Dharia S and Rabbitt RD (2011), Monitoring voltage-dependent charge displacemen...

XB-ART-42884

PLoS One 2011 Feb 04;62:e17363. doi: 10.1371/journal.pone.0017363.

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

Dharia S , Rabbitt RD .

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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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Species referenced: Xenopus laevis
Genes referenced: shb

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	Figure 1. Set-up and circuit model.A) Changes in RF membrane impedance (\|ΔZRF\|) during TEVC were measured by passing RF current from an electrode surrounding the meridian of the cell (black) to a ground electrode (media, above the cell). Contour lines and colors of the saggital cross-section of a cell in the recording chamber, shown here, illustrate the general spatial distribution of the RF electric potential expected based on the Maxwell equations for a passive cell under axisymmetric conditions (π/4 phase shown). B) A circuit model of the chamber including the shunt resistance (Rs,), membrane impedance (Zm), intracellular resistance (Ri), and electrode double layer (Zdl). C) Using the circuit model, the frequency-dependent RF impedance would change (Δ\|ZRF\|) with an increase or decrease in membrane capacitance — a change that would be most easily detectable at frequency ω* where the maxima of the \|ΔZRF\| occurs. The present study reports changes in RF impedance \|ΔZRF\| evoked by TEVC step changes in membrane potential.
	Figure 2. Temporally Resolved RF Measurements.A) RF impedance changes (\|ΔZRF\|) measured during TEVC relative to the impedance at holding potential (−90 mV) in control oocytes, expressing endogenous proteins only (n = 10, left column), and ShB-IR expressing oocytes (n = 9, right column). ShB-IR expressing oocytes elicited a membrane-potential-dependent (Vm) RF response different than control oocytes. RF impedance changes were analyzed in two regions; the RF response during the onset of voltage-step (o, average \|ΔZRF\|o 0–1 ms after voltage step, dVm/dt > 0) and the RF response after membrane potential achieved its command (steady-state) level (s, average \|ΔZRF\|s 5–35 ms after voltage step, dVm*/dt ≅ 0). B) TEVC current measurements were used to verify ion-channel expression and responses (leak current subtracted, capacitive transient unsubtracted) to C) whole-cell voltage-clamp.
	Figure 3. 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).
	Figure 4. 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).
	Figure 5. 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).

References [+] :

Armstrong, Inactivation of the sodium channel. II. Gating current experiments. 1977, Pubmed

	Figure 1. Set-up and circuit model.A) Changes in RF membrane impedance (\|ΔZRF\|) during TEVC were measured by passing RF current from an electrode surrounding the meridian of the cell (black) to a ground electrode (media, above the cell). Contour lines and colors of the saggital cross-section of a cell in the recording chamber, shown here, illustrate the general spatial distribution of the RF electric potential expected based on the Maxwell equations for a passive cell under axisymmetric conditions (π/4 phase shown). B) A circuit model of the chamber including the shunt resistance (Rs,), membrane impedance (Zm), intracellular resistance (Ri), and electrode double layer (Zdl). C) Using the circuit model, the frequency-dependent RF impedance would change (Δ\|ZRF\|) with an increase or decrease in membrane capacitance — a change that would be most easily detectable at frequency ω* where the maxima of the \|ΔZRF\| occurs. The present study reports changes in RF impedance \|ΔZRF\| evoked by TEVC step changes in membrane potential.
	Figure 2. Temporally Resolved RF Measurements.A) RF impedance changes (\|ΔZRF\|) measured during TEVC relative to the impedance at holding potential (−90 mV) in control oocytes, expressing endogenous proteins only (n = 10, left column), and ShB-IR expressing oocytes (n = 9, right column). ShB-IR expressing oocytes elicited a membrane-potential-dependent (Vm) RF response different than control oocytes. RF impedance changes were analyzed in two regions; the RF response during the onset of voltage-step (o, average \|ΔZRF\|o 0–1 ms after voltage step, dVm/dt > 0) and the RF response after membrane potential achieved its command (steady-state) level (s, average \|ΔZRF\|s 5–35 ms after voltage step, dVm*/dt ≅ 0). B) TEVC current measurements were used to verify ion-channel expression and responses (leak current subtracted, capacitive transient unsubtracted) to C) whole-cell voltage-clamp.
	Figure 3. 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).
	Figure 4. 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).
	Figure 5. 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).