domain rotation. A conserved WNФФD motif on the cytoplasmic end of S3Istabilizes the down conformation of VSDI. One GC residue is transferred in VSDII mainly through helix sliding. Accompanying GC transfer in VSDI and VSDII, rearrangement and contraction of the intracellular gate is achieved through concerted movements of adjacent segments, including S4-5I, S4-5II, S5II, and all S6 segments. Our studies provide important insight into the electromechanical coupling mechanism of the single-chain voltage-gated ion channels and afford molecular interpretations for a number of pain-associated mutations whose pathogenic mechanism cannot be revealed from previously reported Nav structures.

Characterization of Inhibitory Capability on Hyperpolarization-Activated Cation Current Caused by Lutein (β,ε-Carotene-3,3'-Diol), a Dietary Xanthophyll Carotenoid.

Lutein (β,ε-carotene-3,3′-diol), a xanthophyll carotenoid, is found in high concentrations in the macula of the human retina. It has been recognized to exert potential effectiveness in antioxidative and anti-inflammatory properties. However, whether and how its modifications on varying types of plasmalemmal ionic currents occur in electrically excitable cells remain incompletely answered. The current hypothesis is that lutein produces any direct adjustments on ionic currents (e.g., hyperpolarization-activated cation current, Ih [or funny current, If]). In the present study, GH3-cell exposure to lutein resulted in a time-, state- and concentration-dependent reduction in Ih amplitude with an IC50 value of 4.1 μM. There was a hyperpolarizing shift along the voltage axis in the steady-state activation curve of Ih in the presence of this compound, despite being void of changes in the gating charge of the curve. Under continued exposure to lutein (3 μM), further addition of oxaliplatin (10 μM) or ivabradine (3 μM) could be effective at either reversing or further decreasing lutein-induced suppression of hyperpolarization-evoked Ih, respectively. The voltage-dependent anti-clockwise hysteresis of Ih responding to long-lasting inverted isosceles-triangular ramp concentration-dependently became diminished by adding this compound. However, the addition of 10 μM lutein caused a mild but significant suppression in the amplitude of erg-mediated or A-type K+ currents. Under current-clamp potential recordings, the sag potential evoked by long-lasting hyperpolarizing current stimulus was reduced under cell exposure to lutein. Altogether, findings from the current observations enabled us to reflect that during cell exposure to lutein used at pharmacologically achievable concentrations, lutein-perturbed inhibition of Ih would be an ionic mechanism underlying its changes in membrane excitability.

Evidence for Dual Activation of IK(M) and IK(Ca) Caused by QO-58 (5-(2,6-Dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-pyrazolol[1,5-a]pyrimidin-7-one).

QO-58 (5-(2,6-dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-pyrazolol[1,5-a]pyrimidin-7-one) has been regarded to be an activator of KV7 channels with analgesic properties. However, whether and how the presence of this compound can result in any modifications of other types of membrane ion channels in native cells are not thoroughly investigated. In this study, we investigated its perturbations on M-type K+ current (IK(M)), Ca2+-activated K+ current (IK(Ca)), large-conductance Ca2+-activated K+ (BKCa) channels, and erg-mediated K+ current (IK(erg)) identified from pituitary tumor (GH3) cells. Addition of QO-58 can increase the amplitude of IK(M) and IK(Ca) in a concentration-dependent fashion, with effective EC50 of 3.1 and 4.2 μM, respectively. This compound could shift the activation curve of IK(M) toward a leftward direction with being void of changes in the gating charge. The strength in voltage-dependent hysteresis (Vhys) of IK(M) evoked by upright triangular ramp pulse (Vramp) was enhanced by adding QO-58. The probabilities of M-type K+ (KM) channels that will be open increased upon the exposure to QO-58, although no modification in single-channel conductance was seen. Furthermore, GH3-cell exposure to QO-58 effectively increased the amplitude of IK(Ca) as well as enhanced the activity of BKCa channels. Under inside-out configuration, QO-58, applied at the cytosolic leaflet of the channel, activated BKCa-channel activity, and its increase could be attenuated by further addition of verruculogen, but not by linopirdine (10 μM). The application of QO-58 could lead to a leftward shift in the activation curve of BKCa channels with neither change in the gating charge nor in single-channel conductance. Moreover, cell exposure of QO-58 (10 μM) resulted in a minor suppression of IK(erg) amplitude in response to membrane hyperpolarization. The docking results also revealed that there are possible interactions of the QO-58 molecule with the KCNQ or KCa1.1 channel. Overall, dual activation of IK(M) and IK(Ca) caused by the presence of QO-58 eventually may have high impacts on the functional activity (e.g., anti-nociceptive effect) residing in electrically excitable cells. Care must be exercised when interpreting data generated with QO-58 as it is not entirely KCNQ/KV7 selective.

Mechanism of voltage sensing in Ca2+- and voltage-activated K+ (BK) channels

In neurosecretion, allosteric communication between voltage sensors and Ca2+ binding in BK channels is crucially involved in damping excitatory stimuli. Nevertheless, the voltage-sensing mechanism of BK channels is still under debate. Here, based on gating current measurements, we demonstrate that two arginines in the transmembrane segment S4 (R210 and R213) function as the BK gating charges. Significantly, the energy landscape of the gating particles is electrostatically tuned by a network of salt bridges contained in the voltage sensor domain (VSD). Molecular dynamics simulations and proton transport experiments in the hyperpolarization-activated R210H mutant suggest that the electric field drops off within a narrow septum whose boundaries are defined by the gating charges. Unlike Kv channels, the charge movement in BK appears to be limited to a small displacement of the guanidinium moieties of R210 and R213, without significant movement of the S4.