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Proc Natl Acad Sci U S A. Jun 6, 2006; 103(23): 8870–8875.
Published online May 30, 2006. doi:  10.1073/pnas.0603376103
PMCID: PMC1472242
Neuroscience

Polarized axonal surface expression of neuronal KCNQ channels is mediated by multiple signals in the KCNQ2 and KCNQ3 C-terminal domains

Abstract

The M channels, important regulators of neuronal excitability, are voltage-gated potassium channels composed of KCNQ2–5 subunits. Mutations in KCNQ2 and KCNQ3 cause benign familial neonatal convulsions (BFNC), dominantly inherited epilepsy and myokymia. Crucial for their functions in controlling neuronal excitability, the M channels must be placed at specific regions of the neuronal membrane. However, the precise distribution of surface KCNQ channels is not known. Here, we show that KCNQ2/KCNQ3 channels are preferentially localized to the surface of axons both at the axonal initial segment and more distally. Whereas axonal initial segment targeting of surface KCNQ channels is mediated by ankyrin-G binding motifs of KCNQ2 and KCNQ3, sequences mediating targeting to more distal portion of the axon reside in the membrane proximal and A domains of the KCNQ2 C-terminal tail. We further show that several BFNC mutations of KCNQ2 and KCNQ3 disrupt surface expression or polarized surface distribution of KCNQ channels, thereby revealing impaired targeting of KCNQ channels to axonal surfaces as a BFNC etiology.

Keywords: axon initial segment, axon targeting, epilepsy, KCNQ potassium channel

Neuronal KCNQ channels, voltage-dependent potassium channels that activate slowly but show no inactivation, correspond to the M channels that exert crucial influence over neuronal excitability (1). Inhibition of M channels by muscarinic agonist (hence the name) and other neurotransmitters enhances action potential firing in central and autonomic neurons (2). M channels are mostly heterotetramers composed of KCNQ2 and KCNQ3 (3, 4). The overlapping expression patterns of KCNQ2 and KCNQ3 include brain areas implicated in seizure development, such as hippocampus, neocortex, and thalamus (3, 5, 6). The critical involvement of KCNQ channels in controlling neuronal excitability is underscored further by the fact that benign familial neonatal convulsions (BFNC) mutations in KCNQ2 and KCNQ3 cause epilepsy (7, 8) and myokymia (9). Consistently, retigabine, a potent KCNQ channel opener (10) can suppress seizures in a number of animal models (1113) and attenuate neuropathic pain (14, 15), whereas linopirdine and XE991, developed as “cognitive enhancer” drugs for treatment of Alzheimer disease and other memory disorders, are potent blockers of KCNQ channels (10).

KCNQ channels contribute to the neuronal resting membrane potentials (16), hippocampal theta oscillation (17, 18), spike frequency adaptation (16, 1820), spike after depolarization (16, 21), and after hyperpolarization (18, 20). Consistent with reports of the ability of KCNQ channels to modulate motor axon excitability (9, 22) and transmitter release (23), KCNQ2 and KCNQ3 proteins have been detected in axons (5, 6, 24, 25), the axonal initial segment (AIS) and nodes of Ranvier (26, 27). It remains possible, however, that the neuronal soma and dendrites express functional KCNQ channels, given the transmitter modulation of KCNQ channels (2) and strong somatodendritic KCNQ2 and KCNQ3 immunoreactivity (3, 5, 6).

The physiological functions of KCNQ channels depend critically on their surface distribution in specific regions of the neuron, such as the axon and dendrites. Because the available antibodies recognize intracellular epitopes of KCNQ2 and KCNQ3, it has not been possible to decipher from immunocytochemical studies which fractions of the total channel protein are on the surface membrane of various neuronal subdomains. By expressing KCNQ channel subunits tagged with an extracellular hemagglutinin (HA) epitope in cultured hippocampal neurons, we demonstrate preferential surface expression of KCNQ channels both at the AIS and more distally on axons. We have identified the key regions in KCNQ2 and KCNQ3 proteins that mediate their axonal targeting. We further examined the effects of BFNC mutations on surface distribution of KCNQ channels.

Results

KCNQ Channels Localize Preferentially on the Axonal Surface.

