scholarly journals MspA Porin as a Local Nanopore Probe for Membrane-bound Proteins

2021 ◽  
Author(s):  
David P. Hoogerheide ◽  
Philip A. Gurnev ◽  
Jens Gundlach ◽  
Andrew Laszlo ◽  
Tatiana K. Rostovtseva ◽  
...  
Keyword(s):  
Protein Detection ◽  
Capture Process ◽  
Neuronal Protein ◽  
Intrinsically Disordered ◽  
Detection Analysis ◽  
Direct Discrimination ◽  
Crossover Behavior ◽  
Voltage Dependent ◽  
Conductance Changes ◽  
Nanopore Confinement

Nanopore sensing is based on detection and analysis of nanopore transient conductance changes induced by analyte capture. We have recently shown that α-Synuclein (αSyn), an intrinsically disordered, membrane-active, neuronal protein implicated in Parkinson disease, can be reversibly captured by the VDAC nanopore. The capture process is a highly voltage dependent complexation of the two proteins where transmembrane potential drives the polyanionic C-terminal domain of αSyn into VDAC--exactly the mechanism by which generic nanopore-based interrogation of proteins and polynucleotides proceeds. The complex formation, and the motion of αSyn in the nanopore, thus may be expected to be only indirectly dependent on the pore identity. Here, we confirm this prediction by demonstrating that when VDAC is replaced with a different transmembrane pore, the engineered mycobacterial porin M2MspA, all the qualitative features of the αSyn/nanopore interaction are preserved. The rate of αSyn capture by M2MspA rises exponentially with the applied field, while the residence time displays a crossover behavior, indicating that at voltages >50 mV M2MspA-bound αSyn largely undergoes translocation to the other side of the membrane. The translocation is directly confirmed using the selectivity tag method, in which the polyanionic C-terminal and neutral N-terminal regions of αSyn alter the selectivity of the M2MspA channel differently, allowing direct discrimination of translocation vs retraction for single αSyn molecules. We thus prove that the physical model of the motion of disordered protein chains in the nanopore confinement and the selectivity tag technique are not limited to VDAC but are broadly applicable to nanopore-based protein detection, analysis, and separation technologies.

scholarly journals Equilibrium properties of a voltage-dependent junctional conductance.

1981 ◽  
Vol 77 (1) ◽  
pp. 77-93 ◽  
Author(s):  
D C Spray ◽  
A L Harris ◽  
M V Bennett
Keyword(s):  
Steady State ◽  
Rana Pipiens ◽  
Energy Difference ◽  
Ambystoma Mexicanum ◽  
Voltage Sensitivity ◽  
Junctional Conductance ◽  
Voltage Dependent ◽  
Accumulation Mechanism ◽  
Conductance Changes ◽  
A Current

The conductance of junctions between amphibian blastomeres is strongly voltage dependent. Isolated pairs of blastomeres from embryos of Ambystoma mexicanum, Xenopus laevis, and Rana pipiens were voltage clamped, and junctional current was measured during transjunctional voltage steps. The steady-state junctional conductance decreases as a steep function of transjunctional voltage of either polarity. A voltage-insensitive conductance less than 5% of the maximum remains at large transjunctional voltages. Equal transjunctional voltages of opposite polarities produce equal conductance changes. The conductance is half maximal at a transjunctional voltage of approximately 15 mV. The junctional conductance is insensitive to the potential between the inside and outside of the cells. The changes in steady-state junctional conductance may be accurately modeled for voltages of each polarity as arising from a reversible two-state system in which voltage linearly affects the energy difference between states. The voltage sensitivity can be accounted for by the movement of about six electron charges through the transjunctional voltage. The changes in junctional conductance are not consistent with a current-controlled or ionic accumulation mechanism. We propose that the intramembrane particles that comprise gap junctions in early amphibian embryos are voltage-sensitive channels.

