scholarly journals The RCK1 domain of the human BKCa channel transduces Ca2+ binding into structural rearrangements

2010 ◽  
Vol 136 (2) ◽  
pp. 189-202 ◽  
Author(s):  
Taleh Yusifov ◽  
Anoosh D. Javaherian ◽  
Antonios Pantazis ◽  
Chris S. Gandhi ◽  
Riccardo Olcese
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Molecular Mechanism ◽  
Conformational Changes ◽  
Bkca Channel ◽  
Structural Rearrangements ◽  
Bkca Channels ◽  
Time Resolved ◽  
High Affinity ◽  
Cytosolic Domains

Large-conductance voltage- and Ca2+-activated K+ (BKCa) channels play a fundamental role in cellular function by integrating information from their voltage and Ca2+ sensors to control membrane potential and Ca2+ homeostasis. The molecular mechanism of Ca2+-dependent regulation of BKCa channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K+ conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BKCa RCK1 domain adopts an α/β fold, binds Ca2+, and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca2+-induced conformational changes in physiologically relevant [Ca2+]. The neutralization of residues known to be involved in high-affinity Ca2+ sensing (D362 and D367) prevented Ca2+-induced structural transitions in RCK1 but did not abolish Ca2+ binding. We provide evidence that the RCK1 domain is a high-affinity Ca2+ sensor that transduces Ca2+ binding into structural rearrangements, likely representing elementary steps in the Ca2+-dependent activation of human BKCa channels.

Sub-Millisecond Time Resolved Imaging of Voltage Changes in Excitable Cells and Tissues Using Multiple Site Optical Recording of Transmembrane Voltage (Msortv) and Molecular Probes

1997 ◽  
Vol 3 (S2) ◽  
pp. 803-804
Author(s):  
B.M. Salzberg ◽  
A.L. Obaid
Keyword(s):  
Membrane Potential ◽  
Conformational Changes ◽  
Molecular Probes ◽  
Optical Recording ◽  
Membrane Voltage ◽  
Excitable Cells ◽  
Time Resolved ◽  
Molecular Weights ◽  
Transmembrane Voltage ◽  
Time Resolved Imaging

Molecular indicators of membrane potential may be used to obtain sub-millisecond time resolved images of transient changes in membrane voltage in a variety of biological systems. These probes are small amphipathic molecules having molecular weights of 400-500, and dimensions on the order of 10 Angstroms, which bind to, but do not cross cell membranes, and change either their absorbance or fluorescence in response to membrane voltage. These extrinsic optical signals depend linearly upon membrane potential, and the best of the dyes respond to a step change in voltage in less than 1.5 μsec at room temperature. The salient properties of fast potentiometric probes will be discussed, and the fidelity of optical recordings to transmembrane voltage changes will be considered.Since voltage changes in excitable cells take place on a time scale that is determined by the kinetics of conformational changes in membrane proteins, and by membrane electrical time constants, these changes tend to be very rapid, and resolving them requires imaging systems that are frequently orders of magnitude faster than usual video rates.

scholarly journals BKCa channel activation increases cardiac contractile recovery following hypothermic ischemia/reperfusion

2015 ◽  
Vol 309 (4) ◽  
pp. H625-H633 ◽  
Author(s):  
Brenda Cordeiro ◽  
Dmitry Terentyev ◽  
Richard T. Clements
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Left Ventricular ◽  
Bkca Channel ◽  
Ros Generation ◽  
H9c2 Cells ◽  
Bkca Channels ◽  
Channel Activation

Mitochondrial Ca2+-activated large-conductance K+ (BKCa) channels are thought to provide protection during ischemic insults in the heart. Rottlerin (mallotoxin) has been implicated as a potent BKCa activator. The purpose of this study was twofold: 1) to investigate the efficacy of BKCa channel activation as a cardioprotective strategy during ischemic cardioplegic arrest and reperfusion (CP/R) and 2) to assess the specificity of rottlerin for BKCa channels. Wild-type (WT) and BKCa knockout (KO) mice were subjected to an isolated heart model of ischemic CP/R. A mechanism of rottlerin-induced cardioprotection was also investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential and reactive oxygen species (ROS) production. CP/R decreased left ventricular developed pressure, positive and negative first derivatives of left ventricular pressure, and coronary flow (CF) in WT mice. Rottlerin dose dependently increased the recovery of left ventricular function and CF to near baseline levels. BKCa KO hearts treated with or without 500 nM rottlerin were similar to WT CP hearts. H9c2 cells subjected to in vitro CP/R displayed reduced mitochondrial membrane potential and increased ROS generation, both of which were significantly normalized by rottlerin. We conclude that activation of BKCa channels rescues ischemic damage associated with CP/R, likely via effects on improved mitochondrial membrane potential and reduced ROS generation.

