scholarly journals Nonsensing residues in S3–S4 linker’s C terminus affect the voltage sensor set point in K+ channels

2018 ◽  
Vol 150 (2) ◽  
pp. 307-321 ◽  
Author(s):  
Joao L. Carvalho-de-Souza ◽  
Francisco Bezanilla
Keyword(s):  
Intermediate State ◽  
Voltage Dependence ◽  
State Model ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
K Channels ◽  
C Terminus ◽  
Voltage Dependent ◽  
Arginine Residues ◽  
Three State Model

Voltage sensitivity in ion channels is a function of highly conserved arginine residues in their voltage-sensing domains (VSDs), but this conservation does not explain the diversity in voltage dependence among different K+ channels. Here we study the non–voltage-sensing residues 353 to 361 in Shaker K+ channels and find that residues 358 and 361 strongly modulate the voltage dependence of the channel. We mutate these two residues into all possible remaining amino acids (AAs) and obtain Q-V and G-V curves. We introduced the nonconducting W434F mutation to record sensing currents in all mutants except L361R, which requires K+ depletion because it is affected by W434F. By fitting Q-Vs with a sequential three-state model for two voltage dependence–related parameters (V0, the voltage-dependent transition from the resting to intermediate state and V1, from the latter to the active state) and G-Vs with a two-state model for the voltage dependence of the pore domain parameter (V1/2), Spearman’s coefficients denoting variable relationships with hydrophobicity, available area, length, width, and volume of the AAs in 358 and 361 positions could be calculated. We find that mutations in residue 358 shift Q-Vs and G-Vs along the voltage axis by affecting V0, V1, and V1/2 according to the hydrophobicity of the AA. Mutations in residue 361 also shift both curves, but V0 is affected by the hydrophobicity of the AA in position 361, whereas V1 and V1/2 are affected by size-related AA indices. Small-to-tiny AAs have opposite effects on V1 and V1/2 in position 358 compared with 361. We hypothesize possible coordination points in the protein that residues 358 and 361 would temporarily and differently interact with in an intermediate state of VSD activation. Our data contribute to the accumulating knowledge of voltage-dependent ion channel activation by adding functional information about the effects of so-called non–voltage-sensing residues on VSD dynamics.

scholarly journals Coupling of Voltage Sensing to Channel Opening Reflects Intrasubunit Interactions in Kv Channels

2004 ◽  
Vol 125 (1) ◽  
pp. 71-80 ◽  
Author(s):  
Alain J. Labro ◽  
Adam L. Raes ◽  
Dirk J. Snyders
Keyword(s):  
Voltage Dependence ◽  
Voltage Sensing ◽  
Α Subunit ◽  
K Channels ◽  
Voltage Gated ◽  
Kv Channels ◽  
Channel Opening ◽  
Subunit Stoichiometry ◽  
Voltage Dependent ◽  
Tetrameric Structure

Voltage-gated K+ channels play a central role in the modulation of excitability. In these channels, the voltage-dependent movement of the voltage sensor (primarily S4) is coupled to the (S6) gate that opens the permeation pathway. Because of the tetrameric structure, such coupling could occur within each subunit or between adjacent subunits. To discriminate between these possibilities, we analyzed various combinations of a S4 mutation (R401N) and a S6 mutation (P511G) in hKv1.5, incorporated into tandem constructs to constrain subunit stoichiometry. R401N shifted the voltage dependence of activation to negative potentials while P511G did the opposite. When both mutations were introduced in the same α-subunit of the tandem, the positive shift of P511G was compensated by the negative shift of R401N. With each mutation in a separate subunit of a tandem, this compensation did not occur. This suggests that for Kv channels, the coupling between voltage sensing and gating reflects primarily an intrasubunit interaction.

scholarly journals Slo3 K+ Channels: Voltage and pH Dependence of Macroscopic Currents

2006 ◽  
Vol 128 (3) ◽  
pp. 317-336 ◽  
Author(s):  
Xue Zhang ◽  
Xuhui Zeng ◽  
Christopher J. Lingle
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Ph Dependence ◽  
K Channel ◽  
Open Probability ◽  
Cytosolic Ph ◽  
Α Subunit ◽  
K Channels ◽  
C Terminus ◽  
Voltage Dependent

