scholarly journals Extracellular protons titrate voltage gating of a ligand-gated ion channel

2010 ◽  
Vol 136 (2) ◽  
pp. 179-187 ◽  
Author(s):  
Juan Ramón Martínez-François ◽  
Yanping Xu ◽  
Zhe Lu
Keyword(s):  
Ion Selectivity ◽  
Low Ph ◽  
Extracellular Ph ◽  
Selectivity Filter ◽  
Voltage Gated ◽  
Cyclic Nucleotide Gated Channels ◽  
Open Conformation ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Electric Signals

Cyclic nucleotide–gated channels mediate transduction of light into electric signals in vertebrate photoreceptors. These channels are primarily controlled by the binding of intracellular cyclic GMP (cGMP). Glutamate residue 363 near the extracellular end of the ion selectivity filter interacts with the pore helix and helps anchor the filter to the helix. Disruption of this interaction by mutations renders the channels essentially fully voltage gated in the presence of saturating concentrations of cGMP. Here, we find that lowering extracellular pH makes the channels conduct in an extremely outwardly rectifying manner, as does a neutral glutamine substitution at E363. A pair of cysteine mutations, E363C and L356C (the latter located midway the pore helix), largely eliminates current rectification at low pH. Therefore, this low pH-induced rectification primarily reflects voltage-dependent gating involving the ion selectivity filter rather than altered electrostatics around the external opening of the ion pore and thus ion conduction. It then follows that protonation of E363, like the E363Q mutation, disrupts the attachment of the selectivity filter to the pore helix. Loosening the selectivity filter from its surrounding structure shifts the gating equilibrium toward closed states. At low extracellular pH, significant channel opening occurs only when positive voltages drive the pore from a low probability open conformation to a second open conformation. Consequently, at low extracellular pH the channels become practically fully voltage gated, even in the presence of a saturating concentration of cGMP.

scholarly journals Mutations reveal voltage gating of CNGA1 channels in saturating cGMP

2009 ◽  
Vol 134 (2) ◽  
pp. 151-164 ◽  
Author(s):  
Juan Ramón Martínez-François ◽  
Yanping Xu ◽  
Zhe Lu
Keyword(s):  
Ion Selectivity ◽  
Selectivity Filter ◽  
Open Probability ◽  
K Channels ◽  
Voltage Gated ◽  
Open Conformation ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Low Probability ◽  
Inherent Voltage

Activity of cyclic nucleotide–gated (CNG) cation channels underlies signal transduction in vertebrate visual receptors. These highly specialized receptor channels open when they bind cyclic GMP (cGMP). Here, we find that certain mutations restricted to the region around the ion selectivity filter render the channels essentially fully voltage gated, in such a manner that the channels remain mostly closed at physiological voltages, even in the presence of saturating concentrations of cGMP. This voltage-dependent gating resembles the selectivity filter-based mechanism seen in KcsA K+ channels, not the S4-based mechanism of voltage-gated K+ channels. Mutations that render CNG channels gated by voltage loosen the attachment of the selectivity filter to its surrounding structure, thereby shifting the channel's gating equilibrium toward closed conformations. Significant pore opening in mutant channels occurs only when positive voltages drive the pore from a low-probability open conformation toward a second open conformation to increase the channels' open probability. Thus, the structure surrounding the selectivity filter has evolved to (nearly completely) suppress the expression of inherent voltage-dependent gating of CNGA1, ensuring that the binding of cGMP by itself suffices to open the channels at physiological voltages.

scholarly journals Regulation of Eag1 gating by its intracellular domains

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Jonathan R Whicher ◽  
Roderick MacKinnon
Keyword(s):  
Potassium Channels ◽  
Membrane Voltage ◽  
Specific Interactions ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Open Conformation ◽  
Channel Opening ◽  
Transmembrane Voltage ◽  
Voltage Dependent ◽  
Intracellular Domains

Voltage-gated potassium channels (Kvs) are gated by transmembrane voltage sensors (VS) that move in response to changes in membrane voltage. Kv10.1 or Eag1 also has three intracellular domains: PAS, C-linker, and CNBHD. We demonstrate that the Eag1 intracellular domains are not required for voltage-dependent gating but likely interact with the VS to modulate gating. We identified specific interactions between the PAS, CNBHD, and VS that modulate voltage-dependent gating and provide evidence that VS movement destabilizes these interactions to promote channel opening. Additionally, mutation of these interactions renders Eag1 insensitive to calmodulin inhibition. The structure of the calmodulin insensitive mutant in a pre-open conformation suggests that channel opening may occur through a rotation of the intracellular domains and calmodulin may prevent this rotation by stabilizing interactions between the VS and intracellular domains. Intracellular domains likely play a similar modulatory role in voltage-dependent gating of the related Kv11-12 channels.

scholarly journals Structural Basis for pH-Gating of the K+ Channel TWIK1 at the Selectivity Filter