scholarly journals Molecular mechanism of depolarization-dependent inactivation in W366F mutant of Kv1.2

2018 ◽  
Author(s):  
H. X. Kondo ◽  
N. Yoshida ◽  
M. Shirota ◽  
K. Kinoshita
Keyword(s):  
Membrane Potential ◽  
Potassium Channels ◽  
Molecular Mechanism ◽  
Conformational Changes ◽  
Structural Changes ◽  
Membrane Depolarization ◽  
Potassium Ions ◽  
Selective Filter ◽  
Dependent Inactivation ◽  
Dynamics Simulations

ABSTRACTVoltage-gated potassium channels play crucial roles in regulating membrane potential. They are activated by membrane depolarization, allowing the selective permeation of potassium ions across the plasma membrane, and enter a nonconducting state after lasting depolarization of membrane potential, a process known as inactivation. Inactivation in voltage-activated potassium channels occurs through two distinct mechanisms, N-type inactivation and C-type inactivation. C-type inactivation is caused by conformational changes in the extracellular mouth of the channel, while N-type inactivation is elicited by changes in the cytoplasmic mouth of the protein. The W434F-mutated Shaker channel is known as a nonconducting mutant and is in a C-type inactivation state at a depolarizing membrane potential. To clarify the structural properties of C-type inactivated protein, we performed molecular dynamics simulations of the wild-type and W366F (corresponding to W434F in Shaker) mutant of the Kv1.2-2.1 chimera channel. The W366F mutant was in a nearly nonconducting state with a depolarizing voltage and recovered from inactivation with a reverse voltage. Our simulations and 3D-RISM analysis suggested that structural changes in the selective filter upon membrane depolarization trap potassium ions around the entrance of the selectivity filter and prevent ion permeation. This pore restriction is involved in the molecular mechanism of C-type inactivation.

scholarly journals Revisiting the Large-Conductance Calcium-Activated Potassium (BKCa) Channels in the Pulmonary Circulation

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1629
Author(s):  
Divya Guntur ◽  
Horst Olschewski ◽  
Péter Enyedi ◽  
Réka Csáki ◽  
Andrea Olschewski ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Potassium Channels ◽  
Pulmonary Circulation ◽  
Hypoxic Pulmonary Vasoconstriction ◽  
Free Calcium ◽  
Bkca Channel ◽  
Lung Pathology ◽  
Open Probability ◽  
Bkca Channels ◽  
Genes Encoding

Potassium ion concentrations, controlled by ion pumps and potassium channels, predominantly govern a cell′s membrane potential and the tone in the vessels. Calcium-activated potassium channels respond to two different stimuli-changes in voltage and/or changes in intracellular free calcium. Large conductance calcium-activated potassium (BKCa) channels assemble from pore forming and various modulatory and auxiliary subunits. They are of vital significance due to their very high unitary conductance and hence their ability to rapidly cause extreme changes in the membrane potential. The pathophysiology of lung diseases in general and pulmonary hypertension, in particular, show the implication of either decreased expression and partial inactivation of BKCa channel and its subunits or mutations in the genes encoding different subunits of the channel. Signaling molecules, circulating humoral molecules, vasorelaxant agents, etc., have an influence on the open probability of the channel in pulmonary arterial vascular cells. BKCa channel is a possible therapeutic target, aimed to cause vasodilation in constricted or chronically stiffened vessels, as shown in various animal models. This review is a comprehensive collation of studies on BKCa channels in the pulmonary circulation under hypoxia (hypoxic pulmonary vasoconstriction; HPV), lung pathology, and fetal to neonatal transition, emphasising pharmacological interventions as viable therapeutic options.

