scholarly journals Mechanism of single- and double-sided inhibition of dual topology fluoride channels by synthetic monobodies

2017 ◽  
Vol 149 (4) ◽  
pp. 511-522 ◽  
Author(s):  
Daniel L. Turman ◽  
Randy B. Stockbridge
Keyword(s):  
Lipid Bilayers ◽  
Symmetry Axis ◽  
Voltage Dependence ◽  
Electrophysiological Study ◽  
Side Chain ◽  
Negative Cooperativity ◽  
Pore Blocking ◽  
Axis Parallel ◽  
Twofold Symmetry Axis ◽  
Blocking Mechanism

The Fluc family of proteins comprises small, electrodiffusive fluoride channels, which prevent accumulation of toxic F− ions in microorganisms. Recent crystal structures have confirmed their unusual architecture, in which a pair of antiparallel subunits convenes to form a dimer with a twofold symmetry axis parallel to the plane of the membrane. These structures have also revealed the interactions between Fluc channels and several different fibronectin domain monobodies that inhibit Fluc-mediated F− currents; in all structures, each channel binds to two monobodies symmetrically, one on either side of the membrane. However, these structures do not reveal the mechanism of monobody inhibition. Moreover, the results appear to diverge from a recent electrophysiological study indicating that monobody binding is negatively cooperative; that is, a bound monobody on one side of a Fluc channel decreases the affinity of an oppositely bound monobody by ∼10-fold. In this study, we reconcile these observations by probing the mechanism of monobody binding and its negative cooperativity using electrophysiological experiments in planar lipid bilayers. Our results indicate that monobody inhibition occurs via a pore-blocking mechanism and that negative cooperativity arises from electrostatic repulsion between the oppositely bound monobodies. A single glutamate residue, on a loop of the monobody that extends into the channel interior, is responsible for negatively cooperative binding. This glutamate side chain also confers voltage dependence and sensitivity to the concentration of trans-F− ion to monobody binding. Neutralization by mutation to glutamine abolishes these electrostatic effects. Monobodies that are amenable to cocrystallization with Fluc channels lack an analogous negatively charged side chain and bind independently to opposite sides of the channel. Thus, this work reveals the source of voltage dependence and negative cooperativity of monobody binding to Fluc channels along with the pore-blocking mechanism.

Effect of Sterol Side Chain on Ion Channel Formation by Amphotericin B in Lipid Bilayers

Biochemistry ◽  
2014 ◽  
Vol 53 (19) ◽  
pp. 3088-3094 ◽  
Author(s):  
Yasuo Nakagawa ◽  
Yuichi Umegawa ◽  
Tetsuro Takano ◽  
Hiroshi Tsuchikawa ◽  
Nobuaki Matsumori ◽  
...  
Keyword(s):  
Ion Channel ◽  
Amphotericin B ◽  
Lipid Bilayers ◽  
Side Chain ◽  
Channel Formation

A new approach to model asphaltene induced permeability damage with emphasis on pore blocking mechanism

2020 ◽  
Vol 184 ◽  
pp. 106512 ◽  
Author(s):  
Marzieh Ghadimi ◽  
Mojtaba Ghaedi ◽  
Mohammad R. Malayeri ◽  
Mohammad J. Amani
Keyword(s):  
New Approach ◽  
Pore Blocking ◽  
Permeability Damage ◽  
Blocking Mechanism

Heat Transfer in the Entrance Region of Tubes that Rotate about a Parallel Axis

1978 ◽  
Vol 20 (6) ◽  
pp. 319-325 ◽  
Author(s):  
W. D. Morris ◽  
J. L. Woods
Keyword(s):  
Heat Transfer ◽  
Symmetry Axis ◽  
Laminar Flows ◽  
Electrical Machines ◽  
Entrance Region ◽  
Axis Parallel ◽  
Prime Movers ◽  
Correlating Equations ◽  
Parallel Axis ◽  
Heat Transfers

The paper presents the results of an investigation into the effect of rotation on heat transfer in the entrance region of circular tubes that are constrained to rotate about an axis parallel to the symmetry axis. This rotating geometry is an important feature of flow systems designed for cooling the rotor windings and armature drums of electrical machines and other prime movers. It is demonstrated that rotation tends to improve local and mean heat transfers, and correlating equations are proposed for turbulent and laminar flows.