2021 ◽  
Author(s):  
Toby S Turney ◽  
Vivian Li ◽  
Stephen G Brohawn
Keyword(s):  
Conformational Changes ◽  
Pancreatic Beta Cells ◽  
Low Ph ◽  
Selectivity Filter ◽  
Structural Basis ◽  
K Channel ◽  
Open Conformation ◽  
Gating Mechanism ◽  
Lipid Nanodiscs ◽  
Pore Domain

TWIK1 is a widely expressed pH-gated two-pore domain K+ channel (K2P) that contributes to cardiac rhythm generation and insulin release from pancreatic beta cells. TWIK1 displays unique properties among K2Ps including low basal activity and inhibition by extracellular protons through incompletely understood mechanisms. Here, we present cryo-EM structures of TWIK1 in lipid nanodiscs at high and low pH that reveal a novel gating mechanism at the K+ selectivity filter. At high pH, TWIK1 adopts an open conformation. At low pH, protonation of an extracellular histidine results in a cascade of conformational changes that close the channel by sealing the top of the selectivity filter, displacing the helical cap to block extracellular ion access pathways, and opening gaps for lipid block of the intracellular cavity. These data provide a mechanistic understanding for extracellular pH-gating of TWIK1 and show how diverse mechanisms have evolved to gate the selectivity filter of K+ channels.

scholarly journals Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

2019 ◽  
Vol 116 (38) ◽  
pp. 18951-18961 ◽  
Author(s):  
Richard Banh ◽  
Vladimir V. Cherny ◽  
Deri Morgan ◽  
Boris Musset ◽  
Sarah Thomas ◽  
...  
Keyword(s):  
Proton Channel ◽  
Voltage Gated ◽  
Closed State ◽  
Channel Opening ◽  
Proton Current ◽  
Voltage Dependent ◽  
Current Flows ◽  
Voltage Gated Ion Channels ◽  
Dynamics Simulations ◽  
Voltage Sensing Domain

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this “gating pore” when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

scholarly journals Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac

2016 ◽  
Vol 113 (48) ◽  
pp. 13762-13767 ◽  
Author(s):  
Monica N. Kinde ◽  
Vasyl Bondarenko ◽  
Daniele Granata ◽  
Weiming Bu ◽  
Kimberly C. Grasty ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Binding Sites ◽  
Ion Selectivity ◽  
Selectivity Filter ◽  
Inactivation Kinetics ◽  
Voltage Gated Sodium Channel ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Binding Data ◽  
Quantitative Analyses

Voltage-gated sodium channels (NaV) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit NaVby stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in NaV, but experimental binding data are lacking. Here we use site-directed placement of19F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac.19F probes were introduced individually to S129 and L150 near the S4–S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of19F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150. We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: (i) as a channel blocker at the base of the selectivity filter, and (ii) as a modulator to restrict the pivot motion at the S4–S5 linker and at a critical hinge that controls the gating and inactivation motion of S6.

scholarly journals Mechanism of the Voltage Sensitivity of IRK1 Inward-rectifier K+ Channel Block by the Polyamine Spermine

2005 ◽  
Vol 125 (4) ◽  
pp. 413-426 ◽  
Author(s):  
Hyeon-Gyu Shin ◽  
Zhe Lu
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Ion Selectivity ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
K Channel ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Head Groups ◽  
Voltage Dependent

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence ∼5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QAC10) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider (∼6 Å) than the ion selectivity filter (∼3 Å). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QAC10 should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.

scholarly journals Voltage Clamp Fluorimetry Reveals a Novel Outer Pore Instability in a Mammalian Voltage-gated Potassium Channel

2008 ◽  
Vol 132 (2) ◽  
pp. 209-222 ◽  
Author(s):  
Moninder Vaid ◽  
Thomas W. Claydon ◽  
Saman Rezazadeh ◽  
David Fedida
Keyword(s):  
Voltage Clamp ◽  
Time Course ◽  
Ionic Current ◽  
Voltage Sensor ◽  
Selectivity Filter ◽  
Similar Time ◽  
Voltage Gated ◽  
Kv Channels ◽  
Channel Opening ◽  
Outer Pore

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K+ ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3–S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 ± 3% of the total signal and occurred with a τ of 3.4 ± 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K+ to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.

scholarly journals Intrinsic versus extrinsic voltage sensitivity of blocker interaction with an ion channel pore

2010 ◽  
Vol 135 (2) ◽  
pp. 149-167 ◽  
Author(s):  
Juan Ramón Martínez-François ◽  
Zhe Lu
Keyword(s):  
Electric Field ◽  
Voltage Dependence ◽  
Ion Selectivity ◽  
Systematic Investigation ◽  
Selectivity Filter ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Boltzmann Function ◽  
Voltage Dependent ◽  
Gated Channel

