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. 2023 Jul;53(8):427-434.
doi: 10.1016/j.ijpara.2022.11.004. Epub 2023 Jan 4.

Use the force, fluke: Ligand-independent gating of Schistosoma mansoni ion channel TRPMPZQ

Evgeny G Chulkov  1 , Elena Isaeva  1 , Cheryl L Stucky  1 , Jonathan S Marchant  2
Affiliations

Use the force, fluke: Ligand-independent gating of Schistosoma mansoni ion channel TRPMPZQ

Evgeny G Chulkov et al. Int J Parasitol. 2023 Jul.

Abstract

The parasitic flatworm ion channel, TRPMPZQ, is a non-selective cation channel that mediates Ca2+ entry and membrane depolarization when activated by the anthelmintic drug, praziquantel (PZQ). TRPMPZQ is conserved in all platyhelminth genomes scrutinized to date, with the sensitivity of TRPMPZQ in any particular flatworm correlating with the overall sensitivity of the worm to PZQ. Conservation of this channel suggests it plays a role in flatworm physiology, but the nature of the endogenous cues that activate this channel are currently unknown. Here, we demonstrate that TRPMPZQ is activated in a ligand-independent manner by membrane stretch, with the electrophysiological signature of channel opening events being identical whether evoked by negative pressure, or by PZQ. TRPMPZQ is therefore a multimodal ion channel gated by both physical and chemical cues. The mechanosensitivity of TRPMPZQ is one route for endogenous activation of this ion channel that holds relevance for schistosome physiology given the persistent pressures and mechanical cues experienced throughout the parasite life cycle.

Keywords: Anthelmintic; Electrophysiology; Ion channel; Mechanosensitivity; Parasite.

