Abstract
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.
Highlights
Voltage-gated K (Kv) channels are quintessential regulators of excitability in brain, heart and muscle
Clay Armstrong used quaternary ammonium (QA) ions to deduce the intracellular location of a critical gate in squid Kv channels[7,8]. He demonstrated that quaternary ammonium (QA) ions occlude the pore at a deep site and are subsequently trapped in a pore cavity by the closing of an intracellular gate
A Shaker Kv channel lacking N-type inactivation (Shaker-IR) but capable of undergoing P/C-type inactivation at the external selectivity filter might exhibit blockade by internally applied QA ions but would not be able to trap them by inactivation
Summary
Voltage-gated K (Kv) channels are quintessential regulators of excitability in brain, heart and muscle. Clay Armstrong used quaternary ammonium (QA) ions to deduce the intracellular location of a critical gate in squid Kv channels[7,8] He demonstrated that quaternary ammonium (QA) ions occlude the pore at a deep site and are subsequently trapped in a pore cavity by the closing of an intracellular gate (trap-door paradigm). The equivalent mutations in the Kv1.4 channel (a close mammalian relative of the Drosophila Shaker) only slowed pore closing without affecting inactivation[24] These findings suggest that distinct interactions control classical inactivation and CSI in Kv1.4 and Kv4.1 channels, respectively[21]. We exploited the trap-door paradigm for QA ions to investigate a possible novel role of the intracellular activation gate acting as inactivation gate in the Kv4.1 channel with a full complement of accessory subunits[21]
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