The mouse Slo3 gene (KCNMA3) encodes a K+ channel that is regulated by changes in cytosolic pH. Like Slo1 subunits responsible for the Ca2+ and voltage-activated BK-type channel, the Slo3 α subunit contains a pore module with homology to voltage-gated K+ channels and also an extensive cytosolic C terminus thought to be responsible for ligand dependence. For the Slo3 K+ channel, increases in cytosolic pH promote channel activation, but very little is known about many fundamental properties of Slo3 currents. Here we define the dependence of macroscopic conductance on voltage and pH and, in particular, examine Slo3 conductance activated at negative potentials. Using this information, the ability of a Horrigan-Aldrich–type of general allosteric model to account for Slo3 gating is examined. Finally, the pH and voltage dependence of Slo3 activation and deactivation kinetics is reported. The results indicate that Slo3 differs from Slo1 in several important ways. The limiting conductance activated at the most positive potentials exhibits a pH-dependent maximum, suggesting differences in the limiting open probability at different pH. Furthermore, over a 600 mV range of voltages (−300 to +300 mV), Slo3 conductance shifts only about two to three orders of magnitude, and the limiting conductance at negative potentials is relatively voltage independent compared to Slo1. Within the context of the Horrigan-Aldrich model, these results indicate that the intrinsic voltage dependence (zL) of the Slo3 closed–open equilibrium and the coupling (D) between voltage sensor movement are less than in Slo1. The kinetic behavior of Slo3 currents also differs markedly from Slo1. Both activation and deactivation are best described by two exponential components, both of which are only weakly voltage dependent. Qualitatively, the properties of the two kinetic components in the activation time course suggest that increases in pH increase the fraction of more rapidly opening channels.

scholarly journals Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels

2016 ◽  
Vol 147 (6) ◽  
pp. 437-449 ◽  
Author(s):  
Petronel Tuluc ◽  
Bruno Benedetti ◽  
Pierre Coste de Bagneaux ◽  
Manfred Grabner ◽  
Bernhard E. Flucher
Keyword(s):  
Calcium Channels ◽  
Molecular Mechanisms ◽  
Voltage Dependence ◽  
Specific Interaction ◽  
Site Directed Mutagenesis ◽  
Control Voltage ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
Activation Kinetics ◽  
Transmembrane Segments

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.

Voltage dependence of conductance changes evoked by glycine release in the zebrafish brain

1995 ◽  
Vol 73 (6) ◽  
pp. 2404-2412 ◽  
Author(s):  
P. Legendre ◽  
H. Korn
Keyword(s):  
Voltage Dependence ◽  
Reversal Potential ◽  
Voltage Sensitivity ◽  
M Cell ◽  
Open Probability ◽  
Similar Time ◽  
Whole Cell ◽  
Glycine Release ◽  
Cell Recording ◽  
Voltage Dependent

1. The kinetics and mechanisms underlying the voltage dependence of inhibitory postsynaptic currents (IPSCs) recorded in the Mauthner cell (M cell) were investigated in the isolated medulla of 52-h-old zebrafish larvae, with the use of whole cell and outside-out patch-clamp recordings. 2. Spontaneous miniature IPSCs (mIPSCs) were recorded in the presence of 10(-6) M tetrodotoxin (TTX), 10 mM MgCl2, and 0.1 mM [CaCl2]o. Depolarizing the cell from -50 to +50 mV did not evoke any significant change in the distribution of mIPSC amplitudes, whereas synaptic currents were prolonged at positive voltages. The average decay time constant was increased twofold at +50 mV. 3. The voltage dependence of the kinetics of glycine-activated channels was first investigated during whole cell recording experiments. Currents evoked by voltage steps in the presence of glycine (50 microM) were compared with those obtained without glycine. The increase in chloride conductance (gCl-) evoked by glycine was time and voltage dependent. Inactivation and reactivation of the chloride current were observed during voltage pulses from 0 to -50 mV and from -50 to 0 mV, respectively, and they occurred with similar time constants (2-3 s). During glycine application, voltage-ramp analysis revealed a shift in the reversal potential (ECl-) occurring at all [Cl-]i tested. 4. The basis of the voltage sensitivity of glycine-evoked gCl- was first analyzed by measuring the relative changes in the total open probability (NPo) of glycine-activated channels with voltage.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Differential voltage-dependent modulation of the ACh-gated K+ current by adenosine and acetylcholine

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261960
Author(s):  
Ana Laura López-Serrano ◽  
Rodrigo Zamora-Cárdenas ◽  
Iván A. Aréchiga-Figueroa ◽  
Pedro D. Salazar-Fajardo ◽  
Tania Ferrer ◽  
...  
Keyword(s):  
Voltage Dependence ◽  
Gradual Increase ◽  
Relaxation Property ◽  
Voltage Sensitivity ◽  
Response Curves ◽  
Deactivation Kinetics ◽  
Relative Affinity ◽  
Voltage Dependent ◽  
K Current ◽  
Inhibitory Regulation

Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the ‘relaxation’ property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.

scholarly journals Interaction Mechanisms between Polyamines and IRK1 Inward Rectifier K+ Channels

2003 ◽  
Vol 122 (5) ◽  
pp. 485-500 ◽  
Author(s):  
Donglin Guo ◽  
Zhe Lu
Keyword(s):  
Voltage Dependence ◽  
Divalent Cations ◽  
Inward Rectifier ◽  
Affinity Binding ◽  
Channel Block ◽  
K Channels ◽  
High Affinity ◽  
Voltage Dependent ◽  
Varying Length ◽  
Amine Groups

Rectification of macroscopic current through inward-rectifier K+ (Kir) channels reflects strong voltage dependence of channel block by intracellular cations such as polyamines. The voltage dependence results primarily from the movement of K+ ions across the transmembrane electric field, which accompanies the binding–unbinding of a blocker. Residues D172, E224, and E299 in IRK1 are critical for high-affinity binding of blockers. D172 appears to be located somewhat internal to the narrow K+ selectivity filter, whereas E224 and E299 form a ring at a more intracellular site. Using a series of alkyl-bis-amines of varying length as calibration, we investigated how the acidic residues in IRK1 interact with amine groups in the natural polyamines (putrescine, spermidine, and spermine) that cause rectification in cells. To block the pore, the leading amine of bis-amines of increasing length penetrates ever deeper into the pore toward D172, while the trailing amine in every bis-amine binds near a more intracellular site and interacts with E224 and E299. The leading amine in nonamethylene-bis-amine (bis-C9) makes the closest approach to D172, displacing the maximal number of K+ ions and exhibiting the strongest voltage dependence. Cells do not synthesize bis-amines longer than putrescine (bis-C4) but generate the polyamines spermidine and spermine by attaching an amino-propyl group to one or both ends of putrescine. Voltage dependence of channel block by the tetra-amine spermine is comparable to that of block by the bis-amines bis-C9 (shorter) or bis-C12 (equally long), but spermine binds to IRK1 with much higher affinity than either bis-amine does. Thus, counterintuitively, the multiple amines in spermine primarily confer the high affinity but not the strong voltage dependence of channel block. Tetravalent spermine achieves a stronger interaction with the pore by effectively behaving like a pair of tethered divalent cations, two amine groups in its leading half interacting primarily with D172, whereas the other two in the trailing half interact primarily with E224 and E299. Thus, nature has optimized not only the blocker but also, in a complementary manner, the channel for producing rapid, high-affinity, and strongly voltage-dependent channel block, giving rise to exceedingly sharp rectification.

scholarly journals Voltage Sensing in Bacterial Protein Translocation

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 78 ◽  
Author(s):  
Denis G. Knyazev ◽  
Roland Kuttner ◽  
Ana-Nicoleta Bondar ◽  
Mirjam Zimmerman ◽  
Christine Siligan ◽  
...  
Keyword(s):  
Protein Translocation ◽  
Voltage Sensor ◽  
Voltage Sensitivity ◽  
Bacterial Protein ◽  
Voltage Sensing ◽  
Voltage Dependent ◽  
Membrane Spanning ◽  
Dipolar Orientation ◽  
Polypeptide Chains ◽  
Gating Charges

The bacterial channel SecYEG efficiently translocates both hydrophobic and hydrophilic proteins across the plasma membrane. Translocating polypeptide chains may dislodge the plug, a half helix that blocks the permeation of small molecules, from its position in the middle of the aqueous translocation channel. Instead of the plug, six isoleucines in the middle of the membrane supposedly seal the channel, by forming a gasket around the translocating polypeptide. However, this hypothesis does not explain how the tightness of the gasket may depend on membrane potential. Here, we demonstrate voltage-dependent closings of the purified and reconstituted channel in the presence of ligands, suggesting that voltage sensitivity may be conferred by motor protein SecA, ribosomes, signal peptides, and/or translocating peptides. Yet, the presence of a voltage sensor intrinsic to SecYEG was indicated by voltage driven closure of pores that were forced-open either by crosslinking the plug to SecE or by plug deletion. We tested the involvement of SecY’s half-helix 2b (TM2b) in voltage sensing, since clearly identifiable gating charges are missing. The mutation L80D accelerated voltage driven closings by reversing TM2b’s dipolar orientation. In contrast, the L80K mutation decelerated voltage induced closings by increasing TM2b’s dipole moment. The observations suggest that TM2b is part of a larger voltage sensor. By partly aligning the combined dipole of this sensor with the orientation of the membrane-spanning electric field, voltage may drive channel closure.

