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 Role of Charged Residues in the S1–S4 Voltage Sensor of BK Channels

2006 ◽  
Vol 127 (3) ◽  
pp. 309-328 ◽  
Author(s):  
Zhongming Ma ◽  
Xing Jian Lou ◽  
Frank T. Horrigan
Keyword(s):  
Voltage Dependence ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Voltage Gating ◽  
Voltage Sensing ◽  
Kv Channels ◽  
Gating Charge ◽  
Charged Residues ◽  
Sensor Activation

The activation of large conductance Ca2+-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K+ (KV) channels. Yet BK and KV channels share many conserved charged residues in transmembrane segments S1–S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (Po) and IK kinetics (τ(IK)) over an extended voltage range in 0–50 μM [Ca2+]i. mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of PO. The voltage dependence of PO and τ(IK) at extreme negative potentials was also reduced, implying that the closed–open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and KV channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to KV channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1–S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3–7 kcal mol−1, indicating a strong contribution of non–voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.

scholarly journals Intermediate state trapping of a voltage sensor

2012 ◽  
Vol 140 (6) ◽  
pp. 635-652 ◽  
Author(s):  
Jérôme J. Lacroix ◽  
Stephan A. Pless ◽  
Luca Maragliano ◽  
Fabiana V. Campos ◽  
Jason D. Galpin ◽  
...  
Keyword(s):  
Ion Channels ◽  
Conformational Changes ◽  
Intermediate State ◽  
Molecular Mechanisms ◽  
Voltage Sensor ◽  
Functional Coupling ◽  
Kv Channel ◽  
New Information ◽  
Helical Turn ◽  
Voltage Gated Ion Channels

Voltage sensor domains (VSDs) regulate ion channels and enzymes by undergoing conformational changes depending on membrane electrical signals. The molecular mechanisms underlying the VSD transitions are not fully understood. Here, we show that some mutations of I241 in the S1 segment of the Shaker Kv channel positively shift the voltage dependence of the VSD movement and alter the functional coupling between VSD and pore domains. Among the I241 mutants, I241W immobilized the VSD movement during activation and deactivation, approximately halfway between the resting and active states, and drastically shifted the voltage activation of the ionic conductance. This phenotype, which is consistent with a stabilization of an intermediate VSD conformation by the I241W mutation, was diminished by the charge-conserving R2K mutation but not by the charge-neutralizing R2Q mutation. Interestingly, most of these effects were reproduced by the F244W mutation located one helical turn above I241. Electrophysiology recordings using nonnatural indole derivatives ruled out the involvement of cation-Π interactions for the effects of the Trp inserted at positions I241 and F244 on the channel’s conductance, but showed that the indole nitrogen was important for the I241W phenotype. Insight into the molecular mechanisms responsible for the stabilization of the intermediate state were investigated by creating in silico the mutations I241W, I241W/R2K, and F244W in intermediate conformations obtained from a computational VSD transition pathway determined using the string method. The experimental results and computational analysis suggest that the phenotype of I241W may originate in the formation of a hydrogen bond between the indole nitrogen atom and the backbone carbonyl of R2. This work provides new information on intermediate states in voltage-gated ion channels with an approach that produces minimum chemical perturbation.

scholarly journals Epilepsy-associated mutations in the voltage sensor of KCNQ3 affect voltage dependence of channel opening

2018 ◽  
Vol 151 (2) ◽  
pp. 247-257 ◽  
Author(s):  
Rene Barro-Soria
Keyword(s):  
Unnatural Amino Acids ◽  
Molecular Mechanisms ◽  
Neuronal Excitability ◽  
Voltage Dependence ◽  
Therapeutic Potential ◽  
Voltage Sensor ◽  
Voltage Range ◽  
M Current ◽  
Channel Opening ◽  
Major Factors

One of the major factors known to cause neuronal hyperexcitability is malfunction of the potassium channels formed by KCNQ2 and KCNQ3. These channel subunits underlie the M current, which regulates neuronal excitability. Here, I investigate the molecular mechanisms by which epilepsy-associated mutations in the voltage sensor (S4) of KCNQ3 cause channel malfunction. Voltage clamp fluorometry reveals that the R230C mutation in KCNQ3 allows S4 movement but shifts the open/closed transition of the gate to very negative potentials. This results in the mutated channel remaining open throughout the physiological voltage range. Substitution of R230 with natural and unnatural amino acids indicates that the functional effect of the arginine residue at position 230 depends on both its positive charge and the size of its side chain. I find that KCNQ3-R230C is hard to close, but it is capable of being closed at strong negative voltages. I suggest that compounds that shift the voltage dependence of S4 activation to more positive potentials would promote gate closure and thus have therapeutic potential.