Separable phases of light-evoked depolarizations in the retina of Strombus

1980 ◽  
Vol 84 (1) ◽  
pp. 137-148
Author(s):  
F. N. Quandt ◽  
H. L. Gillary
Keyword(s):  
Synaptic Transmission ◽  
Voltage Clamp ◽  
Stimulus Intensity ◽  
Interstimulus Interval ◽  
Direct Action ◽  
Resting Potential ◽  
Second Phase ◽  
Reverse Polarity ◽  
Voltage Dependent ◽  
Conductance Changes

The waveforms of light-evoked depolarizations in Strombus retinal neurones can exhibit two sequential peaks or phases, the relative amplitudes of which vary with changes in stimulus intensity and interstimulus interval. Experiments employing either the passage of constant intracellular current or voltage clamp techniques indicate that both phases reverse polarity at intracellular potentials less negative than the resting potential. The potential at which the first phase reverses its polarity is considerably more positive than that of the second phase. The results indicate that the light-evoked depolarizations are generated by at least two different processes; these appear to be separate conductance changes, neither of which is voltage dependent. Under certain conditions, the second phase was inhibited by high extracellular concentrations of Mg2+, indicating that it may arise as a result of chemically mediated synaptic transmission. The first phase did not show such inhibition and appears to be caused by the direct action of light on the cell.

scholarly journals Monazomycin-induced single channels. I. Characterization of the elementary conductance events.

1982 ◽  
Vol 80 (3) ◽  
pp. 403-426 ◽  
Author(s):  
O S Andersen ◽  
R U Muller
Keyword(s):  
Lipid Bilayer ◽  
Single Channels ◽  
Lipid Bilayer Membranes ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Conductance Changes ◽  
Current Voltage Characteristics ◽  
The Individual ◽  
Positively Charged

Monazomycin (a positively charged, polyene-like antibiotic) induces voltage-dependent conductance changes in lipid bilayer membranes when added to one of the bathing solutions. These conductance changes have generally been attributed to the existence of channels spanning the membrane. In this article we characterize the behavior of the individual conductance events observed when adding small amounts of monazomycin to one side of a lipid bilayer. We find that there are several apparent channel types with one or sometimes two amplitudes predominating. We find further that these fairly similar amplitudes represent two different states of the same fundamental channel entity, presumed to be the monazomycin channel. The current-voltage characteristics of these channels are weakly hyperbolic functions of applied potential. The average lifetimes are essentially voltage independent (between 50 and 400 mV). The average channel intervals, on the other hand, can be strongly voltage dependent, and we can show that the time-averaged conductance of a membrane is proportional to the average channel frequency.

scholarly journals Probing the Motion of the Intrinsically Disordered Neuronal Protein Alpha-Synuclein through the VDAC Pore using a Single-Molecule Approach

2015 ◽  
Vol 108 (2) ◽  
pp. 185a
Author(s):  
David P. Hoogerheide ◽  
Philip A. Gurnev ◽  
Tatiana K. Rostovtseva ◽  
Sergey M. Bezrukov
Keyword(s):  
Single Molecule ◽  
Alpha Synuclein ◽  
Neuronal Protein ◽  
Intrinsically Disordered

scholarly journals The intrinsically disordered N-terminus of the voltage-dependent anion channel

2021 ◽  
Vol 17 (2) ◽  
pp. e1008750
Author(s):  
Jordane Preto ◽  
Isabelle Krimm
Keyword(s):  
Force Field ◽  
Disordered Systems ◽  
Anion Channel ◽  
Dynamics Simulation ◽  
Mitochondrial Outer Membrane ◽  
Voltage Dependent Anion Channel ◽  
Intrinsically Disordered ◽  
Gating Mechanism ◽  
N Terminus ◽  
Voltage Dependent

The voltage-dependent anion channel (VDAC) is a critical β-barrel membrane protein of the mitochondrial outer membrane, which regulates the transport of ions and ATP between mitochondria and the cytoplasm. In addition, VDAC plays a central role in the control of apoptosis and is therefore of great interest in both cancer and neurodegenerative diseases. Although not fully understood, it is presumed that the gating mechanism of VDAC is governed by its N-terminal region which, in the open state of the channel, exhibits an α-helical structure positioned midway inside the pore and strongly interacting with the β-barrel wall. In the present work, we performed molecular simulations with a recently developed force field for disordered systems to shed new light on known experimental results, showing that the N-terminus of VDAC is an intrinsically disordered region (IDR). First, simulation of the N-terminal segment as a free peptide highlighted its disordered nature and the importance of using an IDR-specific force field to properly sample its conformational landscape. Secondly, accelerated dynamics simulation of a double cysteine VDAC mutant under applied voltage revealed metastable low conducting states of the channel representative of closed states observed experimentally. Related structures were characterized by partial unfolding and rearrangement of the N-terminal tail, that led to steric hindrance of the pore. Our results indicate that the disordered properties of the N-terminus are crucial to properly account for the gating mechanism of VDAC.

