A common ankyrin-G-based mechanism retains KCNQ and NaV channels at electrically active domains of the axon

Abstract

KCNQ (KV7) potassium channels underlie subthreshold M-currents that stabilize the neuronal resting potential and prevent repetitive firing of action potentials. Here, antibodies against four different KCNQ2 and KCNQ3 polypeptide epitopes show these subunits concentrated at the axonal initial segment (AIS) and node of Ranvier. AIS concentration of KCNQ2 and KCNQ3, like that of voltage-gated sodium (NaV) channels, is abolished in ankyrin-G knock-out mice. A short motif, common to KCNQ2 and KCNQ3, mediates both in vivo ankyrin-G interaction and retention of the subunits at the AIS. This KCNQ2/KCNQ3 motif is nearly identical to the sequence on NaV alpha subunits that serves these functions. All identified NaV and KCNQ genes of worms, insects, and molluscs lack the ankyrin-G binding motif. In contrast, vertebrate orthologs of NaV alpha subunits, KCNQ2, and KCNQ3 (including from bony fish, birds, and mammals) all possess the motif. Thus, concerted ankyrin-G interaction with KCNQ and NaV channels appears to have arisen through convergent molecular evolution, after the division between invertebrate and vertebrate lineages, but before the appearance of the last common jawed vertebrate ancestor. This includes the historical period when myelin also evolved.

Figure 1.

KCNQ2, KCNQ3, and Na_V channels are concentrated at the node of Ranvier and AIS. A, Teased sciatic nerve fibers show staining for KCNQ3 at nodes of Ranvier. The KCNQ3-C immunostain (red) is located in patches, flanked by staining for Kv1.2 (green), a marker of the juxtaparanodes. B, Superimposed DIC and immunofluorescence images show several teased sciatic nerve fibers, including two nodes of Ranvier (dashed boxed regions in main image). As indicated, the Kv1.2 antibody (green in main image, bottom panels in insets) labels both juxtaparanodes (jpn) strongly. KCNQ3-C antibodies (red in main image, top panels in insets) label the upper node strongly but show only a tiny area of weak staining at the lower node. C, Single teased fiber showing colocalized KCNQ3-C (red) and Na_V (green) staining at a node (n). The paranode (p) is partially demyelinated, as seen in the DIC image. D, Longitudinal optical z-section through the center of a single sciatic nerve fiber stained using KCNQ2-C (green) and KCNQ3-C (red) antibodies, obtained by wide-field deconvolution microscopy (see Materials and Methods). Both antibodies show strong colabeling at the membrane of node of Ranvier, but only KCNQ3-C shows light additional staining of myelin. The bottom panels show the nodal region at higher magnification. E, Strong colabeling at AIS of a large motoneuron in the spinal cord ventral horn by KCNQ2-C (red) and KCNQ3-C (green). F, A Purkinje cell AIS is intensely colabeled by Na_V (red) and KCNQ3-C (green) antibodies. KCNQ3-C also stains the nucleus of this cell type (pc nuc). Cerebellar cortical layers (mol, molecular; pc, Purkinje cell; gc, granule cell) are indicated. G, Colabeling of cerebellar white matter nodes of Ranvier by Na_V (red) and KCNQ3-C (green) antibodies, one with ring appearance. H, The diagram at the left shows the arrangement of somata, dendrites, and axons in hippocampal area CA1. Pyramidal cells, with somata clustered in the stratum pyrimidale (pyr) and apical dendrites (den) in the stratum radiatum (rad), give off axons (green) that enter stratum oriens (ori), acquire myelin, and travel to the subiculum within the alveus (alv). The color micrographs show that CA1 AISs are labeled by KCNQ2-N and KCNQ3b-N antibodies (green), as indicated. Grayscale insets show colabeling of AISs in boxed regions by Na_V antibodies. I, Colabeling of CA1 pyramidal cell AISs by KCNQ2-C (green) and Na_V antibodies (red). J, Colabeling of AISs of cortical layer 5 neurons by KCNQ3-C (green) and Na_V antibodies (red). In these neurons, Na_V staining begins more proximally on the axon than does KCNQ3-C (arrows), as previously shown for KCNQ2 (Devaux et al., 2004). Scale bars: A, 10 μm; B, main image, 5 μm; inset, 3 μm; C, 5 μm; D, top image, 3 μm; bottom images, 1 μm; E, 10 μm; F, 5 μm; G, 2.5 μm; H, 20 μm; I, J, 10 μm.

Figure 2.

