A-type voltage-gated K+ (Kv) channels self-regulate their activity by inactivating directly from the open state (open-state inactivation [OSI]) or by inactivating before they open (closed-state inactivation [CSI]). To determine the inactivation pathways, it is often necessary to apply several pulse protocols, pore blockers, single-channel recording, and kinetic modeling. However, intrinsic hurdles may preclude the standardized application of these methods. Here, we implemented a simple method inspired by earlier studies of Na+ channels to analyze macroscopic inactivation and conclusively deduce the pathways of inactivation of recombinant and native A-type Kv channels. We investigated two distinct A-type Kv channels expressed heterologously (Kv3.4 and Kv4.2 with accessory subunits) and their native counterparts in dorsal root ganglion and cerebellar granule neurons. This approach applies two conventional pulse protocols to examine inactivation induced by (a) a simple step (single-pulse inactivation) and (b) a conditioning step (double-pulse inactivation). Consistent with OSI, the rate of Kv3.4 inactivation (i.e., the negative first derivative of double-pulse inactivation) precisely superimposes on the profile of the Kv3.4 current evoked by a single pulse because the channels must open to inactivate. In contrast, the rate of Kv4.2 inactivation is asynchronous, already changing at earlier times relative to the profile of the Kv4.2 current evoked by a single pulse. Thus, Kv4.2 inactivation occurs uncoupled from channel opening, indicating CSI. Furthermore, the inactivation time constant versus voltage relation of Kv3.4 decreases monotonically with depolarization and levels off, whereas that of Kv4.2 exhibits a J-shape profile. We also manipulated the inactivation phenotype by changing the subunit composition and show how CSI and CSI combined with OSI might affect spiking properties in a full computational model of the hippocampal CA1 neuron. This work unambiguously elucidates contrasting inactivation pathways in neuronal A-type Kv channels and demonstrates how distinct pathways might impact neurophysiological activity.

Voltage-gated K+ (Kv) channel activation depends on interactions between voltage sensors and an intracellular activation gate that controls access to a central pore cavity. Here, we hypothesize that this gate is additionally responsible for closed-state inactivation (CSI) in Kv4.x channels. These Kv channels undergo CSI by a mechanism that is still poorly understood. To test the hypothesis, we deduced the state of the Kv4.1 channel intracellular gate by exploiting the trap-door paradigm of pore blockade by internally applied quaternary ammonium (QA) ions exhibiting slow blocking kinetics and high-affinity for a blocking site. We found that inactivation gating seemingly traps benzyl-tributylammonium (bTBuA) when it enters the central pore cavity in the open state. However, bTBuA fails to block inactivated Kv4.1 channels, suggesting gated access involving an internal gate. In contrast, bTBuA blockade of a Shaker Kv channel that undergoes open-state P/C-type inactivation exhibits fast onset and recovery inconsistent with bTBuA trapping. Furthermore, the inactivated Shaker Kv channel is readily blocked by bTBuA. We conclude that Kv4.1 closed-state inactivation modulates pore blockade by QA ions in a manner that depends on the state of the internal activation gate.