An Ankyrin-G N-terminal gate and casein kinase 2 dually regulate binding of voltage-gated sodium and KCNQ2/3 potassium channels [Cell Biology]

May 21st, 2015 by Xu, M., Cooper, E. C.

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 conductances at the distal axon initial segment (AIS) and nodes of Ranvier (NR) in a ratio of approximately 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 NRs. Here, we show that the channel anchors bind at overlapping but distinct sites near the AnkG N-terminus. In pull down assays, the rank order of AnkG binding strength is Nav1.2>>KCNQ3>KCNQ2. Phosphorylation of KCNQ2 and KCNQ3 anchor domains by casein kinase 2 (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 R47, R50) in the first repeat 1 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.
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