Voltage-dependent membrane displacements measured by atomic force microscopy

Abstract

Cells use polar molecules in the membrane to sense changes in the transmembrane potential. The opening of voltage-gated ion channels and membrane bending due to the inverse flexoelectric effect are two examples of such electromechanical coupling. We have looked for membrane motions in an electric field using atomic (or scanning) force microscopy (AFM) with the intent of studying voltage-dependent conformational changes of ion channels. Voltage-clamped HEK293 cells were either untransfected controls or transfected with Shaker K+ channels. Using a +/- 10-mV peak-peak AC carrier stimulus, untransfected cells moved 0.5-15 nm normal to the plane of the membrane. These movements tracked the voltage at frequencies >1 kHz with a phase lead of 60-120 degrees, as expected of a displacement current. The movement was outward with depolarization, but the holding potential only weakly influenced the amplitude of the movement. In contrast, cells transfected with a noninactivating mutant of Shaker K+channels showed similar movements, but these were sensitive to the holding potential; decreasing with depolarization between -80 and 0 mV. Searching for artifactual origins of these movements, we used open or sealed pipettes and AFM cantilever placements just above the cells. These results were negative, suggesting that the observed movements were produced by the cell membrane rather than by movement of the patch pipette, or by acoustic or electrical interactions of the membrane with the AFM tip. In control cells, the electrical motor may arise from the flexoelectric effect, where changes in potential induce changes in curvature. In transfected cells, it appears that channel-specific movements also occurred. These experiments demonstrate that the AFM may be able to exploit voltage-dependent movements as a source of contrast for imaging membrane components. The electrically induced motility will cause twitching during action potentials, and may have physiological consequences.

Figure 1

A shows a schematic top view drawing (not to scale) of the set up. The patch pipette holding the HEK cell was attached to the tubular piezo ceramic used for x, y, z scanning. The holding potential, V_h, and the AC carrier voltage were applied to the cell by the patch-clamp amplifier. The cantilever movement was translated into a voltage, V_det, by the laser and quadrant detector. The output was proportional to the height difference Δh of the surface. In B, force–distance plots are shown for a cantilever approaching glass and an (untransfected) HEK293 cell. Forward and backward movements of the cantilever are indicated by arrows. C shows a calibration curve for the cantilever. The piezo holding a sealed, stiff pipette was moved over a defined distance with an AC signal, and the corresponding peak of the PSD was the displacement measured. A linear regression line gave a slope of 16 μV_rms/√ Hz)/nm. A PSD of a typical experiment is shown in C. The (transfected) cell was held at V_h = −60 mV and an AC voltage of ± 10 mV at 66 Hz (vertical arrow) was applied by the patch amplifier. The corresponding peak in the PSD of the detector signal had an amplitude of 2.8 nm. E shows a PSD without an electrical stimulus (same cell). F shows the effect of restoring the AC carrier but placing the cantilever tip just above the surface of the cell.

Figure 2

The voltage-induced movements had a higher bandwidth than did movements coupled from movement of the patch pipette. In A, a series of spectra are superimposed that show the frequency dependence of cantilever movement induced by 11-nm oscillations of the patch pipette (± 5 mV sinusoidal stimulation applied to the piezo). The cell was simultaneously voltage clamped with ± 30 mV at 66 Hz and V_h = −45 mV (untransfected cell). For comparison, B shows several superimposed spectra where the frequency of the voltage clamp carrier was changed while the movement of the clamping pipette was kept at 66 Hz. This series was recorded subsequent to the one in A. (To permit the measurement at higher frequencies, B was recorded with three different PSD bandwidths, which are printed in different grey scales. This accounts for the varying width of the peaks at 66 Hz.)

Figure 3

Frequency dependence of the voltage-induced movement of six cells normalized to the amplitude at the lowest frequency tested (66 [3 cells], 85 [2 cells], and 166 [1 cell] Hz). In three experiments (bold markers), we used a stiffer cantilever with k = 0.02 N/m (normal cantilever, k = 0.01 N/m). The mean sensitivity at the lowest frequency was (0.15 ± 0.05) nm/mV_pp (mean ± SEM, n = 6). Points measured above the resonance frequency of the set-up (∼2 kHz) are drawn in grey to indicate that the decrease in signal amplitude is also affected by the detection system.

Figure 5

Currents and conductance-voltage relation of untransfected control cells and cells transfected with a noninactivating mutant of Shaker H4 K⁺ channel. A and C show 20 superimposed traces of currents for 50-ms step changes in potential from a holding potential of −80 mV in steps of 5 mV for untransfected and transfected HEK293 cells, respectively. B and D show the chord-conductance/voltage behavior for both cells as calculated by the steady state current during the last 20 ms of the 50-ms depolarizing pulse.

Figure 4

The effect of holding potential on the movement of untransfected versus transfected cells. Movement of the transfected cells had a unique dependence on the holding potential. (A) Pooled and normalized data from four untransfected cells. The displacements were normalized to their largest value (16.7 nm at 45 mV ± 50 mV, 10.4 nm at 20 mV ± 15 mV, 4.5 nm at 70 mV ± 10 mV, and 3.7 nm at −100 mV ± 25 mV AC stimulus) and plotted against the holding potential of the cell. (B) Data for three transfected cells. Maximal values were 3.0 nm at −80 mV (± 5 mV AC stimulus), 5.9 nm at −80 mV (± 10 mV AC stimulus), and 9.9 nm at −80 mV (± 10 mV AC stimulus). (The holding potential of the cells was only changed from hyperpolarized to depolarized because the slow recovery of the movement signal after activation of the current in transfected cells interfered with running the experiment.)

Figure 6

(A) Mechanical equivalent model showing the effect of cytoskeletal (k_cyt) and cantilever (k_cant) stiffness to reduce the true voltage-induced displacement (d) to the observed displacement (see text). (B–D) Cartoons of an AFM tip indenting the membrane and moving with the applied voltage. B is for the dipole rotation model, C is for the Shaker channel model, and D is for the flexoelectric effect where the minus signs represent fixed charges on the outer monolayer.