In many mammalian neurons, fidelity and robustness of action potential generation and conduction depends on the co-localization of voltage-gated sodium (Nav) and KCNQ2/3 potassium channel conductance at the distal axon initial segment (AIS) and nodes of Ranvier in a ratio of ∼40 to 1. Analogous "anchor" peptides within intracellular domains of vertebrate KCNQ2, KCNQ3, and Nav channel α-subunits bind Ankyrin-G (AnkG), thereby mediating concentration of those channels at AISs and nodes of Ranvier. Here, we show that the channel anchors bind at overlapping but distinct sites near the AnkG N terminus. In pulldown assays, the rank order of AnkG binding strength is Nav1.2 ≫ KCNQ3 > KCNQ2. Phosphorylation of KCNQ2 and KCNQ3 anchor domains by protein kinase CK2 (CK2) augments binding, as previously shown for Nav1.2. An AnkG fragment comprising ankyrin repeats 1 through 7 (R1-7) binds phosphorylated Nav or KCNQ anchors robustly. However, mutational analysis of R1-7 reveals differences in binding mechanisms. A smaller fragment, R1-6, exhibits much-diminished KCNQ3 binding but binds Nav1.2 well. Two lysine residues at the tip of repeat 2-3 β-hairpin (residues 105-106) are critical for Nav1.2 but not KCNQ3 channel binding. Another dibasic motif (residues Arg-47, Arg-50) in the repeat 1 front α-helix is crucial for KCNQ2/3 but not Nav1.2 binding. AnkG's alternatively spliced N terminus selectively gates access to those sites, blocking KCNQ but not Nav channel binding. These findings suggest that the 40:1 Nav:KCNQ channel conductance ratio at the distal AIS and nodes arises from the relative strength of binding to AnkG.
Keywords: M-current; ankyrin; axon; axon initial segment; evolution; node of Ranvier; potassium channel; sodium channel.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.