Fluorescence Anisotropy and Mobility of Dansyl Fluorophore in Labelled Homologous Alkanes

1999 ◽  
Vol 64 (9) ◽  
pp. 1369-1384 ◽  
Author(s):  
Drahomír Výprachtický ◽  
Veronika Pokorná ◽  
Jan Pecka ◽  
František Mikeš
Keyword(s):  
Relaxation Time ◽  
Steady State ◽  
Conformational Changes ◽  
Fluorescence Anisotropy ◽  
Relaxation Spectrum ◽  
Rotational Relaxation ◽  
Aliphatic Chain ◽  
Time Resolved ◽  
Methylene Groups ◽  
Rotational Relaxation Time

Using the steady-state and time-resolved fluorescence anisotropy, the mobility of 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl) fluorophore in homologous 1-[2-acetamido-3-(1H-indol-3-yl)propanamido]-n-[5-(dimethylamino)naphthalene-1-sulfonamido]alkanes 1 was studied in binary solvents glycerol-water. Steady-state fluorescence data were evaluated by the generalized Perrin equation and the micro-Brownian motion of dansyl fluorophore was described by means of average characteristics (rotational relaxation times) of the rotational relaxation spectrum. The rotational relaxation time of "fast" motions caused by torsional vibrations of single bonds within the rotational-isomeric states decreases with increasing number of methylene groups in homologous compounds. The rotational relaxation time of "slow" motions due to conformational changes of the chain between the tryptophane and dansyl fluorophore remains at first approximately constant with increasing number of methylene groups but increases considerably for long aliphatic chains. The observed decrease in the rate of conformational changes of a long aliphatic chain is probably due to intramolecular interaction of parts of the methylene chain in a medium with high water content. The values of activation enthalpy ∆H≠ and activation entropy ∆S≠ calculated from experimental data corroborate such interpretation. Time-resolved anisotropy of dansyl fluorophore at a particular binary solvent composition confirmed the shape of rotational relaxation spectrum and the measured rotational correlation times have been discussed. The time-dependent decays of anisotropy supported our previous interpretation in terms of intramolecular association of the long aliphatic chain in polar medium.

scholarly journals Molecular mechanism of multispecific recognition of Calmodulin through conformational changes

2017 ◽  
Vol 114 (20) ◽  
pp. E3927-E3934 ◽  
Author(s):  
Fei Liu ◽  
Xiakun Chu ◽  
H. Peter Lu ◽  
Jin Wang
Keyword(s):  
Protein Binding ◽  
Conformational Change ◽  
Molecular Mechanism ◽  
Conformational Changes ◽  
Electrostatic Interaction ◽  
Hydrophobic Interaction ◽  
High Specificity ◽  
Binding Peptide ◽  
Conformational Selection ◽  
High Affinity

Calmodulin (CaM) is found to have the capability to bind multiple targets. Investigations on the association mechanism of CaM to its targets are crucial for understanding protein–protein binding and recognition. Here, we developed a structure-based model to explore the binding process between CaM and skMLCK binding peptide. We found the cooperation between nonnative electrostatic interaction and nonnative hydrophobic interaction plays an important role in nonspecific recognition between CaM and its target. We also found that the conserved hydrophobic anchors of skMLCK and binding patches of CaM are crucial for the transition from high affinity to high specificity. Furthermore, this association process involves simultaneously both local conformational change of CaM and global conformational changes of the skMLCK binding peptide. We found a landscape with a mixture of the atypical “induced fit,” the atypical “conformational selection,” and “simultaneously binding–folding,” depending on the synchronization of folding and binding. Finally, we extend our discussions on multispecific binding between CaM and its targets. These association characteristics proposed for CaM and skMLCK can provide insights into multispecific binding of CaM.

scholarly journals Providing a molecular mechanism for P-glycoprotein; why would I bother?

2015 ◽  
Vol 43 (5) ◽  
pp. 995-1002 ◽  
Author(s):  
Richard Callaghan
Keyword(s):  
Cancer Cells ◽  
Molecular Mechanism ◽  
Conformational Changes ◽  
Efflux Pump ◽  
Structural Data ◽  
Multi Drug Resistance ◽  
P Glycoprotein ◽  
Complex Protein ◽  
Cytosolic Domains ◽  
Drug Efflux Pump

It is almost 40 years since the drug efflux pump P-glycoprotein (permeability glycoprotein or P-gp) was shown to confer multi-drug resistance in cancer cells. This protein has been one of the most extensively investigated transport proteins due to its intriguing mechanism and its affect in oncology. P-gp is known to interact with over 300 compounds and the ability to achieve this has not yet been revealed. Following the binding of substrate and nucleotide, a complex series of conformational changes in the membrane and cytosolic domains translocates substrate across the membrane. Despite over 30 years of biochemical investigation, the availability of structural data and a plethora of chemical tools to modulate its function, the molecular mechanism remains a mystery. In addition, overcoming its activity in resistant cancer cells has not been achieved in the clinic, thereby garnering some degree of pessimism in the field. This review highlights the progress that has been achieved in understanding this complex protein and the value of undertaking molecular studies.

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