Figure 3.

Experimental strategy to investigate closed-state inactivation in T449A/V474C Shaker-IR ion channels. (A) The pulse protocol consisted of three 5-ms-long depolarizations to +50 mV (P₁, P₂, and P₃). The corresponding peak currents evoked by these pulses are represented as I₁, I₂, and I₃ in B. Between P₁ and P₂, patches were held in Cd²⁺ containing intracellular solution at −90 mV holding potential for 20 s. To avoid the Cd²⁺ exposure of the open-state channels during P₁ and P₂, 30-ms-long steps to −120 mV were applied directly after P₁ and before P₂, indicated by symbol ×. The holding potential of −120 mV was applied between P₂ and P₃ for 60 s. (B) The accessibility of cysteines at position 474 (yellow spheres) located between the activation (intracellular part of S6 helices, dark green) and C-type inactivation (red spheres) gates during inactivation was determined by applying Cd²⁺ (gray spheres) from the intracellular side. Upon binding to 474C, Cd²⁺ inhibits K⁺ currents, thus tracking precisely the incidental opening of the A-gate during the development of C-type inactivation. Model predictions for RCF are in the text; I₁, I₂, and I₃ are defined in A.