Positive contribution of 95C in TM1 to Cd²⁺ binding to 1141C in TM12. (A) Reversible inhibition of 95C inside-out currents by Cd²⁺. (B) After Cd²⁺-induced inhibition, 95C/1141C currents can recover at a discernable rate back to the preinhibition level by simple Cd²⁺ removal; in the same inside-out patch, current recovery is dramatically accelerated by DTT. Time constants were generated from fitting both current relaxations with a single exponential function (red and blue curves). (C) Summary of the relaxation time constants for current recovery in the presence or absence of DTT. Mean ± SEM is shown. **, P < 0.01. Here, we need to point out that, although another study drew the same conclusion that position 95 is close to position 1141 in the pore (El Hiani and Linsdell, 2014), observations made between the current study and that study are somewhat different. Specifically, in El Hiani and Linsdell (2014), both 95C and 95C/1141C currents were increased upon Cd²⁺ exposure, opposite to what we observed. The reason behind this discrepancy is unclear, but numerous differences in SCAM results have been noted before in the literature (Bai et al., 2010, 2011; El Hiani and Linsdell, 2010; Qian et al., 2011; Wang et al., 2011; Gao et al., 2013). Regardless, because the interaction between Cd²⁺ and the binding site constructed by 95C and 1141C is relatively weak judged from a relatively faster relaxation time constant, when compared with other positive pairs in the current study (e.g., 102C/341C), we wondered whether moving the cysteine at position 1141 to its neighboring positions (i.e., 1140C or 1142C) could potentially identify a tighter binding site for Cd²⁺ with the 95C background. Unfortunately, although the 95C/1142C double mutant failed to yield any current in our experiments (n > 10), 95C/1140C seems less likely to compose such a tight binding site we sought (see Fig. S8).