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.

scholarly journals Structural determinants of voltage-gating properties in calcium channels

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Monica L Fernández-Quintero ◽  
Yousra El Ghaleb ◽  
Petronel Tuluc ◽  
Marta Campiglio ◽  
Klaus R Liedl ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Molecular Mechanisms ◽  
Mechanistic Model ◽  
Ion Pair ◽  
Pair Formation ◽  
Structural Determinants ◽  
Voltage Sensing ◽  
Voltage Gated ◽  
Ion Pair Formation ◽  
Activated State

Voltage-gated calcium channels control key functions of excitable cells, like synaptic transmission in neurons and the contraction of heart and skeletal muscles. To accomplish such diverse functions, different calcium channels activate at different voltages and with distinct kinetics. To identify the molecular mechanisms governing specific voltage-sensing properties we combined structure modeling, mutagenesis, and electrophysiology to analyze the structures, free energy, and transition kinetics of the activated and resting states of two functionally distinct voltage-sensing domains (VSDs) of the eukaryotic calcium channel CaV1.1. Both VSDs displayed the typical features of the sliding helix model; however, they greatly differed in ion-pair formation of the outer gating charges. Specifically, stabilization of the activated state enhanced the voltage-dependence of activation, while stabilization of resting states slowed the kinetics. This mechanism provides a mechanistic model explaining how specific ion-pair formation in separate VSDs can realize the characteristic gating properties of voltage-gated cation channels.

scholarly journals The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop

2012 ◽  
Vol 139 (4) ◽  
pp. 285-294 ◽  
Author(s):  
Valentina Cenedese ◽  
Giulia Betto ◽  
Fulvio Celsi ◽  
O. Lijo Cherian ◽  
Simone Pifferi ◽  
...  
Keyword(s):  
Structure Function ◽  
Sensory Neurons ◽  
Molecular Mechanisms ◽  
Voltage Dependence ◽  
Cell Voltage ◽  
Site Directed Mutagenesis ◽  
Initial Structure ◽  
Intracellular Loop ◽  
Retinal Photoreceptors ◽  
Cl Channels

Ca2+-activated Cl− channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure–function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca2+ dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca2+ concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates 386EEEEE390 and 399EYE401. The EYE deletion did not significantly modify the apparent Ca2+ dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca2+ affinity but modified the voltage dependence, shifting the conductance–voltage relations toward more positive voltages. These findings indicate that glutamates E367 and 386EEEEE390 in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure–function study for this channel.

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 Conserved amino acids residing outside the voltage field can shift the voltage sensitivity and increase the signal size of Ciona based GEVIs

2020 ◽  
Author(s):  
Masoud Sepehri Rad ◽  
Lawrence B. Cohen ◽  
Bradley J. Baker
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Optical Signal ◽  
Voltage Sensitivity ◽  
Intracellular Loop ◽  
Voltage Sensing ◽  
Voltage Range ◽  
Transmembrane Segments ◽  
Voltage Sensing Domain

AbstractTo identify potential regions of the voltage-sensing domain that could shift the voltage sensitivity of Ciona intestinalis based Genetically Encoded Voltage Indicators (GEVIs), we aligned 183 voltage-gated sodium channels from different organisms. Conserved polar residues were identified at multiple transmembrane loop junctions in the voltage sensing domain. Similar conservation of polar amino acids was found in the voltage sensing domain of the voltage-sensing phosphatase gene family. These conserved residues were mutated to nonpolar or oppositely charged amino acids in a GEVI that utilizes the voltage sensing domain of the voltage sensing phosphatase from Ciona fused to the fluorescent protein, super ecliptic pHlorin (A227D). Different mutations shifted the voltage sensitivity in a more positive or a more negative direction. Double mutants were then created by selecting constructs that shifted the optical signal to more negative potentials resulting in an improved signal in the physiological voltage range. The combination of two mutations, Y172A (intracellular loop between transmembrane segments S2 and S3) and D204K (extracellular loop between transmembrane segments S3 and S4) showed the increased the signal size from 2.5% to 13.8% for a 100 mV depolarization. Introduction of these mutations into previously developed GEVIs improved the dynamic range to 40% ΔF/F/100 mV.

