Extended Data Fig. 5: Transient effects of crosslinking cysteine pairs on in vitro NOMPC function.
From: NOMPC ion channel hinge forms a gating spring that initiates mechanosensation
a, Representative traces of spontaneous currents recorded before and at different times after application of the crosslinking agent MTS6. b, Corresponding traces of pressure-evoked currents. MTS6 transiently increases spontaneous and pressure-evoked currents of LH+LHCys-NOMPC::GFP carrying three cysteine pairs (I1161C–C1203, K1177C–N1241C, A1212C–H1249C), but not of NOMPC::GFP, LH-LH-NOMPC::GFP, and also LH+LHCys-ctrl-NOMPC::GFP, in which two of the three cysteine pairs of LH+LHCys-NOMPC::GFP (K1177C–N1241C and A1212C–H1249C) are reverted back to single cysteines (K1177C and A1212C). c, Corresponding pressure\(\,\left({\rm{P}}\right)\)-dependent open probability \({{\rm{p}}}_{{\rm{o}}}\left({\rm{P}}\right)\) before (hatched lines) and immediately after (solid lines) MTS6 application (N = 5 cells per construct). For each construct, \({{\rm{p}}}_{{\rm{o}},\ge 1}\left({\rm{P}}\right)={1-(1-{{\rm{p}}}_{{\rm{o}}}\left({\rm{P}}\right))}^{{\rm{N}}}\) (left) and \({{\rm{p}}}_{{\rm{o}}}\left({\rm{P}}\right)\) (right) are shown. MTS6 shifts \({{\rm{p}}}_{{\rm{o}}}\left({\rm{P}}\right)\) to lower pressure amplitudes for LH+LHCys-NOMPC::GFP, but not NOMPC::GFP, LH + LH-NOMPC::GFP, and LH+LHCys-ctrl-NOMPC::GFP. This narrows down the effect to modification of the two cysteine pairs that are present only in LH+LHCys-NOMPC::GFP (K1177C–N1241C and A1212C–H1249C) and that, according to our molecular dynamics (MD) simulations, change distance in response to force (Fig. 5b). At the same time, it shows that this increase arises neither from modification of the cysteine pair that is present in both constructs (I1161C–C1203, which does not change distance in our MD simulations (Fig. 5b)), nor from modification of the two single cysteines K1177C and A1212C. In principle, the effect could arise from modification of the two single cysteines N1241C and H1249C, yet this possibility seems unlikely, not only because MTS6 crosslinks cysteine pairs, but because MTS6 does not randomly impair LH+LHCys-NOMPC::GFP, but shifts its \({p}_{o}\left(P\right)\) to that of NOMPC::GFP.
