Structural Determinants of Closed-State Inactivation Studied with Channel Chimeras

Abstract

Shal-gene-related voltage-gated potassium (Kv4) channels exhibit a prominent low-voltage-induced closed-state inactivation. Recent experimental results show that the S4 voltage sensor drives closed-state inactivation (Dougherty et al., J Gen Physiol 131: 257-273, 2008), and that the S4S5 linker and the main S6 activation gate are instrumental in the installment of closed-state inactivation (Barghaan and Bahring, J Gen Physiol 133: 205-224, 2009). In particular, an inactivated voltage sensor conformation correlates with a temporary uncoupling between the S4S5 linker and the S6 gate. We used two different chimeric approaches to further study the structural determinants of Kv4 channel closed-state inactivation: First, chimeric swapping of S4S5 linker and distal S6 sequences between N-terminally truncated Kv4.2delta2-40 A-type channels and non-inactivating ShakerIR channels; Second, chimeric insertion of the Kv4.3 cytoplasmic C-terminus or the Kv4.3 T1S1 linker in Kv4.1. The first approach was pursued to possibly prevent inactivation in Kv4.2delta2-40 or introduce inactivation in ShakerIR. The second approach was pursued to possibly transfer slow Kv4.3 inactivation kinetics to Kv4.1. By two-electrode voltage-clamp on cRNA-injected Xenopus oocytes and kinetic analysis of the recorded currents we found that Shaker sequences slowed Kv4.2delta2-40 inactivation, and that Kv4.2 sequences introduced a novel form of inactivation in ShakerIR. Furthermore, we found that, rather than Kv4.3 C-terminal sequences, chimeric introduction of Kv4.3 T1S1 linker sequences made Kv4.1 channels inactivate slower. Our data confirm a model of temporary uncoubling between S4S5 and S6 as a mechanism involved in closed-state inactivation. Furthermore, our data suggest that the T1S1 linker region plays a role in closed-state inactivation.