Piezo1 Gating: Comparison Between Whole Cell Currents and the Patch

Abstract

Piezo1 channels gate with mechanical stress in the membrane and gating involves both activation and inactivation. In HEK293 cells transfected with Piezo1 and subjected to pressure stimuli, cell-attached patch recordings showed that the inactivation rate slowed as extracellular divalent ions were reduced. With >1mM Mg+2, activation had no measurable latency and the inactivation rate was rapid but stress dependent, suggesting that Mg+2 may act as an open channel blocker (the effects of Ca+2 are in progress). Without divalents there was no inactivation, but surprisingly, activation now had a pronounced latency (∼500 ms). Inactivation may actually represent adaptation of the local stimulus by the cytoskeleton and not overt channel closure. To disrupt the cytoskeleton we treated cells with cytochalasin D before patching and found inactivation was unaffected suggesting cytoskeletal adaption was not the cause. Attempting the inverse experiment, we increased cytoskeletal stress by swelling the cells osmotically, but that too didn't affect the inactivation rate.For analogy close to the in situ situation, we evoked whole cell Piezo1 currents by indenting cells with a glass probe. Like the patch, removing extracellular divalent ions reversibly reduced the inactivation rate. However, in contrast to patch recordings, Cytochalasin D caused a loss of whole cell current and cell swelling increased the evoked currents. These results suggest that the forces that gate Piezo1 in whole cell mode propagate through the cytoskeleton, and that divalent ion block may be responsible for inactivation.Supported by the NIH and CDMRP