KCNQ2 and KCNQ3 polypeptides share a homologous domain near their C termini, harboring a candidate ankyrin-G binding motif. A, Topology map of KCNQ2. Transmembrane domains, the subunit interaction domain (SID), and sites for interaction with PIP2, calmodulin (CM), and A-kinase anchoring protein 79/150 (AKAP79/150) are indicated in the proximal portion of the large C-terminal intracellular portion of the polypeptide. The C3 domain (boxed) is at the distal C-terminal region. B, Alignment of functional domains of five human KCNQ subunits, with residue number positions of transmembrane (6 TM), and highly conserved C-terminal regions (C1, C2/SID, and C3) regions indicated. Only KCNQ2 and KCNQ3 have C3 domains. C, The C3 domains contain a putative ankyrin-G binding loop motif. Clustal alignment of KCNQ2, KCNQ3, and KCNQ5 shows high homology of KCNQ2 and KCNQ3 within the C3 domain. Within C3, the arrows identify the beginning and end of the sequence with high homology to the previously identified Na_V channel ankyrin-G binding motif. At positions within the alignment where two or more residues are identical, the background is shown as black. At positions where two or more sequences show conservative substitutions, the background is shown as light gray.

Figure 3.

Concentration of KCNQ2, KCNQ3, and Na_V channels at AISs of cerebellar cortical neurons requires ankyrin-G. A, Cerebellum of wild-type mice shows intense staining of AISs and nodes using antibodies against Na_V channels (i, red in iii and iv), KCNQ2 (ii, green in iii), or KCNQ3 (green in iv). In iii and iv, nuclei are counterstained with the DNA-binding dye DAPI (blue). AISs of stellate cells (single-headed arrow), basket cells (double-headed arrows), Purkinje cells (triple-headed arrows), and granule cells (four-headed arrows) exhibit distinctive morphologies, and are all colabeled by the three antibodies. In white matter, nodes (arrowheads) are also labeled. KCNQ3-C (iv) but not KCNQ2-C (ii) (also, KCNQ3b-N, but not KCNQ2-N, not shown), labels ∼5 μm² structures in the molecular layer only. The location, appearance, and distribution of these structures suggest they may correspond to cerebellar glomeruli (Glom). B, KCNQ2, KCNQ3, and Na_V channel targeting to AISs is absent in ankyrin-G mutant mice. No stellate, basket, Purkinje, or granule cell AISs are seen. Rare AISs are seen in the granule cell layer (circles). These are considerably larger and thicker than granule cell AISs. In white matter, some nodes still stain for all markers; in granule cell layer, putative mossy fiber presynaptic termini also stain for KCNQ3. ML, Molecular layer; PC, Purkinje cell layer; GC, granule cell layer; WM, white matter. Scale bar, 20 μm.

Figure 4.

The KCNQ2 and KCNQ3 C3 domain motifs confer ability to bind ankyrin-G. The subcellular distribution of AnkG-MB-GFP fusion protein was detected in cells expressing AnkG-MB-GFP only (A), or coexpressed with HA-NF (B), HA-NF with C-terminal truncation eliminating intracellular ankyrin-G binding site (HA-NF-del) (C), NF-KCNQ2 C3 domain fusion (HA-NF-Q2C) (D), NF-KCNQ2 C3 domain fusion with C3 motif ESD mutated to AAA [HA-NF-Q2C(AAA)], NF-KCNQ3 C3 domain fusion (HA-NF-Q3C), NF-KCNQ3 C3 domain fusion with C3 motif ETD mutated to AAA [HA-NF-Q3C(AAA)]. i, Representative optical sections, obtained by wide-field deconvolution immunofluorescence microscopy, showing distribution of HA-tagged constructs (red) and AnkG-MB-GFP (green). ii, Intensity histograms for images in i. Histograms are shown partially overlapping to facilitate comparison of distribution of the two fluorophores. HA-NF, HA-NF-Q2C3, and HA-NF-Q3C3 strongly redistributed AnkG-MB-GFP to the cell surface, but constructs with truncation of NF or mutation of the C3 domains lack this ability. iii, Counts of cells with and without surface-redistributed GFP fluorescence. In each trial, 100 cells/condition were classified visually as possessing or lacking a detectable cytoplasmic clearing and surface redistribution, or edge. Bar graphs indicate average (±SEM) of three experiments. cyto, Cytoplasm; nuc, nucleus. Scale bar, 10 μm.

Figure 5.

Functional KCNQ2 and KCNQ2/KCNQ3 tetramers interact with ankyrin-G. A, B, FRAP measurement of AnkG-MB-GFP mobility. AnkG-MB-GFP fluorescence in a bleached region recovers quickly when it is expressed alone or coexpressed with KCNQ2/KCNQ3 channels with mutated C3 motifs, but more slowly when coexpressed with either wild-type heteromeric or KCNQ2 homomeric channels. A, Images of GFP fluorescence during the FRAP experiments. The arrows show the bleached region (see Materials and Methods) at indicated times before or after exposure to a high-intensity bleaching light. B, Averaged recovery time courses and bar graph showing immobile fraction for each transection condition (±SEM). C, Western blots show that mutant and wild-type channels are expressed at equal protein levels. Top, Representative blots; bottom, average densitometry results (±SEM). Scale bar, 2.5 μm.

