Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels

Abstract

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs+, Na+ and K+ had chord conductances in the range of 35-55 pS with the exception of Li+, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K+, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba2+ and 15 pS for Ca2+ at -80 mV and 10 pS for Mg2+ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K+ currents much like the divalents. As expected, the channel K+ conductance increased with K+ concentration saturating at ~ 45 pS and the KD of K+ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.

Fig 1. Extracellular K⁺ currents through hPIEZO1 channels.

(A) Unitary currents from hPIEZO1 cDNA transfected cells in response to pressure steps (pipette suction) applied via a cell-attached pipette containing K⁺. A linear fit of the I/V relationship yielded a slope conductance of 47 pS (n = 6). (B) Amplitude histograms of the unitary open channel current for the indicated voltages (n = 6). The calculated chord conductance was 47 pS at -80 mV and 53 pS at -100 mV. (C) I/V relationships for monovalent ions Cs⁺ (n = 3), Na⁺ (n = 6) and Li⁺ (n = 5); the linear fits were extrapolated to intersect the abscissa. K⁺ is our standard for comparison. The unitary current with Cs⁺ and Na⁺ were lower than that with K⁺. With Li⁺ as the current carrying ion the unitary current was approximately halved.

Fig 2. Permeability ratio of monovalent ions and the relationship between [K⁺] and conductance.

(A) Permeability ratios were determined by using the Goldman-Hodgkin-Katz (GHK) equation for monovalent cations. I/V relationships were obtained from outside-out patches with Cs⁺, Na⁺ or Li⁺ on the cytoplasmic side and K⁺ on the extracellular side. The reversal potentials were compensated a posteriori for the liquid junction potentials resulting from assymetric ion composition across the outside-out patch. The monovalent pipette solutions did not contain Ca²⁺ or Mg²⁺ ions. (B) Conductance as a function of K⁺ ion concentration ([K⁺]) in cell-attached patches (n = 3 patches for each concentration). The pipette solutions did not contain Ca²⁺ or Mg²⁺. The comparison across concentrations was performed for a fixed driving force of 40 mV. The conductance versus [K⁺] data were curve-fitted using the Michealis-Menten equation: ${\mathrm{\gamma }}_s=\frac{{\mathrm{\gamma }}_{\max }}{1+\frac{K_D}{[K^{+}]}}$. The junction potentials were ignored because they were within 0–2 mV. Single channel traces corresponding to 10 mM, 30 mM, 100 mM and 300 mM are shown in the inset.

Fig 3. Divalent ions traverse hPIEZO1 channels.

(A) Currents from channels activated in response to suction in a cell-attached patch with 90 mM extracellular Ba²⁺ held at -80 mV (n = 10). (B) Current from a cell (pre-exposed to cytochalasin D) showing multiple channels activated by suction with 90 mM extracellular (in the pipette) Ca²⁺ (n = 10). The inset shows a single opening from which the calculated conductance was ~15 pS at -80 mV. (B) The conductance of the channel using 90 mM extracellular Mg²⁺ was ~10 pS at -50 mV (n = 4). Correcting for the junction potentials (-7.8 mV, -8.3 mV and -9.1 mV for 90 mM Ba²⁺, Ca²⁺ and Mg²⁺, respectively) would cause a minor increase in the chord conductance.

Fig 4. K⁺ unitary current amplitude is reduced by 1 mM divalent ions.

(A) Unitary currents in the presence of 1 mM Ba²⁺ in the pipette with K⁺ saline (right). There is a leftward shift in the mean current amplitude when the concentration of Ba²⁺ is increased from 100 μM to 1 mM as shown by the amplitude histogram (at -80 mV). A similar shift in unitary current is observed with an increase in Ca²⁺ (B) and with an increase in Mg²⁺ (C). Three patches with multiple openings were analyzed for each concentration.

Fig 5. Current-Voltage (I/V) relationships with a mix of divalents and K⁺ ions.

I/V plots for mixtures of K⁺ with Ba²⁺ (A), Ca²⁺ (B) or Mg²⁺ (C) in the pipette saline of cell-attached patches. Three concentrations (squares: 10 μM, circles: 100 μM and triangles: 1 mM) were used for each divalent ion used in the mixture (D) Unitary amplitude in the presence of each divalent (i_dival) has been normalized to that of pure K⁺ current in divalent-free pipette solution (i_dival-free) at -80 mV. Student’s t-tests were performed to examine if reductions in current amplitudes were statistically significant (*: P < 0.01; n.s.: not significant). The reduction of unitary K⁺ current is greatest with Ca²⁺. The junction potentials were in the range of 0.3–0.5 mV for 1 mM Ba²⁺, Ca²⁺ or Mg²⁺ (highest concentration) included in the pipette solution.

Fig 6. Permeation of TMA and TEA in outside-out patches.

Current elicited from outside-out patches in response to 500 ms positive pressure pulses. Pipette solutions contained either 150 mM TMA or 150 mM TEA, 10 HEPES at pH 7.4. Divalent ions were not included in the pipette solution. For 150 mM TMA, the current would reverse near +45 mV, whereas with 150 mM TEA, the reversal potential would be near +8 mV (n = 3 patches) after compensating for the junction potential. The calculated junction potential was -5.0 mV for the 150 mM TMA containing pipette solution and -7.7 mV for the 150 mM TEA containing pipette solution.

Fig 7. Titration of K⁺ currents by TMA and TEA.

(A) Bar graph summarizing the effect of increasing concentrations of TMA or TEA (20 mM to 150 mM) on the unitary conductance when the main permeant ion was K⁺ in cell-attached patches. Student’s t-tests were performed to examine if reductions in current amplitudes (mean ± s.e.m) were statistically significant (*: P < 0.01; n.s.: not significant). As Fig 5 shows, both TMA and TEA can traverse the channel. The current traces shown below the bar graph (left: TMA, right: TEA) highlight the decrease in unitary current with TEA compared to TMA, suggesting TEA is the better blocker (n = 3 patches for each organic ion concentration). The junction potentials were ignored (for mixed solutions of TMA and TEA) because they were between -0.5mV and -3.7 mV for 20–150 mM TMA; for 20–150 mM TEA they were between -0.7 mV and -5.3 mV. (B) Current from outside-out patches with mixed solutions of K⁺ with either TMA or TEA (n = 3). The current is due to the permeation of both organic cations and K⁺. As expected, the amplitude of current produced with mixed solutions is lower compared to that with pure K⁺.