scholarly journals PIP2-dependent coupling of voltage sensor and pore domains in Kv7.2 channel

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shashank Pant ◽  
Jiaren Zhang ◽  
Eung Chang Kim ◽  
Kin Lam ◽  
Hee Jung Chung ◽  
...  
Keyword(s):  
Binding Sites ◽  
Epileptic Encephalopathy ◽  
Channel Activation ◽  
Voltage Gated ◽  
Molecular Action ◽  
Correlated Motion ◽  
Dynamics Simulations ◽  
Voltage Sensing Domain ◽  
Pore Domain

AbstractPhosphatidylinositol-4,5-bisphosphate (PIP2) is a signaling lipid which regulates voltage-gated Kv7/KCNQ potassium channels. Altered PIP2 sensitivity of neuronal Kv7.2 channel is involved in KCNQ2 epileptic encephalopathy. However, the molecular action of PIP2 on Kv7.2 gating remains largely elusive. Here, we use molecular dynamics simulations and electrophysiology to characterize PIP2 binding sites in a human Kv7.2 channel. In the closed state, PIP2 localizes to the periphery of the voltage-sensing domain (VSD). In the open state, PIP2 binds to 4 distinct interfaces formed by the cytoplasmic ends of the VSD, the gate, intracellular helices A and B and their linkers. PIP2 binding induces bilayer-interacting conformation of helices A and B and the correlated motion of the VSD and the pore domain, whereas charge-neutralizing mutations block this coupling and reduce PIP2 sensitivity of Kv7.2 channels by disrupting PIP2 binding. These findings reveal the allosteric role of PIP2 in Kv7.2 channel activation.

scholarly journals PIP2-dependent coupling of voltage sensor and pore domains in Kv7.2

2021 ◽  
Author(s):  
Shashank Pant ◽  
Jiaren Zhang ◽  
Eung Chang Kim ◽  
Hee Jung Chung ◽  
Emad Tajkhorshid
Keyword(s):  
Binding Sites ◽  
Epileptic Encephalopathy ◽  
Channel Activation ◽  
Voltage Gated ◽  
Molecular Action ◽  
Correlated Motion ◽  
Dynamics Simulations ◽  
Voltage Sensing Domain ◽  
Pore Domain

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a signaling lipid which regulates voltage-gated Kv7/KCNQ potassium channels. Altered PIP2 sensitivity of neuronal Kv7.2 channel is involved in KCNQ2 epileptic encephalopathy. However, the molecular action of PIP2 on Kv7.2 gating remains largely elusive. Here, we use molecular dynamics simulations and electrophysiology to characterize PIP2 binding sites in a human Kv7.2 channel. In the closed state, PIP2 localizes to the periphery of the voltage-sensing domain (VSD). In the open state, PIP2 binds to 4 distinct interfaces formed by the cytoplasmic ends of the VSD, the gate, intracellular helices A and B and their linkers. PIP2 binding induces bilayer-interacting conformation of helices A and B and the correlated motion of the VSD and the pore domain, whereas charge-neutralizing mutations block this coupling and reduce PIP2 sensitivity of Kv7.2 channels by disrupting PIP2 binding. These findings reveal the allosteric role of PIP2 in Kv7.2 channel activation.

scholarly journals Propofol inhibits the voltage-gated sodium channel NaChBac at multiple sites

2018 ◽  
Vol 150 (9) ◽  
pp. 1317-1331 ◽  
Author(s):  
Yali Wang ◽  
Elaine Yang ◽  
Marta M. Wells ◽  
Vasyl Bondarenko ◽  
Kellie Woll ◽  
...  
Keyword(s):  
Binding Sites ◽  
Molecular Mechanisms ◽  
General Anesthetics ◽  
Computational Docking ◽  
Voltage Gated ◽  
Nav Channels ◽  
Dynamics Simulations ◽  
Voltage Sensing Domain