Mutations throughout the S6 region of the hKv1.5 channel alter the stability of the activation gate

2002 ◽  
Vol 282 (1) ◽  
pp. C161-C171 ◽  
Author(s):  
Thomas C. Rich ◽  
Sarita W. Yeola ◽  
Michael M. Tamkun ◽  
Dirk J. Snyders
Keyword(s):  
Voltage Dependence ◽  
Channel Gating ◽  
Temperature Sensitive ◽  
Voltage Sensitivity ◽  
Wild Type ◽  
Activation Kinetics ◽  
Deactivation Kinetics ◽  
Activation Gate ◽  
The Stability ◽  
Kinetics Of

First published September 21, 2001; 10.1152/ ajpcell.00232.2001.—The S6 segment of voltage-gated K+ channels is thought to contribute to the gate that opens the central permeation pathway. Here we present evidence that mutations throughout the cytoplasmic end of S6 strongly influence hKv1.5 channel gating characteristics. Modification of hKv1.5 at positions T505, V512, and S515 resulted in large negative shifts in the voltage dependence of activation, whereas modifications at position Y519 resulted in negative (Y519N) and positive (Y519F) shifts. When adjusted for the altered voltage sensitivity, activation kinetics of mutated channels were similar to those of the wild-type (WT) channel; however, deactivation kinetics of mutations T505I, T505V, V512A, and V512M [time constant (τ) = 35, 250, 170, and 420 ms, respectively] were still slower than WT (τ = 8.3 ms). In addition, deactivation of WT channels was highly temperature sensitive. However, deactivation of T505I and V512A channels was largely temperature insensitive. Together, these data suggest that mutations in S6 decouple activation from deactivation by altering the open-state stability and that residues on both sides of the highly conserved Pro-X-Pro sequence influence the movement of S6 during channel gating.

scholarly journals Coupling between the voltage-sensing and phosphatase domains of Ci-VSP

2009 ◽  
Vol 134 (1) ◽  
pp. 5-14 ◽  
Author(s):  
Carlos A. Villalba-Galea ◽  
Francesco Miceli ◽  
Maurizio Taglialatela ◽  
Francisco Bezanilla
Keyword(s):  
Phosphatase Activity ◽  
Voltage Dependence ◽  
Specific Interaction ◽  
Ionic Currents ◽  
Voltage Sensor ◽  
Voltage Sensing ◽  
Phosphatase And Tensin Homologue ◽  
S4 Segment ◽  
Phosphate Groups ◽  
Phosphatase Enzyme

The Ciona intestinalis voltage sensor–containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.

scholarly journals Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

2012 ◽  
Vol 86 (16) ◽  
pp. 8375-8387 ◽  
Author(s):  
Marie Galloux ◽  
Bogdan Tarus ◽  
Ilfad Blazevic ◽  
Jenna Fix ◽  
Stéphane Duquerroy ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Specific Interaction ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Interaction Domain ◽  
Rna Complex

The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (PCTD) and N. However, the P binding region on N remains to be identified. In this study, glutathioneS-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (NNTD) as a P binding domain. A biochemical characterization of the PCTDand molecular modeling of the NNTDallowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the PCTDinteraction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolishedin vitroandin vivoP-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.

scholarly journals Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.

1991 ◽  
Vol 98 (1) ◽  
pp. 77-93 ◽  
Author(s):  
C K Abrams ◽  
K S Jakes ◽  
A Finkelstein ◽  
S L Slatin
Keyword(s):  
Voltage Dependence ◽  
Ion Selectivity ◽  
Lipid Vesicles ◽  
Voltage Gating ◽  
Site Directed Mutagenesis ◽  
Phospholipid Bilayer ◽  
Voltage Gated ◽  
Colicin E1 ◽  
Gating Charge ◽  
Ion Conducting

The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.

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.

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