A combined computational and experimental study of the interaction between the voltage-gated potassium channel Kv4.3 and its auxiliary protein KChIP

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Voltage-gated potassium (Kv) channels of the Kv4 subfamily are central regulators of neuronal and cardiac excitability. In particular, Kv4.3 plays a vital role in shaping the timing, frequency, and backpropagation of electrical signals in the brain and heart by generating fast transient currents at subthreshold membrane potentials in repetitive firing neurons. Kv4.3 function is critically modulated by auxiliary Kv channel-interacting proteins (KChIPs), leading to higher current density, modulation of channel inactivation, and faster recovery from inactivation. The loss of KChIP function may lead to severe pathological states. Previous crystallographic and functional studies focusing on the Kv4.3-KChIP1 complex have identified two main interaction interfaces between the channel and its auxiliary subunit responsible for the formation and stabilization of the protein assembly. In this work, combining molecular dynamics (MD) simulations, mutagenesis analysis, and electrophysiology measurements, a further interface between Kv4 and KChIP1, which was overlooked in past studies, is identified and its structural role well assessed. In particular, this interaction region is formed by a network of salt bridges between the H2 helix of KChIP (residues R51, R58, and E63) and the Kv4 T1 domain (residues R37, D39, E40, and R60). Results show that such a third interface is fundamental for KChIP to recover Kv4 channel stability and function in mutant subunits lacking the zinc-binding site, thus highlighting its crucial role in the formation of the KChIP-assisted tetrameric complex. Overall, this study enhances our understanding of the Kv4-KChIP1 interaction at the molecular level.