Closed-State Inactivation of a Voltage-Gated Potassium Channel Involves Inter-Subunit Interactions

Abstract

We study the structural determinants of inactivation in voltage-gated potassium (Kv) channels of the Kv4 subfamily, which exhibit preferential closed-state inactivation (CSI). There is strong evidence that dynamic coupling between the S4S5 linker (S4S5) and the main S6 activation gate (S6) plays a central role in CSI (Barghaan and Bähring, J Gen Physiol 133: 205-224, 2009). In particular, absence or loss of contact between S4S5 and S6 is thought to uncouple the activation gate from the voltage sensor. Here we examined the role of dynamic coupling between the S4S5 residue Phe 326 and the S6 residue Val 404 in CSI. We expressed Kv4.2 wild-type (wt), the single mutants Kv4.2 F326A and Kv4.2 V404A, and the double mutant Kv4.2 F326A:V404A in Xenopus oocytes and measured CSI parameters of these constructs under two-electrode voltage-clamp. Double-mutant cycle analysis revealed strong thermodynamic coupling between Phe 326 in S4S5 and Val 404 in S6 during CSI (ln Omega at −65 mV = 1.85). Next we asked whether this dynamic coupling occurs between S4S5 and S6 of individual alpha-subunits (loss of intra-subunit contact) or two neighboring alpha-subunits (loss of inter-subunit contact). To answer this question we created dimer constructs of Kv4.2 (1) with a point mutation in S6 of one subunit and a point mutation in S4S5 of the second subunit (Kv4.2 V404A_F326A), and (2) with a double point mutation in one subunit and no mutation in the second subunit (Kv4.2 F326A:V404A_wt). Notably, only the dimer Kv4.2 V404A_F326A was able to reproduce the CSI properties observed when Kv4.2 F326A:V404A monomers were expressed, whereas the dimer Kv4.2 F326A:V404A_wt resembled wild-type. Our data support the notion that CSI in Kv4.2 channels involves dynamic coupling between neighboring subunits. Supported by the Deutsche Forschungsgemeinschaft (DFG).