To examine surface and total expression of KCNQ channels in axons and dendrites, we expressed in cultured hippocampal neurons recombinant KCNQ proteins containing an extracellular HA tag (HA-KCNQ), whose surface expression and function in Xenopus oocytes have been demonstrated in ref. 28. Surface HA-KCNQ proteins were labeled with a mouse anti-HA antibody without permeabilization of neurons, whereas total HA-KCNQ proteins were visualized by immunostaining with a rat anti-HA antibody subsequent to neuronal permeabilization. Recapitulating the endogenous channel protein distribution (Fig. 7, which is published as supporting information on the PNAS web site), total HA-KCNQ2/KCNQ3 (Fig. 1 AC) and HA-KCNQ3/KCNQ2 (Fig. 8 AC, which is published as supporting information on the PNAS web site) channels resided in soma, dendrites, and axons. In contrast, the surface density of KCNQ channels was higher on axons than soma and dendrites, with the highest concentrations in proximal regions near the soma (Figs. 1 AC and 8 AC). In some transfected neurons, surface KCNQ channels were found on distal axons (Figs. 1D and 8D).

Fig. 1
KCNQ2 and KCNQ3 localize preferentially on the axonal surface. Hippocampal neurons (10 DIV) transfected with HA-KCNQ2 and KCNQ3 were immunostained first with mouse anti-HA antibody before permeabilization (surface HA) and then rat anti-HA antibody (total ...

To quantify the surface and total expression, we measured the background-subtracted mean intensity of surface HA and total HA fluorescence in axons and dendrites. Axonal surface expression of HA-KCNQ2/KCNQ3 (Fig. 1E) and HA-KCNQ3/KCNQ2 (Fig. 8E) was much higher than HA-KCNQ2 or HA-KCNQ3 alone despite similar levels of the total protein expression in axons and dendrites. In contrast to the robust axonal surface expression, the dendritic surface HA fluorescence intensities of neurons expressing one or both channel subunits were not statistically different from the background fluorescence intensities of untransfected neuronal dendrites (Figs. 1E and 8E).

Enrichment of Surface KCNQ Channels at the AIS.

Because endogeneous KCNQ channels are localized to the AIS (26, 27), we tested whether surface KCNQ channels are concentrated at the AIS by costaining with antibody that recognize epitopes common to voltage-gated sodium channels (Nav), which are highly concentrated at the AIS (29). Our immunostaining revealed that surface HA-KCNQ2/KCNQ3 (Fig. 2A; see also Fig. 9A, which is published as supporting information on the PNAS web site) and HA-KCNQ3/KCNQ2 channels (Figs. 2B and 9B) are enriched at but not restricted to the AIS, where they colocalized with endogeneous Nav channels. Moreover, in 75% of the transfected neurons, the first 2- to 4-μm portion of the AIS stained positive for Nav channels but not for surface HA-KCNQ channels (Fig. 2 A and B).

Fig. 2
Enrichment of KCNQ2 and KCNQ3 on the AIS surface. (A and B) The Nav channels, which localize to the AIS, show overlapping distribution with surface HA-KCNQ2/KCNQ3 (A) and HA-KCNQ3/KCNQ2 (B) in hippocampal neurons (DIV 10). (Scale bars: 20 μm.) ...

To quantify the polarized surface expression of KCNQ channels, we calculated the ratio of the mean surface fluorescence intensity for major axonal and dendritic branches. The axon/dendrite ratios for HA-KCNQ2/KCNQ3, and HA-KCNQ3/KCNQ2 were significantly larger than that for CD4 with equal distribution in axons and dendrites (Fig. 2C). To quantify the enrichment of surface KCNQ channels at the AIS, we calculated the ratio of the mean surface fluorescence intensity of the proximal axon including the AIS (0–30 μm from soma) to the distal axon (50–80 μm from soma). The AIS/distal axon ratios for surface HA-KCNQ2/KCNQ3, surface HA-KCNQ3/KCNQ2, and Nav channels were 2- to 3-fold larger than that for surface HA-Kv1.2 (Fig. 2D), which is not enriched at the AIS and is uniformly distributed along the axon.

Mutation of the Ankyrin-G Binding Motifs Reduces but Does Not Abolish Preferential Targeting of KCNQ Channels to Axonal Membranes.

