Quinidine is a commonly used antiarrhythmic agent and a tool to study ion channels. Here it is reported that quinidine equilibrates within seconds across the Sf9 plasma membrane, blocking the open pore of Shab channels from the intracellular side of the membrane in a voltage‐dependent manner with 1:1 stoichiometry. On binding to the channels, quinidine interacts with pore K⁺ions in a mutually destabilizing manner. As a result, when the channels are blocked by quinidine with the cell bathed in an external medium lacking K⁺, the Shab conductanceG_Kcollapses irreversibly, despite the presence of a physiological [K⁺] in the intracellular solution. The quinidine‐promoted collapse of ShabG_Kresembles the collapse of ShakerG_Kobserved with 0 K⁺solutions on both sides of the membrane: thus the extent ofG_Kdrop depends on the number of activating pulses applied in the presence of quinidine, but is independent of the pulse duration. Taken together the observations indicate that, as in Shaker, the quinidine‐promoted collapse of ShabG_Koccurs during deactivation of the channels, at the end of each activating pulse, with a probability of 0.1 per pulse at −80 mV. It appears that when Shab channels are open, the pore conformation able to conduct is stable in the absence of K⁺, but on deactivation this conformation collapses irreversibly.

Herein, we report the first characterization of Shab slow inactivation. Open Shab channels inactivate within seconds, with two voltage-independent time constants. Additionally, Shab presents significant closed-state inactivation. We found that with short depolarizing pulses, shorter than the slowest inactivation time constant, the resulting inactivation curve has a marked U-shape, but as pulse duration increases, approaching steady-state conditions, the U-shape vanishes, and the resulting inactivation curves converge to the classical Boltzmann h∞ curve. Regarding the mechanism of inactivation, we found that external K+ and TEA facilitate both open- and closed-state inactivation, while the cavity blocker quinidine hinders inactivation. These results together with our previous observations regarding the K+-dependent stability of the K+ conductance, suggest the novel hypothesis that inactivation of Shab channels, and possibly that of other Kv channels whose inactivation is facilitated by K+, does not involve a significant narrowing of the extracellular entry of the pore. Instead, we hypothesize that there is only a rearrangement of a more internal segment of the pore that affects the central cavity and halts K+ conduction.

Potassium channels allow the selective flux of K+ excluding the smaller, and more abundant in the extracellular solution, Na+ ions. Here we show that Shab is a typical K+ channel that excludes Na+ under bi-ionic, Nao/Ki or Nao/Rbi, conditions. However, when internal K+ is replaced by Cs+ (Nao/Csi), stable inward Na+ and outward Cs+ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca2+ ions, and compare the effect that internal K+ replacement exerts on both Ca2+ and TEA block. Our observations indicate that Ca2+ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na+ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca2+ is probably coordinated by main chain carbonyls of the pore´s first K+-binding site.