To determine the effect of membrane stretch at NRs of myelinated sensory nerves, we performed a pressure-clamped patch-clamp on intact NRs of rat sciatic nerve [11] (Fig. 1A). We first analyzed single-channel activities at the NRs by applying various negative pressures on the axonal membrane with a high-speed pressure-clamp device under the cell-attached configuration (Fig. 1B). The single-channel conductance was approximately 90 pS at 80 mV, which was consistent with the channel conductance of the heteromeric TREK-1/TRAAK channels [4] (Fig. 1C). The single-channel conductance was not affected by membrane stretch (Fig. 1C). The single-channel event numbers and open probability increased in a pressure-dependent manner and showed a significant difference from the negative pressure of 60 mmHg or more (Fig. 1D, E). These results show that TREK-1/TRAAK channels are highly sensitive to mechanical membrane stretch, and suggest that TREK-1/TRAAK activation by membrane stretch may modulate neuronal excitability at the NRs.
At NRs, strong leak potassium outward (lKleak) currents following depolarizing voltage steps mainly mediated by activation of TREK-1/TRAAK channels [4]. To determine whether membrane stretch enhances the IKleak currents by this activation at the NRs, we applied positive pressure into cells through a recording pipette under the whole-cell configuration (Fig. 1F). By applying positive pressure in this configuration, we applied membrane tension in the same direction from inside to outside of the cell as cell-attached configuration. Because the seal between the cell membrane and patch pipette is easily broken by internal positive pressure, we applied a maximum pressure of 10 mmHg for the experiment. This positive pressure enhanced the outward currents in response to the voltage steps (Fig. 1F). Consistent with single-channel activities, intra-pipette positive pressure significantly increased IKleak conductance compared to that at 0 mmHg (Fig. 1G). To confirm whether the enhanced outward currents were mediated by TREK-1/TRAAK channels, we pharmacologically blocked these channels by applying Ba2+. The bath application of Ba2+ significantly suppressed pressure-enhanced outward currents and IKleak conductance (Fig. 1G, H). These results clearly show that intra-pipette pressure enhances K + conductance by activating TREK-1/TRAAK channels at the NRs.
We further confirmed whether membrane stretch modulates nodal membrane excitability in NRs in the current-clamp mode. The input resistance was significantly decreased by 10 mmHg intra-pipette positive pressure (Fig. 1I, J). The resting membrane potential was significantly decreased (Fig. 1K) and the rheobase was significantly increased by an intra-pipette pressure of 10 mmHg (Fig. 1L). The blockage of TREK-1/TRAAK channels by Ba2+ reversed the changes in the intrinsic electrophysiological properties induced by intra-pipette pressure (Fig. 1J–L).
In conclusion, our study demonstrates that mechanosensitive TREK-1/TRAAK channels may be activated by axonal stretch. The activation could suppress neuronal excitability. We previously found that TREK-1 and TRAAK are highly expressed in the NRs of mammalian sensory nerves, which leads to AP repolarization instead of voltage-gated K+ channels [4]. In the present study, single-channel activities with channel conductance of 90 pS (TREK-1/TRAAK-like) were increased by membrane stretch in a pressure-dependent manner. Consistent with single-channel activity, intra-pipette positive pressure increased IKleak currents and suppressed membrane excitability. Furthermore, pharmacological blockage of TREK-1/TRAAK channels reversed the changes in intrinsic electrophysiological properties induced by intra-pipette pressure. These results indicate the importance of TREK-1/TRAAK channels in the prevention of ectopic AP discharge at the axon by intense mechanical nerve stretch under physiological conditions.
As a limitation of this study, we used intra-pipette pressure to study ion channel function at the NRs instead of axonal stretch, because the membrane seal for patch-clamp recording is easily disrupted by membrane displacement. Thus, the application of mechanical pressure with a high-speed pressure-clamp device may not fully represent axonal stretch.
In conclusion, our study demonstrates the effect of membrane stretching on the intrinsic electrophysiological properties of NR. The findings provide important insights into the pathology of diseases, such as demyelination.