scholarly journals A Programmable DNA Origami Platform for Organizing Intrinsically Disordered Nucleoporins within Nanopore Confinement

ACS Nano ◽  
2018 ◽  
Vol 12 (2) ◽  
pp. 1508-1518 ◽  
Author(s):  
Patrick D. Ellis Fisher ◽  
Qi Shen ◽  
Bernice Akpinar ◽  
Luke K. Davis ◽  
Kenny Kwok Hin Chung ◽  
...  
Keyword(s):  
Dna Origami ◽  
Intrinsically Disordered ◽  
Nanopore Confinement

Presynaptic inhibition produced by an identified presynaptic inhibitory neuron. II. Presynaptic conductance changes caused by histamine

1986 ◽  
Vol 55 (1) ◽  
pp. 131-146 ◽  
Author(s):  
R. Kretz ◽  
E. Shapiro ◽  
C. H. Bailey ◽  
M. Chen ◽  
E. R. Kandel
Keyword(s):  
Transmitter Release ◽  
Presynaptic Inhibition ◽  
Inhibitory Neuron ◽  
Abdominal Ganglion ◽  
Presynaptic Neuron ◽  
Voltage Dependent ◽  
K Current ◽  
Conductance Changes ◽  
Electron Dense Core ◽  
Histaminergic Neuron

We have examined the morphology and pharmacology of the L32 neurons, identified cells that mediate presynaptic inhibition in the Aplysia abdominal ganglion, to gain insight into the putative transmitter released by the L32 cells. We analyzed the fine structure of the synaptic release sites of L32 cells stained with horseradish peroxidase. Each varicosity of L32 was found to contain two general classes of vesicles. One class of vesicles is large (mean long diameter of 98 nm) and contains an electron-dense core that typically filled or nearly filled each vesicle profile. The second class of vesicles is smaller (mean long diameter of 67 nm) and relatively electron lucent. The size, distribution, and morphology of the vesicle population in L32's terminals was similar to that described at the synapses of the identified histaminergic neuron C2 in Aplysia (2). These morphological observations suggested that L32 cells might be histaminergic. Among the various putative transmitters tested, histamine was most effective in mimicking the postsynaptic effects of L32 cells onto L10, and onto other follower cells of L32 in the abdominal ganglion. Histamine also caused inhibition of transmitter output from L10. Both the IPSP produced by L32 in L10 and the response of L10 to histamine could be reversibly blocked by cimetidine, a histamine antagonist in Aplysia (14). These results support, but do not establish the identification of histamine as the putative transmitter of L32 cells. Histamine mimics the action of L32 in mediating presynaptic inhibition allowing us to examine in more detail the conductance changes in L10 underlying presynaptic inhibition. Voltage-clamp analysis revealed that histamine blocked the voltage-dependent Ca2+ current and increased a voltage-dependent K+ current in L10, much as did L32. Both of these changes are likely to act synergistically to inhibit transmitter release. Reduction of Ca2+ current in L10 would directly inhibit transmitter release from L10 directly by decreasing the amount of Ca2+ entering during spike depolarization. The increase in K+ current would act indirectly to reduce transmitter release from L10, by hyperpolarizing L10 and decreasing the amplitude and duration of spikes in L10, as well as reducing the steady-state Ca2+ influx. These results support the idea that in Aplysia presynaptic inhibition is caused primarily by a direct transmitter-mediated reduction in presynaptic Ca2+ current and secondarily by a hyperpolarization of the presynaptic neuron due to a transmitter-mediated increase in a K+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The intrinsically disordered N-terminus of the voltage-dependent anion channel

2020 ◽  
Author(s):  
Jordane Preto ◽  
Isabelle Krimm
Keyword(s):  
Anion Channel ◽  
Mitochondrial Outer Membrane ◽  
Voltage Dependent Anion Channel ◽  
Free Peptide ◽  
Intrinsically Disordered ◽  
Gating Mechanism ◽  
N Terminus ◽  
Intrinsically Disordered Region ◽  
Voltage Dependent ◽  
Transport Of Ions

