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Monitoring voltage-dependent charge displacement of Shaker B-IR K+ ion channels using radio frequency interrogation.

Dharia S, Rabbitt RD - PLoS ONE (2011)

Bottom Line: Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K(+) ion channels to express large densities of this protein in the oocyte membranes.Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents.Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains--capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Utah, Salt Lake City, Utah, United States of America. sameera_dharia@yahoo.com

ABSTRACT
Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz) electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K(+) ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents. Simultaneously, RF electric fields were applied to perturb the membrane potential about the TEVC level and to measure voltage-dependent RF displacement currents. ShB-IR expressing oocytes showed significantly larger changes in RF displacement currents upon membrane depolarization than control oocytes. Voltage-dependent changes in RF displacement currents further increased in ShB-IR expressing oocytes after ∼120 µM Cu(2+) addition to the external bath. Cu(2+) is known to bind to the ShB-IR ion channel and inhibit Shaker K(+) conductance, indicating that changes in the RF displacement current reported here were associated with RF vibration of the Cu(2+)-linked mobile domain of the ShB-IR protein. Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains--capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug-protein interactions.

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Copper treatment and steady-state ShB-IR RF response.A) Voltage-dependent differences in /ΔZRF/s were observed between Shaker expressing oocytes (“ShB-IR + Endo”, green line) and the same cells exposed to ∼120 µM Cu2+ (purple line). A similar effect was apparent, albeit to a lesser extent, for control cells before (“Endo”, green markers)/after Cu2+ treatment (purple markers). Error bars denote +/− standard errors of the mean (SEM). B) Even though RF charge displacements increased in Cu2+-exposed ShB-IR expressing oocytes, TEVC whole-cell current decreased showing that Cu2+ successfully blocked the channels (channel conductance shown as inset).
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pone-0017363-g004: Copper treatment and steady-state ShB-IR RF response.A) Voltage-dependent differences in /ΔZRF/s were observed between Shaker expressing oocytes (“ShB-IR + Endo”, green line) and the same cells exposed to ∼120 µM Cu2+ (purple line). A similar effect was apparent, albeit to a lesser extent, for control cells before (“Endo”, green markers)/after Cu2+ treatment (purple markers). Error bars denote +/− standard errors of the mean (SEM). B) Even though RF charge displacements increased in Cu2+-exposed ShB-IR expressing oocytes, TEVC whole-cell current decreased showing that Cu2+ successfully blocked the channels (channel conductance shown as inset).

Mentions: A subset of ShB-IR expressing oocytes (n = 4) were treated with Cu2+, a K+ ion-channel modulator, and normalized /ΔZRF/s were determined for these oocytes before and after Cu2+ treatment (exp2). Results are shown in Fig. 4A as a function of steady-state membrane potential (Vm*). Data from each cell were normalized to its RF impedance at +30 mV for the nontreated ShB-IR expressing oocytes, to permit comparisons between cells before/after copper treatment (see Methods). As expected, Cu2+ application greatly reduced the ShB-IR current (Im*) in the voltage-range where ShB-IR channels activate (Fig. 4B). ShB-IR channel conductance before and after copper addition are shown in the inset (Fig. 4B). The change in RF impedance after copper treatment was larger than the untreated condition (Fig. 4A). To examine statistical significance for this relatively small population, data above −60 mV (see Methods) were pooled together. Pooled data showed a statistically significant difference between nontreated vs. Cu2+-treated ShB-IR expressing oocytes (p = .04, U = 1007, Total Points = 80, normalized median-values of the nontreated/treated ShB-IR expressing oocytes are .36 and .90, respectively; see Methods). Results from control cells (n = 2, Fig. 4A) are scaled by the ratio of the control cell to ShB-IR cell data at +30 mV (shown in Fig. 3) to enable inter-cellular comparison, after normalizing the data from each control cell to its nontreated /ΔZRF/s measured at +30 mV (see Methods). A change in the normalized /ΔZRF/s is noticeable in the control cells after Cu2+ application (above −60 mV), indicating that Cu2+ might non-specifically interact with the endogeneous oocyte membrane in addition to the known effect of binding the ShB-IR channels. While non-specific Cu2+ likely contributed to the change in RF response before/after Cu2+ addition, the effect in ShB-IR expressing oocytes was much larger, suggesting that the RF also detected Cu2+ interaction specifically with the Shaker channels. As depolarization level increased, the effect of the bound Cu2+ increased. At 30 mV, the Cu2+-treated ShB-IR expressing oocytes exhibited a response approximately 1.5 times that of the non-treated ShB-IR expressing oocytes, even though the ionic current (TEVC) was approximately one fifth of the non-treated cells.