Voltage-gated sodium (NaV) channels are important targets of general anesthetics, including the intravenous anesthetic propofol. Electrophysiology studies on the prokaryotic NaV channel NaChBac have demonstrated that propofol promotes channel activation and accelerates activation-coupled inactivation, but the molecular mechanisms of these effects are unclear. Here, guided by computational docking and molecular dynamics simulations, we predict several propofol-binding sites in NaChBac. We then strategically place small fluorinated probes at these putative binding sites and experimentally quantify the interaction strengths with a fluorinated propofol analogue, 4-fluoropropofol. In vitro and in vivo measurements show that 4-fluoropropofol and propofol have similar effects on NaChBac function and nearly identical anesthetizing effects on tadpole mobility. Using quantitative analysis by 19F-NMR saturation transfer difference spectroscopy, we reveal strong intermolecular cross-relaxation rate constants between 4-fluoropropofol and four different regions of NaChBac, including the activation gate and selectivity filter in the pore, the voltage sensing domain, and the S4–S5 linker. Unlike volatile anesthetics, 4-fluoropropofol does not bind to the extracellular interface of the pore domain. Collectively, our results show that propofol inhibits NaChBac at multiple sites, likely with distinct modes of action. This study provides a molecular basis for understanding the net inhibitory action of propofol on NaV 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 A pharmacological master key mechanism that unlocks the selectivity filter gate in K+channels

Science ◽  
2019 ◽  
Vol 363 (6429) ◽  
pp. 875-880 ◽  
Author(s):  
Marcus Schewe ◽  
Han Sun ◽  
Ümit Mert ◽  
Alexandra Mackenzie ◽  
Ashley C. W. Pike ◽  
...  
Keyword(s):  
Selectivity Filter ◽  
Rational Drug Design ◽  
Related Gene ◽  
K Channels ◽  
X Ray ◽  
Voltage Gated ◽  
X Ray Crystallography ◽  
Rational Drug ◽  
Dynamics Simulations ◽  
Pore Domain

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+channels gated at their selectivity filter (SF), including many two-pore domain K+(K2P) channels, voltage-gated hERG (human ether-à-go-go–related gene) channels and calcium (Ca2+)–activated big-conductance potassium (BK)–type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+channel activators and highlight a filter gating machinery that is conserved across different families of K+channels with implications for rational drug design.

scholarly journals Interpreting the functional role of a novel interaction motif in prokaryotic sodium channels

2017 ◽  
Vol 149 (6) ◽  
pp. 613-622 ◽  
Author(s):  
Altin Sula ◽  
B.A. Wallace
Keyword(s):  
Sodium Channels ◽  
Functional Role ◽  
Structural Features ◽  
Channel Activation ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Channel Opening ◽  
Activated State ◽  
Electrical Signaling

Voltage-gated sodium channels enable the translocation of sodium ions across cell membranes and play crucial roles in electrical signaling by initiating the action potential. In humans, mutations in sodium channels give rise to several neurological and cardiovascular diseases, and hence they are targets for pharmaceutical drug developments. Prokaryotic sodium channel crystal structures have provided detailed views of sodium channels, which by homology have suggested potentially important functionally related structural features in human sodium channels. A new crystal structure of a full-length prokaryotic channel, NavMs, in a conformation we proposed to represent the open, activated state, has revealed a novel interaction motif associated with channel opening. This motif is associated with disease when mutated in human sodium channels and plays an important and dynamic role in our new model for channel activation.

scholarly journals The propofol binding sites of prokaryotic voltage-gated sodium channels

2020 ◽  
Author(s):  
Elaine Yang ◽  
Weiming Bu ◽  
Antonio Suma ◽  
Vincenzo Carnevale ◽  
Kimberly C. Grasty ◽  
...  
Keyword(s):  
Binding Sites ◽  
Structural Basis ◽  
Dependent Manner ◽  
General Anesthetics ◽  
Neuronal Function ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
State Dependent ◽  
Dynamics Simulations

AbstractPropofol, one of the most commonly used intravenous general anesthetics, modulates neuronal function by interacting with ion channels. The mechanisms that link propofol binding to the modulation of distinct ion channel states, however, are not understood. To tackle this problem, we investigated prokaryotic ancestors of eukaryotic voltage-gated Na+ channels (Navs) using unbiased photoaffinity labeling with a photoacitivatable propofol analog (AziPm), electrophysiological methods and mutagenesis. The results directly demonstrate conserved propofol binding sites involving the S4 voltage sensors and the S4-S5 linkers in NaChBac and NavMs, and also suggest state-dependent changes at these sites. Then, using molecular dynamics simulations to elucidate the structural basis of propofol modulation, we show that the S4 voltage sensors and the S4-S5 linkers shape two distinct propofol binding sites in a conformation-dependent manner. These interactions help explain how propofol binding promotes activation-coupled inactivation to inhibit Nav channel function.

