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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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Steady-state RF response for ShB-IR and Control Cells.A) Significant (xp = .1, *p = .05) voltage-dependent differences in /ΔZRF/ s were observed between control oocytes, expressing endogenous proteins only (“Endo”, orange), and ShB-IR expressing oocytes (express both endogenous and ShB-IR proteins, “ShB-IR + Endo”, brown). The “Endo” response was subtracted from the “ShB-IR + Endo” response to estimate the isolated RF response from the ShB-IR channels only (“ShB-IR only”, blue). Error bars denote +/− standard errors of the mean (SEM). B) The SEM for the isolated ShB-IR proteins (“ShB-IR only”, blue) was also estimated by subtracting the SEM from the control oocytes (“Endo”, orange) from the SEM associated with the ShB-IR expressing oocytes (“ShB-IR + Endo”, brown). The SEM for isolated ShB-IR expressing oocytes was largest near the half-activation potential for these ion channels. C) ShB-IR channel expression and voltage-dependent whole-cell current was verified using TEVC, and this data was used to estimate ShB-IR conductance (G/Gmax, inset).
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pone-0017363-g003: Steady-state RF response for ShB-IR and Control Cells.A) Significant (xp = .1, *p = .05) voltage-dependent differences in /ΔZRF/ s were observed between control oocytes, expressing endogenous proteins only (“Endo”, orange), and ShB-IR expressing oocytes (express both endogenous and ShB-IR proteins, “ShB-IR + Endo”, brown). The “Endo” response was subtracted from the “ShB-IR + Endo” response to estimate the isolated RF response from the ShB-IR channels only (“ShB-IR only”, blue). Error bars denote +/− standard errors of the mean (SEM). B) The SEM for the isolated ShB-IR proteins (“ShB-IR only”, blue) was also estimated by subtracting the SEM from the control oocytes (“Endo”, orange) from the SEM associated with the ShB-IR expressing oocytes (“ShB-IR + Endo”, brown). The SEM for isolated ShB-IR expressing oocytes was largest near the half-activation potential for these ion channels. C) ShB-IR channel expression and voltage-dependent whole-cell current was verified using TEVC, and this data was used to estimate ShB-IR conductance (G/Gmax, inset).

Mentions: To further examine differences between steady-state RF impedance in control (n = 10, “Endo”, orange) vs. ShB-IR expressing oocytes (n = 9, “ShB-IR + Endo”, brown), we averaged /ΔZRF/s 5–35 ms (see Methods) after the onset of voltage step (Methods), and plotted the result against the average membrane potential Vm* (Fig. 3A). ShB-IR expressing oocytes include the exogenously expressed ShB-IR proteins and endogenous membrane proteins and both contribute to the RF data. Averaged control data were subtracted from the ShB-IR expressing oocyte data, to estimate the RF response associated with the ShB-IR protein only (Fig. 3A, “ShB-IR only”, blue line). Error bars denote standard errors of the mean (+/−, SEM), and are shown as a function of Vm* for ShB-IR expressing oocytes and control oocytes in Fig. 3B. To demonstrate successful transfection, steady-state ionic currents are shown in Fig. 3C, and ShB-IR channel conductance is shown in the inset. Like the averaged RF data, the SEM associated with the ShB-IR protein only (Fig. 3B, “ShB-IR only”, blue line) was estimated by subtracting the SEM associated with the ShB-IR expressing oocytes at each membrane potential from the control oocytes SEM(s) (see Methods). Significant differences in /ΔZRF/s were found between Shaker-expressing oocytes and control oocytes at and above −10 mV (Fig. 3A), and differences are easily observable above the half-activation voltage for ShB-IR channels (xp<0.10, *p<0.05, Fig. 3A). Interestingly, the RF ShB-IR-only data (Fig. 3A, blue line) and the corresponding ShB-IR conductance data (shown in Fig. 3C) have a linear correlation coefficient of .94 (and a correlation coefficient of .99 in the voltage range of −45 mV to 2 mV, when the ShB-IR data increase from 5–95% of their final value), indicating a positive association between channel activity and the measured RF response. Furthermore, unlike the control oocytes that showed increased SEM for large Vm*, the ShB-IR-only SEM (Fig. 3B, blue line) showed the largest value near the half-activation voltage, and standard errors for the ShB-IR expressing oocytes were comparable to baseline values when the ensemble of channels in the membrane were predominantly closed or open. These results are consistent with voltage-dependent SEM arising from probabilistic conformational state of the Shaker ion-channels expressed in the membrane.