Chloroquine blocks the Kir4.1 channels by an open-pore blocking mechanism

2017 ◽  
Vol 800 ◽  
pp. 40-47 ◽  
Author(s):  
Leticia G. Marmolejo-Murillo ◽  
Iván A. Aréchiga-Figueroa ◽  
Eloy G. Moreno-Galindo ◽  
Ricardo A. Navarro-Polanco ◽  
Aldo A. Rodríguez-Menchaca ◽  
...  
Keyword(s):  
Pore Blocking ◽  
Blocking Mechanism

Dynamical Diffraction and X-Ray Standing Waves from Atomic Planes Normal to a Twofold Symmetry Axis of the Quasicrystal AlPdMn

1999 ◽  
Vol 82 (14) ◽  
pp. 2904-2907 ◽  
Author(s):  
Terrence Jach ◽  
Y. Zhang ◽  
R. Colella ◽  
M. de Boissieu ◽  
M. Boudard ◽  
...  
Keyword(s):  
Symmetry Axis ◽  
Standing Waves ◽  
Dynamical Diffraction ◽  
X Ray ◽  
Twofold Symmetry Axis

scholarly journals Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel.

1996 ◽  
Vol 108 (4) ◽  
pp. 237-250 ◽  
Author(s):  
T Y Chen ◽  
C Miller
Keyword(s):  
Lipid Bilayers ◽  
Voltage Dependence ◽  
High Rate ◽  
Charge Movement ◽  
Ion Conduction ◽  
Closed State ◽  
Cl Channel ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
Fast Gating

The gating of ClC-0, the voltage-dependent Cl- channel from Torpedo electric organ, is strongly influenced by Cl- ions in the external solution. Raising external Cl- over the range 1-600 mM favors the fast-gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl- than to external Cl-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl- or not, in a weakly voltage-dependent fashion, (c) that the Cl(-)-liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl(-)-liganded closed state opens with a very high rate. According to this picture, Cl- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC-0. Moreover, since the Cl- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessarily coupled to ion conduction, a nonequilibrium process.

scholarly journals Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade.

1983 ◽  
Vol 82 (4) ◽  
pp. 543-568 ◽  
Author(s):  
C Vergara ◽  
R Latorre
Keyword(s):  
Lipid Bilayers ◽  
Voltage Dependence ◽  
K Channel ◽  
Rabbit Muscle ◽  
Planar Bilayers ◽  
Slow Kinetics ◽  
The Mean ◽  
Mean Time ◽  
Kinetics Of ◽  
Burst Time

The interaction of Ca2+ and Ba2+ with a Ca2+-activated K+ channel from rabbit skeletal muscle membranes is studied in planar lipid bilayers. At [Ca2+] greater than or equal to 100 microM in the cis side (the side to which the vesicles are added) and at positive voltages, the channel kinetics consisted of bursts of activity interrupted by long periods of quiescence. We found that the reciprocal of the mean burst time increases linearly with [Ca2+], whereas the mean time for the quiescent (closed) periods is independent of [Ca2+]. The number of quiescent periods is reduced by increasing [K+]. Micromolar amounts of cis Ba2+ do not activate the channel, but induce similar "slow" closings. Also, in this case, the mean burst time is inversely proportional to the [Ba2+] and the mean closed time is independent of [Ba2+]. Raising [K+] either symmetrically or only in the trans side relieved the Ba2+ effect. trans Ba2+ also induces changes in the slow kinetics, but in millimolar amounts. These results suggest that the quiescent periods correspond to a channel blocked by a Ba ion. The voltage dependence of the cis blockade indicates that the Ba2+ binding site is past the middle of the membrane field. The similarities in the slow kinetics induced by Ca2+ and Ba2+ suggest that Ca2+ blocks the channel by binding to the same site. However, binding of Ca2+ to the site is 10(5)-fold weaker.

scholarly journals Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

2020 ◽  
Author(s):  
Kacey Mersch ◽  
Tugba N. Ozturk ◽  
Kunwoong Park ◽  
Hyun-Ho Lim ◽  
Janice L. Robertson
Keyword(s):  
Lipid Bilayers ◽  
Single Molecule ◽  
Close Contact ◽  
Hot Spot ◽  
Side Chain ◽  
Side Chains ◽  
Single Mutation ◽  
Dimerization Interface ◽  
Solvent Environment ◽  
Detergent Micelles

ABSTRACTCLC-ec1 is a Cl-/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mole relative to the 1 subunit/lipid standard state in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

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