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 Cu2+ addition to the external bath. Cu2+ 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 Cu2+-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.
Highlights
Techniques to monitor displacement currents in the proteinrich cell membrane have been used extensively in previous studies to examine ion-channel voltage-sensor movement [1,2,3,4,5,6] and the piezoelectric-like behavior of transmembrane proteins [7,8,9]
In all cases, |DZRF| consisted of an initial onset response when the rate of change of two-electrode voltage clamp (TEVC) membrane potential was large (o, |DZRF|o, dVm*/dt.0) and a steady-state response when TEVC membrane potential was approximately constant (s, |DZRF|s, dVm*/dt > 0)
Steady state RF changes (s) 5–35 ms after the voltage step were observed in both control and Shaker B-IR (ShB-IR) oocytes, but were significantly larger in the ShB-IR expressing oocytes at membrane potentials above 220 mV
Summary
Techniques to monitor displacement currents in the proteinrich cell membrane have been used extensively in previous studies to examine ion-channel voltage-sensor movement [1,2,3,4,5,6] and the piezoelectric-like behavior of transmembrane proteins [7,8,9]. We introduce a new technique to monitor protein-dependent charge displacements by superimposing an extracellularly applied RF interrogation signal on top of a traditional voltage-clamp commanded membrane potential. This RF interrogation technique has its basis in electric impedance spectroscopy, which has been applied previously to probe membrane dielectric properties of isolated cells by measuring the electrical impedance between pairs or groups of extracellular electrodes [10,11,12,13,14,15]. We extend such RF dielectric measurements to study electrical charge displacement arising from electrically excited voltage-sensitive membranebound proteins
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