Molecular and structural bases of the neuronal A-type current

Kv4 channels mediate the somatodendritic A-type current (Isa) and are major regulators of neuronal excitability and firing frequency. Kv4.2 channels preferentially associate with two auxiliary subunits, Kv channel-interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like protein 6 (DPP6), which modulate channel voltage-dependent gating and surface expression. Significantly, DPP6 dysfunction has been associated with a myriad of human brain disorders, presenting the Kv4.2 Isa complex as a promising drug target. Here, a variety of approaches were used to gain insights into Kv4.2 and to understand the molecular basis of DPP6 modulation. A series of constructs and expression strategies were investigated to enable mammalian expression of the human Kv4.2-KChIP3-DPP6 complex. In parallel, electrophysiological characterisation of Kv4.2 C-terminal truncation mutants alone and co-assembled with DPP6 revealed the cytoplasmic portion of the channel S6 helix is important for the generation and expression of functional channels, and that the C-terminus regulates Kv4.2 functional expression, but may play a reduced role in channel gating in the presence of DPP6. Further, mutagenesis of cysteine residues C529 and C530 in the C terminus predicted to be S-palmitoylation sites reduced Kv4.2 current amplitudes supporting S palmitoylation of the C-terminus may regulate Kv4.2 surface expression. Finally, a novel study using homology modelling and mutational analysis of the DPP6 transmembrane helix was conducted to investigate key residues involved in its functional coupling with Kv4.2. This revealed two binding regions important for the modulatory effects of DPP6, but not for increased channel trafficking; and highlighted specific DPP6 residues conferring the accelerated Kv4.2 recovery from inactivation, an essential characteristic of the Isa current. Overall, insights have been gained into the structural and functional properties of Kv4.2 and how DPP6 exerts its modulatory effects on the channel, which provide important details on the pathophysiology of human brain disorders associated with mutations in the DPP6 gene and aid the design of novel Kv4.2-selective modulators.