scholarly journals Mechanism of gating by calcium in connexin hemichannels

2016 ◽  
Vol 113 (49) ◽  
pp. E7986-E7995 ◽  
Author(s):  
William Lopez ◽  
Jayalakshmi Ramachandran ◽  
Abdelaziz Alsamarah ◽  
Yun Luo ◽  
Andrew L. Harris ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Small Molecules ◽  
Binding Sites ◽  
General Mechanism ◽  
Salt Bridges ◽  
Charged Residues ◽  
Dynamics Simulations ◽  
Connexin Hemichannel ◽  
Connexin Hemichannels

Aberrant opening of nonjunctional connexin hemichannels at the plasma membrane is associated with many diseases, including ischemia and muscular dystrophy. Proper control of hemichannel opening is essential to maintain cell viability and is achieved by physiological levels of extracellular Ca2+, which drastically reduce hemichannel activity. Here we examined the role of conserved charged residues that form electrostatic networks near the extracellular entrance of the connexin pore, a region thought to be involved in gating rearrangements of hemichannels. Molecular dynamics simulations indicate discrete sites for Ca2+ interaction and consequent disruption of salt bridges in the open hemichannels. Experimentally, we found that disruption of these salt bridges by mutations facilitates hemichannel closing. Two negatively charged residues in these networks are putative Ca2+ binding sites, forming a Ca2+-gating ring near the extracellular entrance of the pore. Accessibility studies showed that this Ca2+-bound gating ring does not prevent access of ions or small molecules to positions deeper into the pore, indicating that the physical gate is below the Ca2+-gating ring. We conclude that intra- and intersubunit electrostatic networks at the extracellular entrance of the hemichannel pore play critical roles in hemichannel gating reactions and are tightly controlled by extracellular Ca2+. Our findings provide a general mechanism for Ca2+ gating among different connexin hemichannel isoforms.

scholarly journals The voltage-sensing domain of a phosphatase gates the pore of a potassium channel

2013 ◽  
Vol 141 (3) ◽  
pp. 389-395 ◽  
Author(s):  
Cristina Arrigoni ◽  
Indra Schroeder ◽  
Giulia Romani ◽  
James L. Van Etten ◽  
Gerhard Thiel ◽  
...  
Keyword(s):  
Electromechanical Coupling ◽  
K Channel ◽  
Modular Architecture ◽  
Voltage Sensing ◽  
Mode Shift ◽  
Voltage Gated ◽  
Kv Channels ◽  
Pore Properties ◽  
Voltage Sensing Domain

The modular architecture of voltage-gated K+ (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates KvSynth1, a functional voltage-gated, outwardly rectifying K+ channel. KvSynth1 displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V1/2 = +56 mV; z of ∼1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.

scholarly journals Asymmetry of movements in CFTR's two ATP sites during pore opening serves their distinct functions

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Ben Sorum ◽  
Beáta Töröcsik ◽  
László Csanády
Keyword(s):  
Binding Sites ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Catalytic Site ◽  
The Other ◽  
Value Analysis ◽  
Binding Domains ◽  
Pore Opening ◽  
Pore Domain

CFTR, the chloride channel mutated in cystic fibrosis (CF) patients, is opened by ATP binding to two cytosolic nucleotide binding domains (NBDs), but pore-domain mutations may also impair gating. ATP-bound NBDs dimerize occluding two nucleotides at interfacial binding sites; one site hydrolyzes ATP, the other is inactive. The pore opens upon tightening, and closes upon disengagement, of the catalytic site following ATP hydrolysis. Extent, timing, and role of non-catalytic-site movements are unknown. Here we exploit equilibrium gating of a hydrolysis-deficient mutant and apply Φ value analysis to compare timing of opening-associated movements at multiple locations, from the cytoplasmic ATP sites to the extracellular surface. Marked asynchrony of motion in the two ATP sites reveals their distinct roles in channel gating. The results clarify the molecular mechanisms of functional cross-talk between canonical and degenerate ATP sites in asymmetric ABC proteins, and of the gating defects caused by two common CF mutations.

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