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. 2011 Jul 26;50(29):6295-300.
doi: 10.1021/bi200770q. Epub 2011 Jun 29.

The mechanosensitive ion channel Piezo1 is inhibited by the peptide GsMTx4

Chilman Bae  1 , Frederick Sachs, Philip A Gottlieb
Affiliations

The mechanosensitive ion channel Piezo1 is inhibited by the peptide GsMTx4

Chilman Bae et al. Biochemistry. .

Abstract

Cells can respond to mechanical stress by gating mechanosensitive ion channels (MSCs). The cloning of Piezo1, a eukaryotic cation selective MSC, defines a new system for studying mechanical transduction at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous cationic MSCs that are selectively inhibited by the peptide GsMTx4, we tested whether the peptide targets Piezo1 activity. Extracellular GsMTx4 at micromolar concentrations reversibly inhibited ∼80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibition was voltage insensitive, and as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding and unbinding based on Piezo1 current kinetics provided association and dissociation rates of 7.0 × 10(5) M(-1) s(-1) and 0.11 s(-1), respectively, and a K(D) of ∼155 nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier behavior, GsMTx4 produced an ∼30 mmHg rightward shift in the pressure-gating curve and was active on closed channels. In contrast, streptomycin, a nonspecific inhibitor of cationic MSCs, showed the use-dependent inhibition characteristic of open channel block. The peptide did not block currents of the mechanical channel TREK-1 on outside-out patches. Whole-cell Piezo1 currents were also reversibly inhibited by GsMTx4, and although the off rate was nearly identical to that of outside-out patches, differences were observed for the on rate. The ability of GsMTx4 to target the mechanosensitivity of Piezo1 supports the use of this channel in high-throughput screens for pharmacological agents and diagnostic assays.

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Figures

Figure 1
Figure 1
GsMTx4 inhibits Piezo1 currents. Panel A (Left): O-O patch with the baseline response measured at -30 mV with pressure pulses of 500 ms at intervals of 3.5 s (black trace). The mechanical response was inhibited by L-GsMTx4 (2.5 μM, red trace) and washout restored control activity (blue trace). The data are an average of 5-10 pulses. Panel A (Right): A comparison of the -30mV response to the +30mV response shows a mild voltage dependence of inhibition. Panel B demonstrates that the D-enantiomer (3.0 μM) reversibly inhibits Piezo1. Panel C is a summary of the average responses to D (n=3 patches) and L (n=7 patches) enantiomers. Data are normalized to allow comparison between experiments and the error bars are SEM.
Figure 2
Figure 2
GsMTx4 is a gating inhibitor. O-O patches were stimulated at indicated positive pressure pulses at +50 mV in the absence (Panel A) and in the presence of GsMTx4 (3.0 μM, Panel B). Panel C is a plot of the average peak current fit to a Boltzmann equation (black trace). In the absence of GsMTx4, the midpoint of the gating curve was 48.7 ±1.3 mmHg (SD). In the presence of GsMTx4 P1/2= 76.8 ±2.2 mmHg (SD) (assuming a saturation current equal to that of the control).
Figure 3
Figure 3
Equilibrium binding constant was determined by association and dissociation kinetics. The indicated pressure pulse was applied to an outside out patch for the lifetime of the experiment. After achieving steady state, a pulse of L-GsMTx4 (2.5 μM) was perfused and inhibition reflected primarily the association rate. Washout of the peptide restored channel activity, reflecting the peptide’s dissociation. Assuming the binding reaction was two states (open-blocked), we extracted the rate constants for association and dissociation from the time constants for wash-in and wash-out using the curve fitting program of QuB (curve fit shown in red, rate constants are indicated with SD). The stippled line is the baseline. GsMTx4 inhibited currents below the baseline indicating that in the “resting patch”, the channels are active, probably as a result of the adhesion energy of the membrane to the glass in the seal. Note that upon the release of the pressure pulse, there is an under shoot of current caused by a transient wrinkling of the membrane. The bowed membrane under pressure has more area than membrane at equilibrium (flat disk). The wrinkled membrane has little tension and that turns off the channels. Over ~1s the membrane reanneals to the glass and restores the resting tension (see reference (18) for a full description of this effect).
Figure 4
Figure 4
Whole cell currents inhibited by GsMTx4. The cells were indented using the protocol indicated in Panel A. L-GsMTx4 (4.0 μM, red trace) inhibited the mechanosensitive currents (compare to the black trace), and washout returned currents to the original level (Panel A, blue). Notice that GsMTx4 had no effect on the holding current showing that the channels are not active in the resting cell. Panel B, L-GsMTx4 (4.0 μM) inhibited the mechanosensitive currents by 58 ±6% (n=6, S.D.) and the D-GsMTx4 (3.0 μM) inhibited by 70±6% (n=3 S.D.). Panel C demonstrates that whole cell currents increased monotonically with the depth of indentation. Panel D shows the mean dissociation time τd = 10.0 ± 1.7 s (S.D., n=3) was estimated by fitting the recovery time upon washout to a single exponential. This time constant was comparable to that measured for O-O patches. Panel E shows the mean association time constant for 4.0 μM GsMTx4 as τa = 10.4 ± 3.0 s (SD). The rate constant, τd, gave a dissociation rate of kd=0.10 s1. However, τa was dominated by kd, and, unlike the rate constant from outside-out patches, the equilibrium constant calculation was untrustworthy.
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