23 research outputs found
Influence of membrane potentials upon reversible protonation of acidic residues from the OmpF eyelet
Meet me on the other side: trans-bilayer modulation of a model voltage-gated ion channel activity by membrane electrostatics asymmetry.
While it is accepted that biomembrane asymmetry is generated by proteins and phospholipids distribution, little is known about how electric changes manifested in a monolayer influence functional properties of proteins localized on the opposite leaflet. Herein we used single-molecule electrophysiology and investigated how asymmetric changes in the electrostatics of an artificial lipid membrane monolayer, generated oppositely from where alamethicin--a model voltage-gated ion channel--was added, altered peptide activity. We found that phlorizin, a membrane dipole potential lowering amphiphile, augmented alamethicin activity and transport features, whereas the opposite occurred with RH-421, which enhances the monolayer dipole potential. Further, the monolayer surface potential was decreased via adsorption of sodium dodecyl sulfate, and demonstrated that vectorial modification of it also affected the alamethicin activity in a predictive manner. A new paradigm is suggested according to which asymmetric changes in the monolayer dipole and surface potential extend their effects spatially by altering the intramembrane potential, whose gradient is sensed by distantly located peptides
Magnified view of traces recorded at −60 mV showing the current amplitudes mediated by the first (O<sub>1</sub>), second (O<sub>2</sub>) and third (O<sub>3</sub>) sub-conductance states of the alamethicin oligomer under control conditions (no amphiphile added, panel b), and presence on the trans side of the membrane of either 8 µM RH 421 (panel a), or 500 µM phlorizin (panel c).
<p>Shown below are representative I–V diagrams illustrating the ion current at various transmembrane potentials, mediated by the first (O<sub>1</sub>) (panel d), second (O<sub>2</sub>) (panel e) and third (O<sub>3</sub>) (panel f) alamethicin substates in the absence (control) and presence of either RH 421 or phlorizin.</p
Quantitative description of the modulatory effect by exerted RH-421 (panel a) and phlorizin (panel b) on alamethicin activity, added asymmetrically to the trans side of a membrane, via estimations made on the standard deviation (denoted by σ) of the electrical current mediated by alamethicin oligomers at different holding potentials, before and after amphiphile adsorption, as well as the probability of appearance of high-conducting substates on alamethicin aggregates, inferred from the normalized amplitude histogram of current fluctuations seen in the absence (control) and presence of adsorbed amphiphiles at −60 mV.
<p>Due to the inherent thermal noise, the first substate (1) on the alamethicin oligomer is poorly discernable from its closed state (0) on the amplitude histogram. Therefore, the area of convoluted Gaussian component denoted by (0, 1) represents the probability of appearance of either ‘closed’ or first substate on alamethicin's reversible oligomerization. Areas assigned to peaks denoted by ‘2’ and ‘3’ provide a quantitative view of the probabilities to which the second, and respectively third conductive substates appear during alamethicin's reversible oligomerization.</p
Quantitative description of the modulatory effect exerted on alamethicin activity by SDS [25 µM] added on the cis (panel a) or trans-side (panel b) of the membrane, via estimations made on the standard deviation (denoted by σ) of the electrical current through alamethicin oligomers at different holding potentials, before and after SDS adsorption, as well as the probability of appearance of various conducting substates on alamethicin aggregates, inferred from the normalized amplitude histogram of current fluctuations seen in the absence (control) and presence of adsorbed amphiphiles at −55 mV.
<p>Areas assigned to peaks denoted by ‘1’ and ‘2’ provide a quantitative view of the probabilities to which the first, and respectively second conductive substates appear during alamethicin's reversible oligomerization (<i>vide supra</i>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025276#pone-0025276-g002" target="_blank">Fig. 2</a>), before and after SDS injection.</p
Representative current traces measured at −55 mV demonstrating the reversible change in alamethicin activity with respect to control condition following SDS addition in a pre-defined order during the same experiment, first to the cis side only (cis), and than to the trans side (cis/trans), or reverse (i.e., SDS added the trans side only (trans), and than to the cis side (trans/cis)) (panel a).
<p>This is suggestive of the paradigm according to which changes in the intramembrane potential profile caused by asymmetric addition of SDS on either cis or trans side, are partially reversed when a similar amount of detergent is present on the opposite side of the membrane. In panel (b) we present a quantitative analysis of this phenomenon, through estimating the standard deviation (σ) of the electrical current mediated by alamethicin oligomers at various holding potentials, under experimental conditions described above (see also text).</p
Representative current recordings which illustrate the alamethicin activity in lipid membranes under control conditions (no amphiphile added, panel b), and presence on the trans side of the membrane of either RH 421 [8 µM] (panel a), or phlorizin [500 µM] (panel c).
