Intra- and Intersubunit Dynamic Binding in Kv4.2 Channel Closed-State Inactivation

We studied the kinetics and structural determinants of closed-state inactivation (CSI) in Kv4.2 channels, considering a multistep process and the possibility that both intra- and intersubunit dynamic binding (i.e., loss and restoration of physical contact) may occur between the S4-S5 linker, including the initial S5 segment (S4S5), and the S6 gate. We expressed Kv4.2 channels in Xenopus oocytes and measured the onset of low-voltage inactivation under two-electrode voltage clamp. Indicative of a transitory state, the onset kinetics were best described by a double-exponential function. To examine the involvement of individual S4S5 and S6 amino acid residues in dynamic binding, we studied S4S5 and S6 single alanine mutants and corresponding double mutants. Both transitory and steady-state inactivation were modified by these mutations, and we quantified the mutational effects based on apparent affinities for the respective inactivated states. Double-mutant cycle analyses revealed strong functional coupling of the S6 residues V404 and I412 to all tested S4S5 residues. To examine whether dynamic S4S5/S6 binding occurs within individual α-subunits or between neighboring α-subunits, we performed a double-mutant cycle analysis with Kv4.2 tandem-dimer constructs. The constructs carried either an S4S5/S6 double mutation in the first α-subunit and no mutation in the second (concatenated) α-subunit or an S4S5 point mutation in the first α-subunit and an S6 point mutation in the second α-subunit. Our results support the notion that CSI in Kv4.2 channels is a multistep process that involves dynamic binding both within individual α-subunits and between neighboring α-subunits.