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Figures

Fig. 1.
Fig. 1.
The Schistosoma mansoni ion channel TRPMPZQ (Sm.TRPMPZQ) is not activated by canonical Homo sapiens TRPM2 (Hs.TRPM2) or Hs.TRPM8 regulators. (A) Representative current traces of HEK293 cell co-transfected with GFP and Sm.TRPMPZQ or Nematostella vectensis TRPM2 before and after addition of adenosine diphosphate ribose (ADPR) (50 µM), Ca2+ (200 nM) and (±)-PZQ (10 µM for Sm.TRPMPZQ only). Recordings were made at room temperature at +60 mV (Sm.TRPMPZQ) or −40 mV (Nv.TRPM2) in ‘inside-out’ configuration. Pipette solution: 140 mM CsMeSO, 10 mM HEPES-CsOH, 1 mM EGTA, pH 7.4. Bath solution: 145 mM KCl, 10 mM HEPES-KOH, 1 mM EGTA, pH 7.4. Nv.TRPM2 currents were 8-pole Bessel filtered at 3 kHz and digitized at 20 kHz to resolve short lived fluctuations. (B) An oxidative agent causes channel activation in the Nv.TRPM2 expressing cells. Representative current traces of HEK293 cells co-transfected with GFP and Sm.TRPMPZQ or Nv.TRPM2 before and after addition of H2O2 (1 mM) to the recording chamber. Recordings were made at room temperature at +60 mV (Sm.TRPMPZQ) and +40 mV (Nv.TRPM2) in a ‘cell-attached’ configuration. Pipette solution: 140 mM CsMeSO, 10 mM HEPES-CsOH, 1 mM EGTA, pH 7.4. Bath solution: HBSS. Subsequent addition of (±)-PZQ (10 µM) increased transmembrane current in Sm.TRPMPZQ expressing cells. (C) Representative current traces of HEK293 cell co-transfected with GFP and Sm.TRPMPZQ in the presence or absence (‘PZQ-free’) of (±)-PZQ (10 µM) during perfusion with room temperature or chilled solution (4°C). Pipette solution: 140 mM CsMeSO, 10 mM HEPES-CsOH, 1 mM EGTA, pH 7.4. Bath solution: 145 mM KCl, 10 mM HEPES-KOH, 1 mM EGTA, pH 7.4 and 10 µM (±)-PZQ (in ‘+PZQ only’). Recordings are made in the ‘inside-out’ configuration, at +60 mV. The timescale is enlarged in the traces on the right to show single channel fluctuations.
Fig. 2.
Fig. 2.
Application of negative pressure activates the Schistosoma mansoni ion channel TRPMPZQ (Sm.TRPMPZQ) (A) ‘Whole-cell’ transmembrane current traces evoked by different pressure steps (~1 s in duration) in HEK293 cells expressing Sm.TRPMPZQ or GFP. Recordings were made at −60 mV with cells bathed in HBSS (bath solution) with a pipette solution of 140 mM CsMeSO,10 mM HEPES-CsOH, 1 mM EGTA and pH 7.4. (B) ‘Whole-cell’ transmembrane current in HEK293 cells expressing Sm.TRPMPZQ (Sm.TRPMPZQ, blue) or GFP (Ctrl, red) during the indicated pressure steps (mean ± S.E., n=3). Differences were analyzed using Tukey’s test, *P<0.05, **P<0.01, ***P<0.01.
Fig. 3.
Fig. 3.
The Schistosoma mansoni ion channel (Sm.TRPMPZQ) responds to repeated, chronic changes in pressure. (A and B) Pressure pulses repeatedly activated Sm.TRPMPZQ. Recordings were made in ‘cell-attached’ mode (HBSS, bath and pipette solutions) from HEK293 cells transfected with (A) Sm.TRPMPZQ and GFP (Sm.TRPMPZQ), or (B) GFP alone (GFP). The inset in (A) shows an expanded trace of pressure-evoked single channel Sm.TRPMPZQ activity resolved at 60 mm Hg. (C) Responses from a ‘cell-attached’ patch to pressure steps (~1 s) in 10 mm Hg increments in HEK293 cells expressing with Sm.TRPMPZQ and GFP (Sm.TRPMPZQ) or Piezo1-GFP (Piezo1). Traces at 60 mm Hg and 120 mm Hg traces are highlighted. Sm.TRPMPZQ exhibited a delayed response with few step-like fluctuations observed. In contrast, Piezo1 expressing cells showed a large response that increased in a pressure-dependent manner.
Fig. 4.
Fig. 4.
Comparison of responses of the Schistosoma mansoni ion channel, Sm.TRPMPZQ, to mechanical and chemical stimulation. (A) Representative recording from a ‘cell-attached’ patch with (top) or without (bottom) a pressure step followed by application of (±)-PZQ (10 µM) in a HEK293 cell expressing Sm.TRPMPZQ and GFP. Also shown are single channel responses to pressure (blue), (±)-PZQ (10 µM, red), and the lack of single channel fluctuations in the absence of mechanical or chemical stimulation (green). (B) Examples of single channel responses recorded under pressure-activated conditions (60 mm Hg for ~10 min) at various applied voltages. Also shown is an all-points histogram collated from analysis before, during and after pressure application. Recordings were made under the following conditions (bath solution, HBSS; pipette solution, 140 mM CsMeSO,10 mM HEPES-CsOH, 1 mM EGTA, pH 7.4). (C) Current-voltage (I-V) plot for single channel fluctuations recorded in response to membrane stretch (black) and (±)-PZQ (10 µM, red). For estimation of channel conductance, data were fitted with a linear function (adjusted R2>0.99). (D-F) Comparison of Sm.TRPMPZQ single channel when activated by membrane stretch at +60 mV (black) or by (±)-PZQ (10 µM) (red). Characteristics measured were: (D) steady-state open probability (Popen, mean±S.D., n=2657 and 5317 events for pressure and PZQ, respectively), (E) dwell time (τ, mean±S.E.) and (F) dwell time distributions for Sm.TRPMPZQ channel fluctuations under these conditions. The dwell time distributions were fitted by a single exponential function (adjusted R2>0.99) with time constants of 7.8±0.1 ms (pressure, black) and 11.0±0.1 ms (PZQ, red).
Fig. 5.
Fig. 5.
The Schistosoma mansoni ion channel TRPMPZQ (Sm.TRPMPZQ) shows chronic adaptation to membrane deformation. Representative current traces recorded in ‘cell-attached’ mode (bath solution, HBSS; pipette solution, 140 mM CsMeSO,10 mM HEPES-CsOH, 1 mM EGTA, pH 7.4) from a HEK293 cells expressing Sm.TRPMPZQ and GFP. Recordings were made in the absence of (±)-PZQ. At baseline (0 mm Hg), no signal channel activity was observed. Timings of recordings are shown after application of pressure (solid circle, 120 mm Hg), release of pressure, then reapplication of the same pressure stimulus.
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