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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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Onset RF Response in ShB-IR Expressing Oocytes.Changes in RF impedance during the onset of voltage-clamp (/ΔZRF/o, 0–1 ms after whole-cell depolarization) were slightly depressed in control oocytes with the addition of Cu2+ (Cu2+-free - green markers, Cu2+ addition - purple markers), but were significantly greater in ShB-IR expressing oocytes (Cu2+-free - green line, Cu2+ addition - purple line).
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pone-0017363-g005: Onset RF Response in ShB-IR Expressing Oocytes.Changes in RF impedance during the onset of voltage-clamp (/ΔZRF/o, 0–1 ms after whole-cell depolarization) were slightly depressed in control oocytes with the addition of Cu2+ (Cu2+-free - green markers, Cu2+ addition - purple markers), but were significantly greater in ShB-IR expressing oocytes (Cu2+-free - green line, Cu2+ addition - purple line).

Mentions: RF onset responses, occurring during the rise of Vm* (when dVm*/dt was maximum), exhibited trends different than those seen in steady state. Fig. 5 plots the onset response /ΔZRF/o, averaged 0–1 ms after voltage command was applied (see Methods) vs. the rate of change of membrane potential (dVm*/dt) and membrane potential (parenthetically noted below the values of dVm*/dt). Cell data have been normalized using the same values in Fig. 4A, to allow for comparisons between transient and steady-state RF results (see Methods). The fast onset recorded in ShB-IR expressing oocytes and controls both increased with the magnitude of the membrane potential change, but there were no statistically significant differences due to expression of K+ channels (p>.1, data not shown). Onset RF charge displacements in control oocytes treated with Cu2+ slightly decreased as voltage-step increased (Fig. 5, filled circles), and in contrast to ShB-IR expressing oocytes, the average onset response of controls treated with Cu2+ was smaller than the non-treated cells. Cu2+-treated ShB-IR expressing oocytes pooled for all voltage commands > −60 mV exhibited significantly larger onset responses than non-treated ShB-IR expressing oocytes (p = .005, U = 1087, Total Points  =  80, median values untreated/treated ShB-IR expressing oocytes of 1.0/1.4, respectively). Interestingly, increases in the average onset /ΔZRF/o for Cu2+ treated ShB-IR expressing oocytes were about 1.5 times greater than those for Cu2+ treated ShB-IR oocytes in the steady state (/ΔZRF/s) for membrane potentials >−60 mV. One hypothesis that might explain the Cu2+-dependent onset response is that Cu2+ might have stabilized a transitional state in the ion channel during rapid membrane depolarization that momentarily affected the charge distribution on the protein, thereby enhancing the momentary RF charge displacement. The onset response was not detectable for -dVm*/dt at the end of the command pulse (see Fig. 2A), showing a rectification in /ΔZRF/ quite distinct from dVm*/dt driven linear capacitive transients (present in Fig. 2B).


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

Dharia S, Rabbitt RD - PLoS ONE (2011)

Onset RF Response in ShB-IR Expressing Oocytes.Changes in RF impedance during the onset of voltage-clamp (/ΔZRF/o, 0–1 ms after whole-cell depolarization) were slightly depressed in control oocytes with the addition of Cu2+ (Cu2+-free - green markers, Cu2+ addition - purple markers), but were significantly greater in ShB-IR expressing oocytes (Cu2+-free - green line, Cu2+ addition - purple line).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046147&req=5

pone-0017363-g005: Onset RF Response in ShB-IR Expressing Oocytes.Changes in RF impedance during the onset of voltage-clamp (/ΔZRF/o, 0–1 ms after whole-cell depolarization) were slightly depressed in control oocytes with the addition of Cu2+ (Cu2+-free - green markers, Cu2+ addition - purple markers), but were significantly greater in ShB-IR expressing oocytes (Cu2+-free - green line, Cu2+ addition - purple line).
Mentions: RF onset responses, occurring during the rise of Vm* (when dVm*/dt was maximum), exhibited trends different than those seen in steady state. Fig. 5 plots the onset response /ΔZRF/o, averaged 0–1 ms after voltage command was applied (see Methods) vs. the rate of change of membrane potential (dVm*/dt) and membrane potential (parenthetically noted below the values of dVm*/dt). Cell data have been normalized using the same values in Fig. 4A, to allow for comparisons between transient and steady-state RF results (see Methods). The fast onset recorded in ShB-IR expressing oocytes and controls both increased with the magnitude of the membrane potential change, but there were no statistically significant differences due to expression of K+ channels (p>.1, data not shown). Onset RF charge displacements in control oocytes treated with Cu2+ slightly decreased as voltage-step increased (Fig. 5, filled circles), and in contrast to ShB-IR expressing oocytes, the average onset response of controls treated with Cu2+ was smaller than the non-treated cells. Cu2+-treated ShB-IR expressing oocytes pooled for all voltage commands > −60 mV exhibited significantly larger onset responses than non-treated ShB-IR expressing oocytes (p = .005, U = 1087, Total Points  =  80, median values untreated/treated ShB-IR expressing oocytes of 1.0/1.4, respectively). Interestingly, increases in the average onset /ΔZRF/o for Cu2+ treated ShB-IR expressing oocytes were about 1.5 times greater than those for Cu2+ treated ShB-IR oocytes in the steady state (/ΔZRF/s) for membrane potentials >−60 mV. One hypothesis that might explain the Cu2+-dependent onset response is that Cu2+ might have stabilized a transitional state in the ion channel during rapid membrane depolarization that momentarily affected the charge distribution on the protein, thereby enhancing the momentary RF charge displacement. The onset response was not detectable for -dVm*/dt at the end of the command pulse (see Fig. 2A), showing a rectification in /ΔZRF/ quite distinct from dVm*/dt driven linear capacitive transients (present in Fig. 2B).

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