Voltage gating of mechanosensitive PIEZO channels

Mechanosensitive PIEZO ion channels are evolutionarily conserved proteins whose presence is critical for normal physiology in multicellular organisms. Here we show that, in addition to mechanical stimuli, PIEZO channels are also powerfully modulated by voltage and can even switch to a purely voltage-gated mode. Mutations that cause human diseases, such as xerocytosis, profoundly shift voltage sensitivity of PIEZO1 channels toward the resting membrane potential and strongly promote voltage gating. Voltage modulation may be explained by the presence of an inactivation gate in the pore, the opening of which is promoted by outward permeation. Older invertebrate (fly) and vertebrate (fish) PIEZO proteins are also voltage sensitive, but voltage gating is a much more prominent feature of these older channels. We propose that the voltage sensitivity of PIEZO channels is a deep property co-opted to add a regulatory mechanism for PIEZO activation in widely different cellular contexts.

additional step was added between a first pressure step at -60mV and a pressure step at +60mV.
During the second (test) pressure step the voltage was increased from 20mV to 80mV in successive sweeps with a 10mV increment. The amplitude and the rise time of the third step was then plotted (right) against the size of the test voltage step. Notice how the increase in the voltage of the second step is accompanied by an increase both in amplitude and rise time, thus underlying a faster recovery from inactivation with increasing voltage. d) The protocol in C is repeated. The voltage of the second step is kept constant at +60mV, while the duration is increased from 20ms to 160ms (20ms grey, 40ms orange, 60ms green, 80ms light blue, 100ms blue, 120ms magenta, 140ms red, 160ms black). Notice how steps as short as 40ms can recover up to 90% of the channels. For both c and d, amplitudes were measured averaging the amplitude values between

Supplementary Figure 2. PIEZO1 R2482H desensitization and inactivation properties.
(Related to Fig. 4) a) R2482H (red trace) shows a slow kinetic of inactivation in comparison to the wild type (blue trace) as previously reported. b) Repetitive pressure stimulations desensitizes R2482H -60mV (b) but not at +60mV (c). As for the wild type at -60mV the fifth stimulus is much smaller than the first one and the current shows a decreased peak current and low peak/steady state current ratio. d) Alternating pressure pulses at -60mV and +60mV abolishes desensitization of PIEZO1 R2482H. e) Left. 3 pressure pulses (P1, P2,P3) at -60mV were applied to patches expressing PIEZO1 R2482H to drive the channel into a desensitized state, followed by 1 positive pressure pulse (P4) at +60mV, as described in Fig 2. The sequence was repeated twice. As for the wild type outward permeation (P4) recovers PIEZO1 R2482H initial current (P1) and resets its time course for inactivation, as shown by the ratio between the current amplitude of P5/P1, shown in f). The stimulation sequence in e was repeated in absence of pressure to prevent channel opening and permeation when switching to a positive voltage (P4). The recovery from desensitization does not occur in absence of a permeation event. As observed for the wild type note the slow rise time of P4, most likely due to a slow transition out of the desensitized state. (magenta) and alternating -60/+60mV (black) were applied to outside-out patches expressing the chimeric PIEZO1-PIEZO2 construct. As for wild type mPIEZO1 inward currents elicited a fast onset of desensitization at negative voltages, lack of desensitization at positive or alternating voltages. c) As for mPIEZO1 outward permeation facilitated recovery from desensitization induced by repetitive inward currents. 3 pressure pulses (P1, P2,P3) at -60mV were applied to patches expressing the chimeric channel to drive the channel into an desensitized state, followed by 1 positive pressure pulses (P4) at +60mV. The sequence was repeated twice. Outward permeation (P4, black trace) allows the chimeric channel to recover from desensitization as shown by the ratio of P5/P1. Absence of outward permeation (P4, green trace) does not allow recovery from desensitization. Increasing pressure stimuli were applied to outside-out patches pulled from N2A cells overexpressing wt PIEZO1 at a constant voltage of either -60mV (inward currents) and +60mV (outward currents). a) Yoda1 (5M) in the intracellular solution b) Vehicle in the intracellular solution. Note the difference in kinetics in presence of Yoda1. c) The wild type PIEZO1 was subject to a family of pressure stimulations at increasing voltages (from 60 to 160mV) and the deactivation phase was extended to 2 seconds. In less than 5% of the cells and at voltages higher than 140mV it was possible to observe a reactivation phase as for the R2482H/K mutant. d) Reactivation time constants referring to Fig. 6a. Note how the time course of re-activation accelerates at more depolarized voltages. e) R2482K was subject to a similar protocol as in Fig.   6b. A saturating pressure pulse at 80mV was applied to force the channel to reactivate and switch to voltage-gated mode. Following a 2 s deactivation period at -20mV (to induce the inactivation gate to close) a family of 1 s steps at increasing voltages (in absence of pressure) was applied. PIEZO1 R2482K cannot be activated by the sole application of voltage when the deactivation occurs at negative voltages. This event forces the inactivation gate to close and the channel into an inactive state.

Supplementary Figure 6. Pressure sensitivity and I/V relationship of the DrPIEZO1
(zebrafish) (Related to Figure 7) a) Patches pulled from N2a Piezo1 -/cell line overexpressing the ZPIEZO1 were clamped at -60mV and subject to increasing pressure (10 to 110mmHg). Fitting of the pressure response curve gave a P 50 value of 51 ± 2.7 (n=9) mmHg. b) Exponential fitting of the time course of inactivation showed a significantly slower kinetic for ZPIEZO1 in comparison to mPIEZO1 (Student's t-test P = 0.0003). c) I/V relationship for ZPIEZO1. Saturating pressure pulses were applied to patches at voltages ranging from -100mv to +80mV. Peak currents were plotted against voltage and showed a linear I/V relationship.

Supplementary Figure 7. Voltage gating of vertebrates and invertebrates PIEZO1
orthologues (Related to Fig. 7) The hPIEZO1, DPIEZO and DrPIEZO1 were subject to a family of pressure stimulations at increasing voltages (as indicated) and the deactivation phase was extended to 2 seconds. The human PIEZO1 deactivated more slowly at depolarized voltages. After an initial deactivation the DPIEZO and DrPIEZO1, at voltages above 40mV and 20mV respectively, exhibited a sag followed by a reactivation phase.