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Figure 1. Measurement of currents flowing through ion channels in response to ultrasonic stimulation.A Xenopus oocyte expressing an ion channel of interest is placed in a recording bath in a chamber (see Methods), and subjected to an US pressure field generated by a focused US transducer placed below the chamber and submerged in deionized and degassed water. A neoprene absorber is immersed in the recording bath to prevent the formation of a standing wave in the chamber. Transmembrane currents are measured using the two-electrode voltage clamp.
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Figure 2. Robust and repeatable effects of ultrasound on transmembrane currents flowing through cells expressing TREK-1.(a) The current flowing through the membrane of an example cell as a function of time. The membrane voltage was held at â80âmV before stepping to +50âmV (time 0). After 2 s, the membrane voltage returned to â80âmV. To test the repeatability of the US effects, we alternated conditions without and with US in the following sequence: #1: noUS, #2: US, #3: noUS, #4: US, #5: noUS, #6: US, #7: noUS with a 5 s period between the repetitions. The corresponding current waveforms are labeled in the figure. In the US conditions, the US was applied at time 0 and lasted 1 s (see blue bar on the bottom). The driving US waveform was sinusoidal with frequency of 10âMHz and spatial-peak pressure amplitude of 240âkPa (2.0âW/cm2). The figure shows the raw recorded currents. (b) Meanâ±âs.e.m. ultrasound effect, i.e., US minus noUS currents, for the three consecutive applications of the US. The currents were measured between 1.0 s to 1.2 s following the stimulus onset. This plot is based on 10 cells using spatial-peak US pressure amplitude of 240âkPa (2.0âW/cm2) and membrane voltage of +50âmV.
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Figure 3. Ultrasound-mediated currents as a function of membrane voltage, in the absence and presence of TREK-1 blocker.Meanâ±âs.e.m. current flowing through the membrane of the example cell as a function of time. The mean was computed over the individual US (blue, nâ=â3) and noUS (black, nâ=â4) repetitions. The individual rows correspond to the currents measured at voltage steps of +50âmV, â10âmV, â70âmV, and â130âmV. In the data in the right column, same protocol sequence was repeated following the administration of BaCl2 (10âmM) into the bath.
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Figure 4. The effects of ultrasound on TREK-1 current.Left: Meanâ±âs.e.m. current due to US as a function of membrane voltage, for TREK-1 cells (dark brown; nâ=â10), the same TREK-1 cells with BaCl2 (10âmM) in the bath (light brown), and control cells (black; nâ=â12). The US stimulus pressure amplitude was 120âkPa, corresponding to ISPTAâââ0.5âW/cm2. The currents were measured in the same interval as previously. Right: Meanâ±âs.e.m. relative changes in the transmembrane currents due to US, for the individual membrane voltages, quantified in the same interval as previously.
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Figure 5. Ultrasound-mediated effects at elevated extracellular K+ concentration.Same format as in Fig. 4 left, for 5 TREK-1 cells recorded in ND96 with a 20âmM K+ concentration.
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Figure 6. The effects of ultrasound on TREK-2 and TRAAK currents.Same protocol and format as for TREK-1 (Fig. 4). In the right panel (relative effects), the data can show a large s.e.m. for the −70 mV holding voltage due to the division by small currents at this voltage.
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Figure 7. Summary of the effects of ultrasound on K2P channels.Meanâ±âs.e.m. effects averaged all holding voltages. The values are taken over the same interval as previously. The number of cells are shown below the channel labels.
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Figure 8. Ultrasound-mediated effects increase with stimulus intensity.Meanâ±âs.e.m. relative changes in the transmembrane currents due to US, for the individual membrane voltages, quantified in the same interval as previously. The data are shown separately for a lower (left) and a higher (right) stimulus intensity applied to separate sets of cells expressing TREK-1.
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Figure 9. The effects of US on TREK-1 current when US propagates directly into the cell.(a) An alternative recording setup in which a cell is placed into a 0.7âmm opening within two thin sheets of borosilicate glass. Both the cell and the transducer share the same ND96 bath. (b) Meanâ±âs.e.m. current due to US (nâ=â9 cells) as a function of the membrane voltage, for the TREK-1 cells (brown) and TREK-1 cells with BaCl2 (10âmM) in the bath (light brown). Same format as in Fig. 4. (c) Meanâ±âs.e.m. relative changes in the transmembrane currents due to the US, for the individual membrane voltages. Same format as in Fig. 4. (d) Meanâ±âs.e.m. temperature (nâ=â5 recordings) of cell membrane surface measured by an infrared camera. The 10âMHz 240âkPa (2.0âW/cm2) US stimulus was applied at 10 s, and lasted for 1 s (thick blue bar).
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Figure 10. The effects of US on NaV1.5 current.Average membrane current of a cell expressing NaV1.5 α and 81 subunits in response to 20âms voltage steps of a particular value (see inset). We alternated 4 noUS and 3 US conditions; average noUS (black) or US (blue) current is shown. Pulsed ultrasound (10âMHz, 1âkHz pulse repetition frequency, 50 us pulse duration, ISPTAâ=â0.3âW/cm2) was applied throughout the US condition (see Methods). The light traces show corresponding currents following the introduction of ranolazine (500âμM), a NaV1.5 channel blocker, into the bath.
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Figure 11. Summary of the effects of US on NaV1.5 currents.(a) Right panel: Average peak negative current in NaV1.5-expressing cells (nâ=â8) in response to voltage steps (same as in Fig. 10), separately for the conditions in which there was no US (black) and in which US was applied (blue). The currents of each cell were normalized by the cellâs overall peak negative current before computing the average over cells. For each cell, we alternated 4 noUS and 3 US conditions, and took the average within each condition. The brown curve shows the meanâ±âs.e.m. difference between the US (blue) and noUS (black) currents. Left panel: Same as right panel for the cells that in addition had ranolazine blocker data (nâ=â5). The currents following the application of ranolazine (500âμM) are shown in light colors. (b) Meanâ±âs.e.m. relative effects of US (100 * (IUS-InoUS)/InoUS) for the individual voltages. The figure shows effects for voltages in which currents were substantial to circumvent division by a small number (small currents at â90âmV and â70âmV). Both (a,b) used stimulus intensity ISPTAâ=â4.9âW/cm2. *pâ<â0.05, two-sided t-test, nâ=â8. (c) The effects of US as a function of stimulus intensity. The effects were averaged over the voltage steps; meanâ±âs.e.m. values over cells (nâ=â8) are shown. *pâ<â0.05, two-sided t-test.
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