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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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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.
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pone-0017363-g002: 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.

Mentions: Fig. 2A compares time-resolved changes in RF impedance /ΔZRF/ in control ooyctes expressing endogenous channels (avg. n = 10, left) to oocytes expressing ShB-IR channels (avg. n = 9, right). Voltage-dependent RF charge displacement occurred in both cell expression systems, but differed in magnitude and temporal waveform between the control and ShB-IR expressing oocytes. The left axis reports the magnitude of the change in impedance, /ΔZRF/ =  /ZRF- Z0/, where the reference, Z0, for each record was the average RF impedance 5–40 ms prior to the voltage step. Although signals were noisy in our apparatus, milliohm changes in /ΔZRF/ during depolarization were readily discernable from noise. In all cases, /ΔZRF/ consisted of an initial onset response when the rate of change of TEVC membrane potential was large (o, /ΔZRF/o, dVm*/dt>0) and a steady-state response when TEVC membrane potential was approximately constant (s, /ΔZRF/s, dVm*/dt ≅ 0). The RF onset response (o) occurred in the first millisecond after a voltage step was applied to the cell, and was similar in both the control and ShB-IR expressing oocytes. Interestingly, the RF onset response was rectified and did not occur at the end of the voltage command step. Hence, the fast RF onset response was not causally related to the standard TEVC capacitive transient (current spikes in Fig. 2B). Steady state RF changes (s) 5–35 ms after the voltage step were also observed in both control and ShB-IR oocytes, but were significantly larger in the ShB-IR expressing oocytes at membrane potentials above −20 mV. This indicated a difference in RF response attributable to Shaker activation. Simultaneous TEVC recordings shown in the lower panels (Fig. 2B, current; Fig. 2C, voltage) were used to confirm expression and test whole-cell currents. Darker lines indicate increased levels of depolarization during the voltage step, commanded from a holding potential of −90 mV (Fig. 2C). TEVC whole-cell currents in the control condition (Fig. 2B, left) were small relative to the large currents in ShB-IR expressing cells (Fig. 2B, right). Voltage sensitivity and sustained ionic currents reported here are typical for ShB-IR cells with fast inactivation removed, and indicate successful protein expression and activity.


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

Dharia S, Rabbitt RD - PLoS ONE (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046147&req=5

pone-0017363-g002: 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.
Mentions: Fig. 2A compares time-resolved changes in RF impedance /ΔZRF/ in control ooyctes expressing endogenous channels (avg. n = 10, left) to oocytes expressing ShB-IR channels (avg. n = 9, right). Voltage-dependent RF charge displacement occurred in both cell expression systems, but differed in magnitude and temporal waveform between the control and ShB-IR expressing oocytes. The left axis reports the magnitude of the change in impedance, /ΔZRF/ =  /ZRF- Z0/, where the reference, Z0, for each record was the average RF impedance 5–40 ms prior to the voltage step. Although signals were noisy in our apparatus, milliohm changes in /ΔZRF/ during depolarization were readily discernable from noise. In all cases, /ΔZRF/ consisted of an initial onset response when the rate of change of TEVC membrane potential was large (o, /ΔZRF/o, dVm*/dt>0) and a steady-state response when TEVC membrane potential was approximately constant (s, /ΔZRF/s, dVm*/dt ≅ 0). The RF onset response (o) occurred in the first millisecond after a voltage step was applied to the cell, and was similar in both the control and ShB-IR expressing oocytes. Interestingly, the RF onset response was rectified and did not occur at the end of the voltage command step. Hence, the fast RF onset response was not causally related to the standard TEVC capacitive transient (current spikes in Fig. 2B). Steady state RF changes (s) 5–35 ms after the voltage step were also observed in both control and ShB-IR oocytes, but were significantly larger in the ShB-IR expressing oocytes at membrane potentials above −20 mV. This indicated a difference in RF response attributable to Shaker activation. Simultaneous TEVC recordings shown in the lower panels (Fig. 2B, current; Fig. 2C, voltage) were used to confirm expression and test whole-cell currents. Darker lines indicate increased levels of depolarization during the voltage step, commanded from a holding potential of −90 mV (Fig. 2C). TEVC whole-cell currents in the control condition (Fig. 2B, left) were small relative to the large currents in ShB-IR expressing cells (Fig. 2B, right). Voltage sensitivity and sustained ionic currents reported here are typical for ShB-IR cells with fast inactivation removed, and indicate successful protein expression and activity.

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