Pan NC , Ma JJ , Peng HB .

	Fig. 1. Mechanosensitivity of AChRs in muscle cells. AChR activity was studied in cultured Xenopus myotomal muscle cells (a–c) and C2C12 myotubes (d–f). a Sample traces of ACh-induced single-channel currents from Xenopus muscle cells recorded with the patch-clamp method under negative pipette pressure of different magnitudes. Pipette ACh concentration = 0.2 μM, holding potential = +70 mV, cell-attached mode. b Channel activity NPo and c its difference ΔNPo under different negative pressures. For the NPo plot, values before (black), during (gray), and after (dark gray) negative pressure application are shown. Data are mean ± SEM, number of patches n = 9, 91, 46, 46, 44, 18 for negative pressures of −6, −10, −20, −30, −40, −60 mmHg, respectively. d AChR single channel currents recorded from C2C12 myotubes under different negative pressures. ACh concentration = 0.5 μM. e NPo values before (black), during (gray), and after (dark gray) negative pressure application. f ΔNPo data from C2C12 cells. Data from 20 patches at each negative pressure were pooled. Statistics: *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s paired t test). For NPo plots, comparisons were made between values obtained during and before negative pressure application
	Fig. 2. Mechanosensitivity of AChRs exogenously expressed in HEK293T cells. Cells were transfected with AChR subunits α, β, δ, and γ. a Sample traces of ACh-induced currents under different negative pressures. Included in the pipette was 0.5 μM ACh. b Channel activity NPo and c its difference ΔNPo under different negative pressure levels; the former shows values before (black), during (gray), and after (dark gray) negative pressure application; n = 26, 24, 22, 10 patches for −20, −40, −60, −80 mmHg, respectively. Symbols denoting statistical significance are the same as in Fig. 1. d Mean amplitudes of single channel currents at different negative pressures. These values were calculated from all-points amplitude histograms as shown in (e), taking the difference between the first and second peak as the mean single-channel AChR current amplitude
	Fig. 3. Analyses of AChR mechanosensitivity in HEK293T cells. a Effects of different pharmacological agents on ΔNPo. Cell-attached single-channel recording was conducted. GsMTx-4 (1 μM), a mechanosensitive channel blocker, was included in the recording pipette. Cells were incubated before recordings for 20 min in 10 mM MβCD, a cholesterol-depleting agent; 30 min in 2 μM cytochalasin D, an F-actin inhibitor; or for 2 h in 5 μM latrunculin A, another F-actin inhibitor. Number of patches recorded: 26 (control), 26 (GsMTx-4), 19 (MβCD), 29 (cytochalasin D), and 19 (latrunculin A). b–e Comparison of on-cell and inside-out recordings. Data are mean ± SEM based on n = 10 patches (for each negative pressure). The same membrane patch was first recorded on-cell and then detached from the cell for inside-out measurement with a pipette holding potential of +70 mV and 0.5 μM ACh. *p < 0.05; **p < 0.01; ***p < 0.001
	Fig. 4. Effect of LPC on AChR mechanosensitivity. AChR-expressing HEK293 cells were pretreated with LPC for 10 min before patch-clamp recording. a Channel activity NPo of AChRs before (black), during (gray), and after (dark gray) negative pressure application in control cultures; n = 16 patches. b NPo of LPC-treated AChRs; n = 16 patches. c The difference of NPo for control and LPC-treated AChRs at different negative pressure
	Fig. 5. The effect of rapsyn on AChR mechanosensitivity. HEK293T cells were transfected with cDNAs encoding AChR subunits plus one encoding rapsyn. a Sample current recording of AChRs under different pipette negative pressures in the cell-attached mode; pipette holding potential +70 mV with 0.5 μM ACh. b NPo of ACh-induced single-channel currents from rapsyn-co-expressing cells before (black), during (gray), and after (dark gray) negative pressure application. Note the reduced scale of y-axis compared to Figs. 1 and 2. N = 21, 18, 11, 6 patches for −20, −40, −60, −80 mmHg, respectively. c ΔNPo values of AChRs expressed alone, with rapsyn or with mutant rapsyn lacking the AChR-binding domain (rapsyn-M). Data from 18 to 26 patches were pooled. *p < 0.05; **p < 0.01; ***p < 0.001
	Fig. 6. Ratio of NPo under different experimental conditions. HEK293T cells expressing AChRs alone or together with rapsyn were recorded in the presence or absence of cytochalasin D (CD; 2 μM, 30 min pre-incubation). The NPo values obtained at each negative pressure were divided by the respective basal NPo. Data from more than 20 patches were pooled for each point. Student’s t test showed no significant difference between the results of AChR_CD and AChR/rapsyn_CD (p > 0.2)
	Fig. 7. A model on the regulation of AChR mechanosensitivity by the membrane and the cytoskeleton. a Channel opening under zero negative pressure. As a transmembrane molecule, AChR’s gating property can potentially be influenced by the mechanical properties of the membrane and the cytoskeleton. b Under negative pressure application through the recording pipette, the tension generated along the plane of the membrane causes increased channel activity. c Disruption of the cortical F-actin cytoskeleton by latrunculin A of cytochalasin D reduces the influence of the membrane stretch force on the receptor, leading to a decrease in channel activity. d Membrane lipid modification that reduces its stiffness such as cholesterol depletion by MβCD also reduces the stretch force experienced by the receptor and the mechanosensitivity. e Rapsyn, through its interaction with AChR subunits, anchors the receptor complex to the cytoskeleton to lessen the impact of membrane stress on its gating, thus reducing the mechanosensitivity

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