Many physiological and synthetic agents act by occluding the ion conduction pore of ion channels. A hallmark of charged blockers is that their apparent affinity for the pore usually varies with membrane voltage. Two models have been proposed to explain this voltage sensitivity. One model assumes that the charged blocker itself directly senses the transmembrane electric field, i.e., that blocker binding is intrinsically voltage dependent. In the alternative model, the blocker does not directly interact with the electric field; instead, blocker binding acquires voltage dependence solely through the concurrent movement of permeant ions across the field. This latter model may better explain voltage dependence of channel block by large organic compounds that are too bulky to fit into the narrow (usually ion-selective) part of the pore where the electric field is steep. To date, no systematic investigation has been performed to distinguish between these voltage-dependent mechanisms of channel block. The most fundamental characteristic of the extrinsic mechanism, i.e., that block can be rendered voltage independent, remains to be established and formally analyzed for the case of organic blockers. Here, we observe that the voltage dependence of block of a cyclic nucleotide–gated channel by a series of intracellular quaternary ammonium blockers, which are too bulky to traverse the narrow ion selectivity filter, gradually vanishes with extreme depolarization, a predicted feature of the extrinsic voltage dependence model. In contrast, the voltage dependence of block by an amine blocker, which has a smaller “diameter” and can therefore penetrate into the selectivity filter, follows a Boltzmann function, a predicted feature of the intrinsic voltage dependence model. Additionally, a blocker generates (at least) two blocked states, which, if related serially, may preclude meaningful application of a commonly used approach for investigating channel gating, namely, inferring the properties of the activation gate from the kinetics of channel block.

scholarly journals Biophysical characterization of the honeybee DSC1 orthologue reveals a novel voltage-dependent Ca2+ channel subfamily: CaV4

2016 ◽  
Vol 148 (2) ◽  
pp. 133-145 ◽  
Author(s):  
Pascal Gosselin-Badaroudine ◽  
Adrien Moreau ◽  
Louis Simard ◽  
Thierry Cens ◽  
Matthieu Rousset ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Expression System ◽  
Selectivity Filter ◽  
Inactivation Kinetics ◽  
Na Channel ◽  
Biophysical Characterization ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Nav Channels

Bilaterian voltage-gated Na+ channels (NaV) evolved from voltage-gated Ca2+ channels (CaV). The Drosophila melanogaster Na+ channel 1 (DSC1), which features a D-E-E-A selectivity filter sequence that is intermediate between CaV and NaV channels, is evidence of this evolution. Phylogenetic analysis has classified DSC1 as a Ca2+-permeable Na+ channel belonging to the NaV2 family because of its sequence similarity with NaV channels. This is despite insect NaV2 channels (DSC1 and its orthologue in Blatella germanica, BSC1) being more permeable to Ca2+ than Na+. In this study, we report the cloning and molecular characterization of the honeybee (Apis mellifera) DSC1 orthologue. We reveal several sequence variations caused by alternative splicing, RNA editing, and genomic variations. Using the Xenopus oocyte heterologous expression system and the two-microelectrode voltage-clamp technique, we find that the channel exhibits slow activation and inactivation kinetics, insensitivity to tetrodotoxin, and block by Cd2+ and Zn2+. These characteristics are reminiscent of CaV channels. We also show a strong selectivity for Ca2+ and Ba2+ ions, marginal permeability to Li+, and impermeability to Mg2+ and Na+ ions. Based on current ion channel nomenclature, the D-E-E-A selectivity filter, and the properties we have uncovered, we propose that DSC1 homologues should be classified as CaV4 rather than NaV2. Indeed, channels that contain the D-E-E-A selectivity sequence are likely to feature the same properties as the honeybee’s channel, namely slow activation and inactivation kinetics and strong selectivity for Ca2+ ions.

scholarly journals Transfer of Voltage Independence from a Rat Olfactory Channel to the Drosophila Ether-à-go-go K+ Channel

1997 ◽  
Vol 109 (3) ◽  
pp. 301-311 ◽  
Author(s):  
Chih-Yung Tang ◽  
Diane M. Papazian
Keyword(s):  
Cyclic Nucleotide ◽  
Voltage Sensor ◽  
K Channel ◽  
Voltage Range ◽  
Voltage Gated ◽  
Cyclic Nucleotide Gated Channels ◽  
Voltage Dependent ◽  
Relative Stabilization ◽  
Voltage Gated Ion Channels ◽  
S4 Segment

The S4 segment is an important part of the voltage sensor in voltage-gated ion channels. Cyclic nucleotide-gated channels, which are members of the superfamily of voltage-gated channels, have little inherent sensitivity to voltage despite the presence of an S4 segment. We made chimeras between a voltage-independent rat olfactory channel (rolf) and the voltage-dependent ether-à-go-go K+ channel (eag) to determine the basis of their divergent gating properties. We found that the rolf S4 segment can support a voltage-dependent mechanism of activation in eag, suggesting that rolf has a potentially functional voltage sensor that is silent during gating. In addition, we found that the S3-S4 loop of rolf increases the relative stability of the open conformation of eag, effectively converting eag into a voltage-independent channel. A single charged residue in the loop makes a significant contribution to the relative stabilization of the open state in eag. Our data suggest that cyclic nucleotide-gated channels such as rolf contain a voltage sensor which, in the physiological voltage range, is stabilized in an activated conformation that is permissive for pore opening.

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