Because AIS localization of endogenous KCNQ2 and KCNQ3 requires their binding to ankyrin-G (27), we next tested whether ankyrin-G binding motifs in the distal C-termini of KCNQ2 and KCNQ3 (27) are necessary for the enrichment of HA-tagged KCNQ channels at the AIS surface by replacing the critical acidic residues in these motifs with alanines (E810A/D812A of KCNQ2 and E837A/D839A of KCNQ3). Similar mutations abolish AIS retention of HA tagged-neurofascin fused to the KCNQ2 or KCNQ3 C-terminal domain (27). Mutation of ankyrin-G binding motifs of both subunits abolished the enrichment of HA-KCNQ2/KCNQ3 (Fig. 3A) and HA-KCNQ3/KCNQ2 channels (Fig. 10A, which is published as supporting information on the PNAS web site) at the AIS surface and reduced their surface AIS/distal axon ratio to 1 (Figs. 3C and 10C), confirming that these motifs are AIS-targeting signals. Interestingly, mutating these motifs in both subunits reduced but did not abolish the surface expression of the channels on the more distal axon (Figs. 3C and 10C), suggesting that additional signals exist for axon targeting.

Fig. 3
Mutation of ankyrin-G binding motifs abolishes AIS concentration of surface KCNQ channels. (A and B) Mutations of ankyrin-G binding motifs in both subunits (E810A/D812A for KCNQ2 and E837A/D839A for KCNQ3) completely abolished AIS enrichment (A) and halved ...

Fusion of KCNQ2 C-Terminal Sequences Targets CD4 to Axons and the AIS, Whereas Fusion of KCNQ3 C-Terminal Sequences Concentrates CD4 only to the AIS.

To search for axon-targeting signals, we fused the C-terminal cytoplasmic domains (C-tails) of KCNQ2 and KCNQ3 to the cytoplasmic C terminus of CD4. Compared with the uniform distribution of CD4 on axonal and dendritic surfaces (Fig. 4A), fusion of the KCNQ2 C-tail preferentially targeted CD4 to axon and enriched it at the AIS (Fig. 4A). In contrast, fusion of KCNQ3 C-tail concentrated CD4 only to the AIS without causing other changes in the more distal axonal and dendritic surface expression (Fig. 4A). The surface axon/dendrite ratio was 3.9 ± 1.3 for CD4-KCNQ2 C-tail, and 1.0 ± 0.1 for CD4-KCNQ3 C-tail (Fig. 4C), suggesting that the C-terminal domain of KCNQ2 but not KCNQ3 contains signals for axonal targeting.

Fig. 4
Fusion of KCNQ2 C-terminal tail (C-tail) targets CD4 to axonal and AIS surface, whereas fusion of KCNQ3 C-tail concentrates CD4 to the AIS. (A and B) Surface immunostaining of hippocampal neurons (DIV 10) transfected with CD4, CD4-KCNQ2 C-tail with or ...

Because axonal surface expression of heteromeric KCNQ2/KCNQ3 channels was impaired partially by mutation of ankyrin-G binding motifs of KCNQ2 and KCNQ3 (Fig. 3), we next tested whether these motifs may act as axonal targeting signals. Mutating the ankyrin-G binding motif of either KCNQ2 (E810A/D812A) or KCNQ3 (E837A/D839A) completely eliminated the enrichment of the CD4 fusion proteins on the AIS surface (Fig. 4 B and C). However, the E810A/D812A mutations of KCNQ2 had no effect on axon targeting of CD4-KCNQ2 C-tail (Fig. 4 B and C), indicating that the ankyrin-G binding motif of KCNQ2 is not required for axon targeting.

Localization of the Axonal Targeting Signals in KCNQ2.

