(A) Control recording of wild-type (WT) GluA2 receptors in response to a trapping protocol. Movements of the piezo reflecting the solution exchange are shown in thin, black lines above the current trace. Composition of the solutions is indicated in thick lines above the piezo trace. Downward ticks are 200 ms control jumps from DTT (1 mM) to DTT and glutamate (Glu, 10 mM). Four pre-trap control pulses were followed by a 1 min long trapping pulse (red line) in Glu (10 mM) and M3M (1 μM). After the trapping pulse, the patch was exposed to 10 post-trap control pulses. The first post-trap pulse gives no response because receptors are desensitized (for details, see Materials and methods). (B) Same as in (A), but for V666C mutant. The top two recordings are from the same patch: in the first trace, V666C receptors were exposed only to Glu. Trapping of the same patch in Glu and M1M (1 μM), results in pronounced peak current reduction, which partly recovered with τrecovery = 30 ± 7 s, n = 5 (grey dots; post-trap control pulses extended to 30 for fit). The bottom trace is a different patch trapped in bMTSp (1 μM), showing even stronger peak current reduction without any recovery. (C) As in (B) but for the A665C mutant. The two traces are paired recordings of the same patch. Post-trap control pulses show that A665C cross-links to itself, but most of the current recovers within several seconds after the trap (grey dots; τrecovery = 3.3 ± 0.4 s, n = 17). If the same patch is now trapped in Glu and M8M (1 μM), the peak current reduction is much more pronounced and does not recover. (D) Summary of the trapping effects for WT (black) and V666C (red) for cross-linkers M1M (7 Å) to M10M (18 Å). Trapping effect after 1 min was calculated as the ratio of the post-trap and pre-trap peak current (arrows in A-C). MTSEA is a monofunctional reagent and ‘w/o MTS’ stands for ‘without MTS’ (Glu only, pooled for all experiments). Dashed line indicates no effect. For peak current reduction in a bis-MTS vs. w/o MTS (pooled), p < 10−7, for all cross-linkers. (E) Same as in (D), but for A665C (red) in cross-linkers M3M (9 Å) to M10M (18 Å). A665C mutant in the presence and absence of an MTS cross-linker resulted in p ≤ 0.02, depending on the cross-linker. For statistics vs. WT and between different bis-MTS, see Figure 2—source data 1. (F) Trapping time for V666C receptors in M3M, M6M and M10M. The 1-min trapping protocol was repeated up to six times, resulting in a cumulative exposure of the patch to a bis-MTS of up to 6 min. The data were fit with a monoexponential for each cross-linker (τ indicated in brackets). (G) Trapping profile of desensitized V666C receptors shows the effect of each cross-linker vs. its length, in the first minute of exposure. The data were fit with a parabola (red line): f(x) = K0 + K1*(x – K2)2 (for details, see Materials and methods).