scholarly journals PIP2 mediates functional coupling and pharmacology of neuronal KCNQ channels

2017 ◽  
Vol 114 (45) ◽  
pp. E9702-E9711 ◽  
Author(s):  
Robin Y. Kim ◽  
Stephan A. Pless ◽  
Harley T. Kurata
Keyword(s):  
Conformational Changes ◽  
Neuronal Excitability ◽  
Voltage Dependence ◽  
Voltage Sensor ◽  
Functional Coupling ◽  
Voltage Sensing ◽  
Kcnq Channels ◽  
C Terminus ◽  
Hyperpolarizing Shift ◽  
State Ionic

Retigabine (RTG) is a first-in-class antiepileptic drug that suppresses neuronal excitability through the activation of voltage-gated KCNQ2–5 potassium channels. Retigabine binds to the pore-forming domain, causing a hyperpolarizing shift in the voltage dependence of channel activation. To elucidate how the retigabine binding site is coupled to changes in voltage sensing, we used voltage-clamp fluorometry to track conformational changes of the KCNQ3 voltage-sensing domains (VSDs) in response to voltage, retigabine, and PIP2. Steady-state ionic conductance and voltage sensor fluorescence closely overlap under basal PIP2 conditions. Retigabine stabilizes the conducting conformation of the pore and the activated voltage sensor conformation, leading to dramatic deceleration of current and fluorescence deactivation, but these effects are attenuated upon disruption of channel:PIP2 interactions. These findings reveal an important role for PIP2 in coupling retigabine binding to altered VSD function. We identify a polybasic motif in the proximal C terminus of retigabine-sensitive KCNQ channels that contributes to VSD–pore coupling via PIP2, and thereby influences the unique gating effects of retigabine.

scholarly journals Mechanism of Ba2+ block of M-like K channels of rod photoreceptors of tiger salamanders.

1994 ◽  
Vol 103 (1) ◽  
pp. 45-66 ◽  
Author(s):  
L P Wollmuth
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Cell Voltage ◽  
Voltage Sensitivity ◽  
Rod Photoreceptors ◽  
Voltage Range ◽  
Tiger Salamanders ◽  
High K ◽  
Voltage Dependent ◽  
Time Courses

IKx is a voltage-dependent K+ current in the inner segment of rod photoreceptors that shows many similarities to M-current. The depression of IKx by external Ba2+ was studied with whole-cell voltage clamp. Ba2+ reduced the conductance and voltage sensitivity of IKx tail currents and shifted the voltage range over which they appeared to more positive potentials. These effects showed different sensitivities to Ba2+: conductance was the least sensitive (K0.5 = 7.6 mM), voltage dependence intermediate (K0.5 = 2.4 mM) and voltage sensitivity the most sensitive (K0.5 = 0.2 mM). Ca2+, Co2+, Mn2+, Sr2+, and Zn2+ did not have actions comparable to Ba2+ on the voltage dependence or the voltage sensitivity of IKx tail currents. In high K+ (100 mM), the voltage range of activation of IKx was shifted 20 mV negative, as was the tau-voltage relation. High K+ did not prevent the effect of Ba2+ on conductance, but abolished its ability to affect voltage dependence and voltage sensitivity. Ba2+ also altered the apparent time-course of activation and deactivation of IKx. Low Ba2+ (0.2 mM) slowed both deactivation and activation, with most effect on deactivation; at higher concentrations (1-25 mM), deactivation and activation time courses were equally affected, and at the highest concentrations, 5 and 25 mM Ba2+, the time course became faster than control. Rapid application of 5 mM Ba2+ suggested that the time dependent currents in Ba2+ reflect in part the slow voltage-dependent block and unblock of IKx channels by Ba2+. This blocking action of Ba2+ was steeply voltage-dependent with an apparent electrical distance of 1.07. Ba2+ appears to interact with IKx channels at multiple sites. A model which assumes that Ba2+ has a voltage-independent and a voltage-dependent blocking action on open or closed IKx channels reproduced many aspects of the data; the voltage-dependent component could account for both the Ba(2+)-induced shift in voltage dependence and reduction in voltage sensitivity of IKx tail currents.

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.

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