Differential Nav and KCNQ2/3 channel binding to Ankyrin-G 1 An Ankyrin-G N-terminal gate and casein kinase 2 dually regulate binding of voltage-gated sodium and KCNQ2/3 potassium channels

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 -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 muchdiminished 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 -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. _______________________________________ Action potentials (APs) are the principal, defining, rapid long-distance signal in neurons (1). Although vertebrate APs consist mainly in an abrupt increase and fall in inward Na current, diverse outward K currents powerfully influence AP properties including threshold, shape, and adaptation. A recently appreciated example of such collaboration is the functional interaction of small numbers of voltage-gated KCNQ2/3 and more abundant Nav channels in action potential http://www.jbc.org/cgi/doi/10.1074/jbc.M115.638932 The latest version is at JBC Papers in Press. Published on May 21, 2015 as Manuscript M115.638932 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Jauary 2, 2018 hp://w w w .jb.org/ D ow nladed from Differential Nav and KCNQ2/3 channel binding to Ankyrin-G