Cross-linking decreases Na⁺ self-inhibition. (A) Representative current traces in Xenopus oocytes (voltage clamped to −60 mV) expressing α_L120Cβγ_E455CENaC, before and after treatment with 30 mM DTT for 30 s. To quantitate Na⁺ self-inhibition, extracellular Na⁺ was transiently reduced to 1 mM, as indicated by the black bar, and then rapidly shifted back to 116 mM Na⁺. ENaC current was blocked by 10 µM amiloride (Amil, black bar). (B) Percent change in Na⁺ self-inhibition (SSI) induced by DTT in oocytes expressing αβγENaC (subunits are wild type or the indicated mutants). Mean ± SEM is shown (n = 6–7; *, P < 0.005). (C–F) Representative current traces in oocytes (voltage clamped to −60 mV) expressing α_K477Cβ_V85CγENaC before (C and E) and after (D and F) treatment with MTS-14-O4-MTS or MTS-2-MTS. The extracellular solution contained 1 mM Na⁺ (black bar) or 116 mM Na⁺ (white bar). (G) Percent change in Na⁺ self-inhibition (SSI) for cells expressing αβγ (black), α_K477Cβγ (green), αβ_V85Cγ (red), or α_K477Cβ_V85CγENaC (blue) induced by treatment with cross-linking reagents of different lengths (MTS-x-MTS, where “x” indicates the number of atoms in the linker backbone). Mean ± SEM is shown (n = 3–15; some error bars are hidden by data symbols).