Figure 6.

Expression of ankyrin-G has very small effects on coexpressed KCNQ2/KCNQ3 channels. A, Representative current traces in response to pulse protocols (shown in insets) used for kinetic analysis: i, deactivation protocol; ii, activation protocol. B, Analysis of voltage gating of wild-type KCNQ2/KCNQ3 heteromeric channels, C3 motif mutants, and wild-type channels coexpressed with ankyrin-G-GFP: i, deactivation time constants; ii, conductance/voltage curves; iii, activation time constants. The kinetics of the three conditions is very similar, with some evidence for slight slowing of activation and deactivation gating in the presence of ankyrin-G. Steady-state voltage dependence of gating of wild-type and mutant are indistinguishable, but ankyrin-G confers a ∼6 mV shift toward depolarized voltages.

Figure 7.

The C3 domain mediates retention at AISs of cultured hippocampal neurons. Hippocampal neurons were dissociated at E18, transfected with the indicated HA-tagged constructs, and immunostained with anti-HA and anti-MAP2 antibodies to determine the subcellular localization of surface HA-tagged proteins. The micrographs show anti-HA (left), anti-MAP2 (center), and merged images (right). Histograms (far right) show the intensity of HA (red) and MAP2 (green) immunofluorescence along the line shown in white in the merged image panels (expressed as a percentage of saturating intensity). A, Neuron transfected with HA-NF-Q2C at 8 d in vitro, stained at 15 d in vitro. HA-NF-Q2C is nonspecifically localized at axonal and somatodendritic membranes. B, Neuron electroporated with HA-NF at E18, and then immunostained after 22 d in vitro. HA-NF is selectively retained at the AIS. C, D, Neurons electroporated with HA-NF-Q2C or HA-NF-Q2C(AAA) at E18, and then immunostained after 17 d in vitro. E, F, Neurons electroporated with HA-NF-Q3C or HA-NF-Q3C(AAA) at E18, and then immunostained after 15 d in vitro. Neurons transfected with constructs including the intact C3 domains from KCNQ2 (C) or KCNQ3 (E) exhibit strong labeling restricted to the proximal axon; constructs with the mutated C3 domain motif show reduced and nonpolarized distribution of fusion protein expression (D, F). Ax, Axon; Den, dendrite; AIS, axon initial segment; hil, axon hillock. Scale bars, 10 μm.

Figure 8.

The ankyrin-G binding motifs of Na_V channel α subunits and KCNQ2/KCNQ3 subunits appear first in lower vertebrates, apparently reflecting a process of convergent molecular evolution. A, Clustal alignment was performed on full-length predicted amino acid sequences of invertebrates and vertebrate Na_V α subunits. The positions corresponding to the ankyrin-G binding motifs and the conserved DIII–DIV intracellular linker mediating fast inactivation are shown. Ankyrin-G binding motifs are absent from invertebrates, but are present in teleost fish and mammals. B, Alignments made, as in A, but of KCNQ channel subunits of invertebrates and vertebrates. A portion of the highly conserved KCNQ C2 domain, mediating subunit association, and the ankyrin-G binding motif are shown. The ankyrin-G binding motif is absent from invertebrate genes but is present in KCNQ2 and KCNQ3 of teleost fish, birds, and mammals. For genes with accession numbers AAN63887, CAA11526, CAF90163, and AAV34442, we assigned KCNQ family identity based on high homology with known KCNQ genes identified by BLASTp analysis. For A and B, species name and NCBI protein database accession numbers are indicated in parentheses. C, Phylogenetic tree of Aplysia Na_V and representative KCNQ channels. The analysis confirms the very distant evolutionary relationship between Na_V and KCNQ channels and illustrates the close paralogous relationship between KCNQ2 and KCNQ3. This suggests that the appearance of the similar ankyrin-G binding motifs in Na_V and KCNQ2/KCNQ3 genes took place independently, before the last common ancestor of jawed vertebrates. Scale bar, absolute number of changed residues. D, Diagram showing proposed interactions between Na_V channels, KCNQ channels, ankyrin-G, and the actin–βIV spectrin cortical cytoskeleton at AISs and nodes. For simplicity, other known membrane proteins of nodes and AISs (neurofascin and relate cell adhesion molecules; Na_V channel β subunits) are not depicted. The stoichiometry of interactions between ankyrin-G and membrane proteins is unknown.