Is the Activation Gate Closed in Kv Channel Closed-State Inactivation?

Abstract

Kv4.x channel complexes including auxiliary subunits such as DPP6-S and KChIP1 undergo preferential closed-state inactivation (CSI; Baehring and Covarrubias, 2011, J Physiol, 589.3:461-479; Fineberg et al., 2012, J Gen Physiol, in press). However, the molecular basis of CSI in Kv4.x channels remains poorly understood. A key unanswered question of this problem is whether the channel's activation is actually closed, as CSI implies. We have previously proposed that CSI results when the activated channel fails to open or simply prefers to close. This hypothesis specifically suggests that the intracellular activation gate may adopt a stable closed conformation, even under depolarized conditions that would initially favor the activated “up” state of the voltage sensors. To test this hypothesis, we are exploiting the trapping paradigm previously investigated by Holmgren et al. to test the state of the intracellular activation gate (1997, J Gen Physiol, 109:527-535). Essentially, if the activation gate closes behind a quaternary ammonium (QA) blocker inside the pore, the blocker cannot exit the pore; effectively, it is trapped. We are expressing the ternary Kv4.1/I400C channels (this mutation favors trapping) including DPP6-S and KChIP1 in Xenopus oocytes. Then, in the inside-out patch-clamp configuration, we are using a computer-controlled perfusion system to deliver the QA derivative decyltrimethylammonium bromide (C10) to the bath and probe the ability of the channel's activation gate to trap C10 in the closed, open and inactivated states. Preliminary results show that the trapping paradigm may yield an answer to the central question of this study. If the activation gate is indeed closed during CSI, C10 trapping will only occur when the drug is applied to the conducting channel, but no trapping of C10 will be observed in the closed and inactivated states. Supported by NIH grant R01 NS032337 (MC).