<p>The applied potential was −60 mV. The closed state of alamethicin oligomers is denoted by the dotted line, and downward spikes designate the electrical current mediated by alamethicin pores in their various conductive state. Beneath it is shown an over-simplified geometric view of changes ensued on the overall membrane potential profile, by the increase (panel a, dotted line) or decrease (panel c, dotted line) of the trans-monolayer dipole potential (Δψ<sub>d</sub>) of the <i>trans</i> lipid monolayer only, as compared to control conditions (panels a, b, and c, solid line). When the dipolar electric field - initially similar for both the cis and trans monolayers (E<sub>control</sub>) - is altered in the trans monolayer only, as a consequence of RH-421 (E<sub>RH-421</sub> > E<sub>control</sub>) or phlorizin (E<sub>phlorizin </sub>< E<sub>control</sub>) adsorption, a corresponding change in the intramembrane potential across the hydrophobic region of the membrane will follow. As a result, the net potential difference sensed by the cis-side adsorbed alamethicin over the hydrophobic region while crossing the membrane, will equal that seen under control conditions at a given trans-applied potential, from which a given value must be subtracted (denoted by ΔU<sub>RH-421</sub>; panel a, dashed line) or added to (denoted by ΔU<sub>phlorizin</sub>; panel c, dashed line). Based on the presented simplified geometric and electric considerations, this value (ΔU) should match the change brought about by either amphiphile adsorption on the trans-monolayer dipole potential (Δψ<sub>d</sub>). The solid arrows assigned to P<sub>alamethicin</sub> indicate the orientation of alamethicin monomers dipole moment, while in the transmembrane orientation.</p
Representative current recordings mediated by alamethicin in lipid membranes under control conditions (panel b), and presence of 25 µM SDS on either the trans (panel c) or cis side (panel a) of the membrane.
<p>The closed state of alamethicin is denoted by the dotted line, whereas downward spikes represent electrical current through alamethicin oligomers at an applied potential of −55 mV. As it was used before (<i>vide supra</i>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025276#pone-0025276-g001" target="_blank">Fig. 1</a>), we display schematically beneath a simplified, geometric view of changes ensued on the membrane potential profile by the change of the membrane surface potential initiated by SDS adsorption on the cis (panel a, dotted line) or trans side (panel c, dotted line) of the membrane, as compared to control conditions (panels a, b, and c, solid line). The membrane surface potential in the control state (ψ<sub>s</sub>) decreases as a result of either cis (ψ<sub>s1</sub>) or trans-side (ψ<sub>s2</sub>) adsorption of SDS, whereas the membrane dipole potential remains un-affected. As a result, a change in the intramembrane electric field (E) across the hydrophobic region of the membrane will follow, as shown. The net potential difference sensed by the cis-side adsorbed alamethicin over the hydrophobic region of the membrane, will equal that seen under control conditions at a given trans-applied potential, from which a given value must be subtracted (ΔU<sub>SDS, cis</sub>; panel a, dashed line) or added to (ΔU<sub>SDS, trans</sub>; panel c, dashed line). These values (ΔU<sub>SDS</sub>) should match the change caused by either SDS adsorption on the surface potential of either monolayer (ΔΨ<sub>s</sub>).</p
Illustrative diagrams showing changes in the membrane activity of alamethicin oligomers in the absence and presence of trans-added RH-421 (panel a) and phlorizin (panel b), quantified through estimations made on the standard deviation (σ) of the electrical current mediated by alamethicin at different holding potentials, used to make quantitative estimations of absolute values to which the trans dipole potential changes as a result of amphiphile asymmetric adsorption.
<p>Based on the rationale presented in the text, we posit that the dipole potential increase brought about by the RH-421 [8 µM] adsorption on the trans-monolayer at any given transmembrane potential is equivalent to a decrease with ∼3.7±2 mV (mean ± s.e.m) of the applied holding potential (denoted by ΔU<sub>RH-421</sub>) and no amphiphile added (control conditions), in order to arrive at a similar alamethicin activity. Similarly, and by comparison with control experiments, the trans-monolayer dipole potential decrease mediated by phlorizin adsorption [500 µM] increases the alamethicin activity to an extent as seen under control experiments, whereby the applied transmembrane potential would increase with roughly 2.8±0.8 mV (mean ± s.e.m) (denoted by ΔU<sub>phlorizin</sub>).</p