AbstractThe voltage-dependent anion channel (VDAC) is a critical β-barrel membrane protein of the mitochondrial outer membrane, which regulates the transport of ions and ATP between mitochondria and the cytoplasm. In addition, VDAC plays a central role in the control of apoptosis and is therefore of great interest in both cancer and neurodegenerative diseases. Although not fully understood, it is presumed that the gating mechanism of VDAC is governed by its N-terminal region which, in the open state of the channel, exhibits an α-helical structure positioned midway inside the pore and strongly interacting with the β-barrel wall.In the present work, we performed molecular simulations with a recently developed force field for disordered systems to shed new light on known experimental results, showing that the N-terminus of VDAC is an intrinsically disordered region (IDR). First, simulation of the N-terminal segment as a free peptide highlighted its disordered nature and the importance of using an IDR-specific force field to properly sample its conformational landscape. Secondly, accelerated dynamics simulation of a double cysteine VDAC mutant under applied voltage revealed metastable low conducting states of the channel representative of closed states observed experimentally. Related structures were characterized by partial unfolding and rearrangement of the N-terminal tail, that led to steric hindrance of the pore. Our results indicate that the disordered properties of the N-terminus are crucial to properly account for the gating mechanism of VDAC.Author summaryThe voltage-dependent anion channel (VDAC) is a membrane protein playing a pivotal role in the transport of ions or ATP across the mitochondrial outer membrane as well as in the induction of apoptosis. At high enough membrane potential, VDAC is known to transition from an open state to multiple closed states, reducing the flow of ions through the channel and blocking the passage of large metabolites. While the structure of the open state was resolved more than a decade ago, a molecular description of the gating mechanism of the channel is still missing. Here we show that the N-terminus of VDAC is an intrinsically disordered region and that such a property has a profound impact on its dynamics either as a free peptide or as part of the channel. By taking disordered properties of the N-terminus into account, we managed to generate long-lived closed conformations of the channel at experimental values of the membrane potential. Our results provide new insights into the molecular mechanism driving the gating of VDAC.

Determination of the Location and Magnitude of Synaptic Conductance Changes in Spinal Motoneurons by Impedance Measurements

2004 ◽  
Vol 92 (3) ◽  
pp. 1400-1416 ◽  
Author(s):  
Mitchell G. Maltenfort ◽  
Carrie A. Phillips ◽  
Martha L. McCurdy ◽  
Thomas M. Hamm
Keyword(s):  
Synaptic Conductance ◽  
Published Data ◽  
Impedance Change ◽  
Low Frequencies ◽  
Membrane Resistivity ◽  
Voltage Dependent ◽  
Reversal Frequency ◽  
Conductance Changes ◽  
Conductance Ratio ◽  
Impedance Magnitude

The relation between impedance change and the location and magnitude of a tonic synaptic conductance was examined in compartmental motoneuron models based on previously published data. The dependency of motoneuron impedance on system time constant (τ), electrotonic length (L), and dendritic-to-somatic conductance ratio (ρ) was examined, showing that the relation between impedance phase and ρ differed markedly between models with uniform and nonuniform membrane resistivity. Dendritic synaptic conductances decreased impedance magnitude at low frequencies; at higher frequencies, impedance magnitude increased. The frequency at which the change in impedance magnitude reversed from a decrease to an increase—the reversal frequency, Fr—was a good estimator of electrotonic synaptic location. A measure of the average normalized impedance change at frequencies less than Fr, cuΔZ, estimated relative synaptic conductance. Fr and cuΔZ provided useful estimates of synaptic location and conductance in models with nonuniform (step, sigmoidal) and uniform membrane resistivity. Fr also provided good estimates of spatial synaptic location on the equivalent cable in both step and sigmoidal models. Variability in relations between Fr, cuΔZ, and conductance location and magnitude between neurons was reduced by normalization with ρ and τ. The effects on Fr and cuΔZ of noise in experimental recordings, different synaptic distributions, and voltage-dependent conductances were also assessed. This study indicates that location and conductance of tonic dendritic conductances can be estimated from Fr, cuΔZ, and basic electrotonic motoneuron parameters with the exercise of suitable precautions.

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