To determine which regions in the KCNQ2 C-terminal domain are important for axon targeting, we fused CD4 to four distinct regions of the KCNQ2 C-tail (Fig. 5B): a membrane proximal region (MP; amino acids 323–500), a conserved A domain (amino acids 501–579; refs. 1 and 28), a subunit interaction domain (Sid, amino acids 580–623; ref. 30), and an ankyrin-G binding domain (AIS; amino acids 624–844; ref. 27). Fusion of the KCNQ2 C-tail lacking the AIS domain (MP+A+Sid) or fusion of the A and Sid domains (A+Sid) increased axonal surface expression of CD4 by 2- to 3-fold (Fig. 5C). Fusion of MP or A domain of KCNQ2 also targeted CD4 to axons, indicating that these domains contain axon targeting signals. In contrast, fusion of Sid domain did not target CD4 to axons (Fig. 5 A and C), indicating that interaction with the endogeneous KCNQ2 or KCNQ3 subunits is not likely the cause of the axonal surface expression observed in this study.

Fig. 5
Fusion of A domain or membrane proximal-domain of KCNQ2 targets CD4 to axonal surface. (A) Surface immunostaining of hippocampal neurons (DIV 10) transfected with CD4 fused to different regions of KCNQ2 C-tail (Upper). Camera lucida drawings (A Lower ...

Effect of BFNC Mutations on Surface Expression of KCNQ Channels.

Having established an assay and identified sequences important for surface KCNQ channel expression, we went on to test whether KCNQ channel targeting to the surface of axons and the AIS might be altered by BFNC mutations in KCNQ2 (Fig. 6A) and KCNQ3 (Fig. 6D). We introduced these mutations in the untagged KCNQ2 or KCNQ3 subunits to minimize any possible complication in channel folding induced by both HA tag and these mutations.

Fig. 6
Certain BFNC mutations disrupt polarized axonal surface expression of heteromeric KCNQ2/3 channels. (A) Schematic drawing (not to scale) of KCNQ2 with various BFNC mutations: C-terminal frameshift mutations replacing distal C-termini with completely different ...

The C-terminal frameshift mutations of KCNQ2 (P681-FS and G838-FS) caused by 1-bp deletion replace the distal C-terminal sequences with unrelated amino acid sequences starting from residue P681 (31) or G838 (32). Whereas both frameshift mutations had no effect on the total protein expression in axons, the P681-FS, but not the G838-FS, mutation abolished axonal surface expression and slightly elevated the dendritic total protein level of HA-KCNQ3/KCNQ2 (Fig. 6C).

Of the C-terminal truncation mutations of KCNQ2, the Q323X missense mutation deletes the entire KCNQ2 C-terminal domain (33), whereas the Y534X mutation caused by a 5-bp insertion at amino acid Y534 deletes the channel sequence from the middle of the conserved A domain (7). These two KCNQ2 truncation mutations abolished surface expression of HA-KCNQ3/KCNQ2, whereas Y534X, but not Q323X, mutation modestly reduced total protein expression in axons (Fig. 6C). In addition, the C-terminal missense mutation (K526N) in a conserved A domain of KCNQ2 (34) moderately reduced axonal surface expression of HA-KCNQ3/KCNQ2 (Fig. 6C) and diminished the surface axon/dendrite ratio (Fig. 6B).

Interestingly, the missense mutations in the pore and the sixth transmembrane segment of KCNQ2 (8) dramatically decreased axonal surface expression (by ≈50% for Y284C and ≈75% for A306T) and significantly reduced the surface axon/dendrite ratio but not AIS concentration (Fig. 6C). Total channel protein levels in axons and dendrites were decreased by A306T but not Y284C mutation (Fig. 6C). Finally, the BFNC pore mutation of KCNQ3 (G310V; ref. 35) caused an ≈50% reduction in axonal surface expression and total protein expression in axons and dendrites (Fig. 6E) but had no effect on the AIS localization (Fig. 6F). No significant surface expression of wild-type and mutant channels was detected in dendrites (Fig. 6 C and E).

Discussion

In this study, we show that surface KCNQ2/KCNQ3 heteromeric channels are primarily expressed in axon (Fig. 1) and enriched in AIS (Figs. 2 and and3).3). MP and A domains of KCNQ2 are important for targeting KCNQ channels or the reporter protein CD4 to the axonal surface (Fig. 5), whereas their enrichment at the AIS surface requires the ankyrin-G binding motifs in KCNQ2 and KCNQ3 (Figs. 3 and and4).4). Finally, we demonstrate that some of the BFNC mutations impair polarized surface expression of heteromeric KCNQ2/3 channels in axons and the AIS (Fig. 6).