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 Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

2016 ◽  
Vol 113 (27) ◽  
pp. 7521-7526 ◽  
Author(s):  
Souhei Sakata ◽  
Yuka Jinno ◽  
Akira Kawanabe ◽  
Yasushi Okamura
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Amino Acid ◽  
Conformational Changes ◽  
Unnatural Amino Acid ◽  
Voltage Sensor ◽  
Voltage Sensing ◽  
Cytoplasmic Region ◽  
Voltage Dependent ◽  
Voltage Gated Ion Channels

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

scholarly journals Fast and Slow Voltage Sensor Movements in HERG Potassium Channels

2002 ◽  
Vol 119 (3) ◽  
pp. 275-293 ◽  
Author(s):  
Paula L. Smith ◽  
Gary Yellen
Keyword(s):  
Conformational Changes ◽  
Voltage Dependence ◽  
Voltage Sensor ◽  
Inward Rectification ◽  
Dependent Inactivation ◽  
Activation Gating ◽  
Voltage Dependent ◽  
Probe Voltage ◽  
Inactivation Gating ◽  
Rapid Kinetics

HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH2-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals.

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 Pore- and voltage sensor–targeted KCNQ openers have distinct state-dependent actions

2018 ◽  
Vol 150 (12) ◽  
pp. 1722-1734 ◽  
Author(s):  
Caroline K. Wang ◽  
Shawn M. Lamothe ◽  
Alice W. Wang ◽  
Runying Y. Yang ◽  
Harley T. Kurata
Keyword(s):  
Voltage Dependence ◽  
Drug Effects ◽  
Drug Binding ◽  
Voltage Sensor ◽  
Voltage Sensing ◽  
Channel Activation ◽  
M Current ◽  
State Dependent ◽  
Mechanistic Basis ◽  
Voltage Sensing Domain

Ion channels encoded by KCNQ2-5 generate a prominent K+ conductance in the central nervous system, referred to as the M current, which is controlled by membrane voltage and PIP2. The KCNQ2-5 voltage-gated potassium channels are targeted by a variety of activating compounds that cause negative shifts in the voltage dependence of activation. The underlying pharmacology of these effects is of growing interest because of possible clinical applications. Recent studies have revealed multiple binding sites and mechanisms of action of KCNQ activators. For example, retigabine targets the pore domain, but several compounds have been shown to influence the voltage-sensing domain. An important unexplored feature of these compounds is the influence of channel gating on drug binding or effects. In the present study, we compare the state-dependent actions of retigabine and ICA-069673 (ICA73, a voltage sensor–targeted activator). We assess drug binding to preopen states by applying drugs to homomeric KCNQ2 channels at different holding voltages, demonstrating little or no association of ICA73 with resting states. Using rapid solution switching, we also demonstrate that the rate of onset of ICA73 correlates with the voltage dependence of channel activation. Retigabine actions differ significantly, with prominent drug effects seen at very negative holding voltages and distinct voltage dependences of drug binding versus channel activation. Using similar approaches, we investigate the mechanistic basis for attenuation of ICA73 actions by the voltage-sensing domain mutation KCNQ2[A181P]. Our findings demonstrate different state-dependent actions of pore- versus voltage sensor–targeted KCNQ channel activators, which highlight that subtypes of this drug class operate with distinct mechanisms.

scholarly journals Determinants That Control the Specific Interactions between TAB1 and p38α

2006 ◽  
Vol 26 (10) ◽  
pp. 3824-3834 ◽  
Author(s):  
Huamin Zhou ◽  
Min Zheng ◽  
Jianming Chen ◽  
Changchuan Xie ◽  
Anand R. Kolatkar ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Transforming Growth Factor ◽  
Mutational Analysis ◽  
Specific Binding ◽  
Point Mutations ◽  
Transforming Growth Factor Β ◽  
Mitogen Activated Protein Kinase ◽  
Docking Site ◽  
C Terminus

ABSTRACT Previous studies have revealed that transforming growth factor-β-activated protein kinase 1 (TAB1) interacts with p38α and induces p38α autophosphorylation. Here, we examine the sequence requirements in TAB1 and p38α that drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38α. Furthermore, a cryptic D-domain-like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in the φB+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38α is involved in this interaction, whereas the CD domain and ED domain are not. Furthermore, chimeric analysis with p38β (which does not bind to TAB1) revealed a previously unidentified locus of p38α comprising Thr218 and Ile275 that is essential for specific binding of p38α to TAB1. Converting either of these residues to the corresponding amino acid of p38β abolishes p38α interaction with TAB1. These p38α mutants still can be fully activated by p38α upstream activating kinase mitogen-activated protein kinase kinase 6, but their basal activity and activation in response to some extracellular stimuli are reduced. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38α substrates and activators. This suggests that TAB1-induced autophosphorylation of p38α results from conformational changes that are similar but unique to those seen in p38α interactions with its substrates and activating kinases.

Sign in / Sign up

Export Citation Format

Share Document