Mechanosensitivity of nicotinic receptors

Abstract

Nicotinic acetylcholine receptors (nAChRs) are heteropentameric ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junction (NMJ) and other peripheral and central synapses. At the NMJ, acetylcholine receptors (AChRs) are constantly exposed to mechanical stress resulting from muscle contraction. It is therefore of interest to understand if their function is influenced by mechanical stimuli. In this study, patch-clamp recordings showed that AChR channel activity was enhanced upon membrane stretching in both cultured Xenopus muscle cells and C2C12 myotubes. To examine how this property is physiologically regulated, effects of membrane-intrinsic and membrane-extrinsic factors on AChRs expressed in HEK293T cells were studied. As in muscle cells, AChR single channel currents recorded under cell-attached configuration were significantly increased-without change in current amplitude-when negative pressure was applied through the patch pipette. GsMTx-4, a peptide toxin that blocks mechanically activated cation channels, inhibited this effect on AChRs. The mechanosensitivity decreased when cells were treated with MβCD, latrunculin A or cytochalasin D, but increased when exposed to lysophosphatidylcholine, indicating contributions from both membrane lipids and the cytoskeleton. Rapsyn, which binds to AChRs and mediates their cytoskeletal interaction in muscle, suppressed AChR mechanosensitivity when co-expressed in HEK293T cells, but this influence of rapsyn was impaired following the deletion of rapsyn's AChR-binding domain or upon cytoskeletal disruption by cytochalasin D. These results suggest a mechanism for regulating AChR's mechanosensitivity through its cytoskeletal linkage via rapsyn, which may serve to protect the receptors and sarcolemmal integrity under high mechanical stress encountered by the NMJ.

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