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

Dharia S, Rabbitt RD - PLoS ONE (2011)

Steady-state RF response for ShB-IR and Control Cells.A) Significant (xp = .1, *p = .05) voltage-dependent differences in /ΔZRF/ s were observed between control oocytes, expressing endogenous proteins only (“Endo”, orange), and ShB-IR expressing oocytes (express both endogenous and ShB-IR proteins, “ShB-IR + Endo”, brown). The “Endo” response was subtracted from the “ShB-IR + Endo” response to estimate the isolated RF response from the ShB-IR channels only (“ShB-IR only”, blue). Error bars denote +/− standard errors of the mean (SEM). B) The SEM for the isolated ShB-IR proteins (“ShB-IR only”, blue) was also estimated by subtracting the SEM from the control oocytes (“Endo”, orange) from the SEM associated with the ShB-IR expressing oocytes (“ShB-IR + Endo”, brown). The SEM for isolated ShB-IR expressing oocytes was largest near the half-activation potential for these ion channels. C) ShB-IR channel expression and voltage-dependent whole-cell current was verified using TEVC, and this data was used to estimate ShB-IR conductance (G/Gmax, inset).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046147&req=5

pone-0017363-g003: Steady-state RF response for ShB-IR and Control Cells.A) Significant (xp = .1, *p = .05) voltage-dependent differences in /ΔZRF/ s were observed between control oocytes, expressing endogenous proteins only (“Endo”, orange), and ShB-IR expressing oocytes (express both endogenous and ShB-IR proteins, “ShB-IR + Endo”, brown). The “Endo” response was subtracted from the “ShB-IR + Endo” response to estimate the isolated RF response from the ShB-IR channels only (“ShB-IR only”, blue). Error bars denote +/− standard errors of the mean (SEM). B) The SEM for the isolated ShB-IR proteins (“ShB-IR only”, blue) was also estimated by subtracting the SEM from the control oocytes (“Endo”, orange) from the SEM associated with the ShB-IR expressing oocytes (“ShB-IR + Endo”, brown). The SEM for isolated ShB-IR expressing oocytes was largest near the half-activation potential for these ion channels. C) ShB-IR channel expression and voltage-dependent whole-cell current was verified using TEVC, and this data was used to estimate ShB-IR conductance (G/Gmax, inset).
Mentions: To further examine differences between steady-state RF impedance in control (n = 10, “Endo”, orange) vs. ShB-IR expressing oocytes (n = 9, “ShB-IR + Endo”, brown), we averaged /ΔZRF/s 5–35 ms (see Methods) after the onset of voltage step (Methods), and plotted the result against the average membrane potential Vm* (Fig. 3A). ShB-IR expressing oocytes include the exogenously expressed ShB-IR proteins and endogenous membrane proteins and both contribute to the RF data. Averaged control data were subtracted from the ShB-IR expressing oocyte data, to estimate the RF response associated with the ShB-IR protein only (Fig. 3A, “ShB-IR only”, blue line). Error bars denote standard errors of the mean (+/−, SEM), and are shown as a function of Vm* for ShB-IR expressing oocytes and control oocytes in Fig. 3B. To demonstrate successful transfection, steady-state ionic currents are shown in Fig. 3C, and ShB-IR channel conductance is shown in the inset. Like the averaged RF data, the SEM associated with the ShB-IR protein only (Fig. 3B, “ShB-IR only”, blue line) was estimated by subtracting the SEM associated with the ShB-IR expressing oocytes at each membrane potential from the control oocytes SEM(s) (see Methods). Significant differences in /ΔZRF/s were found between Shaker-expressing oocytes and control oocytes at and above −10 mV (Fig. 3A), and differences are easily observable above the half-activation voltage for ShB-IR channels (xp<0.10, *p<0.05, Fig. 3A). Interestingly, the RF ShB-IR-only data (Fig. 3A, blue line) and the corresponding ShB-IR conductance data (shown in Fig. 3C) have a linear correlation coefficient of .94 (and a correlation coefficient of .99 in the voltage range of −45 mV to 2 mV, when the ShB-IR data increase from 5–95% of their final value), indicating a positive association between channel activity and the measured RF response. Furthermore, unlike the control oocytes that showed increased SEM for large Vm*, the ShB-IR-only SEM (Fig. 3B, blue line) showed the largest value near the half-activation voltage, and standard errors for the ShB-IR expressing oocytes were comparable to baseline values when the ensemble of channels in the membrane were predominantly closed or open. These results are consistent with voltage-dependent SEM arising from probabilistic conformational state of the Shaker ion-channels expressed in the membrane.

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