Monitoring voltage-dependent charge displacement of Shaker B-IR K+ ion channels using radio frequency interrogation.

Dharia S, Rabbitt RD - PLoS ONE (2011)

Copper treatment and steady-state ShB-IR RF response.A) Voltage-dependent differences in /ΔZRF/s were observed between Shaker expressing oocytes (“ShB-IR + Endo”, green line) and the same cells exposed to ∼120 µM Cu2+ (purple line). A similar effect was apparent, albeit to a lesser extent, for control cells before (“Endo”, green markers)/after Cu2+ treatment (purple markers). Error bars denote +/− standard errors of the mean (SEM). B) Even though RF charge displacements increased in Cu2+-exposed ShB-IR expressing oocytes, TEVC whole-cell current decreased showing that Cu2+ successfully blocked the channels (channel conductance shown as inset).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046147&req=5

pone-0017363-g004: Copper treatment and steady-state ShB-IR RF response.A) Voltage-dependent differences in /ΔZRF/s were observed between Shaker expressing oocytes (“ShB-IR + Endo”, green line) and the same cells exposed to ∼120 µM Cu2+ (purple line). A similar effect was apparent, albeit to a lesser extent, for control cells before (“Endo”, green markers)/after Cu2+ treatment (purple markers). Error bars denote +/− standard errors of the mean (SEM). B) Even though RF charge displacements increased in Cu2+-exposed ShB-IR expressing oocytes, TEVC whole-cell current decreased showing that Cu2+ successfully blocked the channels (channel conductance shown as inset).
Mentions: A subset of ShB-IR expressing oocytes (n = 4) were treated with Cu2+, a K+ ion-channel modulator, and normalized /ΔZRF/s were determined for these oocytes before and after Cu2+ treatment (exp2). Results are shown in Fig. 4A as a function of steady-state membrane potential (Vm*). Data from each cell were normalized to its RF impedance at +30 mV for the nontreated ShB-IR expressing oocytes, to permit comparisons between cells before/after copper treatment (see Methods). As expected, Cu2+ application greatly reduced the ShB-IR current (Im*) in the voltage-range where ShB-IR channels activate (Fig. 4B). ShB-IR channel conductance before and after copper addition are shown in the inset (Fig. 4B). The change in RF impedance after copper treatment was larger than the untreated condition (Fig. 4A). To examine statistical significance for this relatively small population, data above −60 mV (see Methods) were pooled together. Pooled data showed a statistically significant difference between nontreated vs. Cu2+-treated ShB-IR expressing oocytes (p = .04, U = 1007, Total Points = 80, normalized median-values of the nontreated/treated ShB-IR expressing oocytes are .36 and .90, respectively; see Methods). Results from control cells (n = 2, Fig. 4A) are scaled by the ratio of the control cell to ShB-IR cell data at +30 mV (shown in Fig. 3) to enable inter-cellular comparison, after normalizing the data from each control cell to its nontreated /ΔZRF/s measured at +30 mV (see Methods). A change in the normalized /ΔZRF/s is noticeable in the control cells after Cu2+ application (above −60 mV), indicating that Cu2+ might non-specifically interact with the endogeneous oocyte membrane in addition to the known effect of binding the ShB-IR channels. While non-specific Cu2+ likely contributed to the change in RF response before/after Cu2+ addition, the effect in ShB-IR expressing oocytes was much larger, suggesting that the RF also detected Cu2+ interaction specifically with the Shaker channels. As depolarization level increased, the effect of the bound Cu2+ increased. At 30 mV, the Cu2+-treated ShB-IR expressing oocytes exhibited a response approximately 1.5 times that of the non-treated ShB-IR expressing oocytes, even though the ionic current (TEVC) was approximately one fifth of the non-treated cells.

Bottom Line: Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K(+) ion channels to express large densities of this protein in the oocyte membranes.Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents.Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains--capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug-protein interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Utah, Salt Lake City, Utah, United States of America. sameera_dharia@yahoo.com

ABSTRACT
Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz) electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K(+) ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents. Simultaneously, RF electric fields were applied to perturb the membrane potential about the TEVC level and to measure voltage-dependent RF displacement currents. ShB-IR expressing oocytes showed significantly larger changes in RF displacement currents upon membrane depolarization than control oocytes. Voltage-dependent changes in RF displacement currents further increased in ShB-IR expressing oocytes after ∼120 µM Cu(2+) addition to the external bath. Cu(2+) is known to bind to the ShB-IR ion channel and inhibit Shaker K(+) conductance, indicating that changes in the RF displacement current reported here were associated with RF vibration of the Cu(2+)-linked mobile domain of the ShB-IR protein. Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains--capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug-protein interactions.

Show MeSH
Related in: MedlinePlus