Flow and shortcuts along the Shaker Kv channel slow inactivation gating cycle. (A) Depiction of the three-pulse protocol devised by Szanto et al. (2020) to assess whether the onset of slow inactivation in the Shaker-ΔI Kv T449A;V474C double-mutant channel at negative membrane potentials occurs through the open or closed states. The voltage and duration of each holding (H) or pulse (P) step is indicated, as is when Cd²⁺ ions (black circle) were applied (solid line above segment H2). Below each H or P segment, the probable channel states are indicated, as principally and qualitatively determined using the law of mass action along the slow inactivation gating cycle of the Shaker-ΔI Kv channel (Fig. 1 B). The red or blue gating sequences along the cycle shown below the −90-mV holding segment (H2) correspond to the onset of slow inactivation through the open or closed state, respectively. Cd²⁺-binding steps or gating transitions among Cd²⁺-bound states are indicated by black arrows. Channel gating states in which Cd²⁺ is bound are indicated by an asterisk. The magnitudes of forward and backward rate constants, determining the prevalence of each state, are qualitatively indicated by the length of each arrow. Note that the timescales of the different holding intervals are not scaled. To monitor whether Cd²⁺modification depends on the probability of the channel being open, the protocol was repeated, setting the H2 holding voltage to either −80 or −70 mV. (B) The protocol used by Szanto et al. (2020) to investigate whether the OI–CI gating transition of the Shaker-ΔI Kv T449A;V476C double-mutant channel is mandatory for recovery from slow inactivation. Locking the channel in the OI state is achieved by applying Cd²⁺ toward the end of the pulse protocol (solid line). The creation of a Cd²⁺-mediated inter-subunit metal ion cross-bridge locks the channel open (O^LI), thus preventing the OI–CI transition and providing a shortcut for flow along the slow inactivation gating cycle (see text for further details). n represents the multiple times the protocol was repeated (represented by the letter x).