Analysis of the State-Dependent Block of Shaker IR by bTBuA

Abstract

Slow inactivation of Shaker-related voltage-gated potassium channels is caused by closure of the inactivation gate in the selectivity filter while the intracellular activation gate (A-gate), that controls ion access to the central pore cavity, is open ([OI] gate configuration). Using internally applied benzyl-tributylammonium (bTBuA) ions and the trap-door paradigm we suggested earlier that the A-gate in Kv4.1 channels might play a novel role as the inactivation gate and being responsible for closed-state inactivation. The current study investigated whether bTBuA shows the characteristics of QA block in slow-inactivated Shaker-IR having open A-gate ([OI]), and therefore, validate the conclusions drawn for Kv4.1. Currents were recorded in excised inside-out patches expressing T449K Shaker-IR channels that displays relatively fast slow inactivation (τi=15 ms). A train of 5.0-s-long depolarizing pulses to +50 mV was applied and the peak currents were determined upon exposure of the intracellular surface of open or open-inactivated channels to 100 µM bTBuA. We found that bTBuA shows a classical open-channel block when applied intracellularly to Shaker channels having both the A-gate and the P-gate open, similarly to when applied to Kv4.1. Next bTBuA was applied to fully inactivated Shaker channels having [OI] configuration (at +50 mV). We found that the compound becomes trapped behind the activation gate upon repolarization: although excess bTBuA was washed out between two pulses, the peak current of the second pulse was greatly reduced indicating trapping of the blocker. On the contrary, bTBuA fails to block inactivated Kv4.1 channels. Our results therefore validate the use of bTBuA as a tool to prove the hypothesis that the A-gate in fully inactivated Kv4.x channels is closed. Futhermore, bTBuA can be locked into Shaker in the absence of the I470C previously shown to be required for trapping of TEA. Support: KTIA_NAP_13-2-2015-0009.