Physiological Implications of Polarized Surface Distribution of KCNQ Channels in Axons and the AIS.

The main objective of our study was to determine surface distribution of KCNQ channels. Because there are no available antibodies that recognize the extracellular regions of these proteins, we determined surface distribution of the recombinant KCNQ2 and KCNQ3 proteins with an extracellular HA tag. HA-KCNQ channels are preferentially expressed on the surface of axons (Fig. 1), consistent with recent studies of endogenous KCNQ2 and KCNQ3 (2427). Importantly, surface HA-KCNQ channels are enriched at the AIS via their interaction with ankyrin-G (Figs. 2 and and3),3), as are endogenous KCNQ2 and KCNQ3 (26, 27). These similarities strongly support the notion that surface distribution of HA-KCNQ channels likely reflects that of endogenous channels.

The AIS is a strategic site for KCNQ channels to shape the waveform of the spike after depolarization (16, 21), modulate spike frequency adaptation (16, 1820), and control action potential firing threshold in response to neurotransmitter actions (2). Surface distribution in distal axons and presynaptic terminals may allow KCNQ channels to regulate action potential propagation along the axon (22, 26) and neurotransmitter release from the nerve terminal (23). Surface KCNQ channels in the AIS and axon also may be important for generating hippocampal theta oscillations (17, 18), which are strongly implicated in sensory-motor behavior, learning and memory, and synaptic plasticity (36). These strategic placements of KCNQ channels are likely important for their ability to prevent intrinsic bursting and epileptiform activity (16, 18), although it remains possible that some KCNQ channels reside in somatodendritic regions as suggested (21, 37). Our study further suggests that reduced KCNQ channel densities in the AIS and axon may contribute to pathological conditions such as BFNC (1), epileptic seizures (12, 13), various pain states (14, 15, 38), myokemia (9), cognitive impairment (39, 40), and, possibly, mental retardation (34).

Some BFNC Mutations Affect Surface Expression and Localization of KCNQ Channels.

Our study highlights the fact that different BFNC mutations disrupt KCNQ channel activity in different ways, by altering channel properties, surface trafficking, or targeting to the axon and AIS. Several BFNC mutations in KCNQ2 C-terminal domain impaired axonal surface expression, underscoring the importance of this cytoplasmic domain in channel folding, assembly, and axonal targeting. Certain BFNC missense mutations of KCNQ2 and KCNQ3 that cause a modest reduction in current or biophysical properties (7, 34, 41) had additional effects in reducing axonal surface expression. Because many genetic and acquired disorders result from altered channel function and impaired channel trafficking (42), it would be important to develop therapies that also correct trafficking defects.

Possible Molecular Mechanisms for Axonal Targeting of KCNQ Channels.

Our findings are compatible with two possible scenarios for targeting KCNQ channels to the surface of axons and the AIS. In the first model, KCNQ channels are directly sorted to the axonal but not somatadendritic plasma membranes, similar to the neuronal glial cell adhesion molecules (43, 44). Here, the axon-targeting signals of KCNQ2 may recruit specific proteins implicated in anterograde axonal transport of cargo along microtubules such as axonal kinesins (4547). Alternatively, the axon targeting signals of KCNQ2 may mediate selective endocytosis of surface KCNQ channels from somatodendritic regions but not axons, as proposed for Nav1.2 (48, 49) and VAMP, a presynaptic vesicle protein (43). Axonal KCNQ channels then are concentrated at the AIS and stabilized at the proximal axons through their interaction with ankyrin-G, an interaction that appears to facilitate channel targeting to distal axons. The channel sequences identified in this study to be important for axonal targeting will be useful in future studies of the axonal-targeting mechanism in both vertebrates and invertebrates, considering that ankyrin-G binding domain is exclusive to vertebrates, whereas MP and A domains are highly conserved between vertebrates and invertebrates (27).

Materials and Methods

Materials.

Reagents used include rabbit anti-KCNQ2 and KCNQ3 antibodies (kind gifts from Ed Cooper, University of Pennsylvania, Philadelphia), mouse anti-HA monoclonal antibody (Covance Research Products, Berkeley, CA), rat anti-HA-monoclonal antibody (Roche Applied Sciences), mouse anti-human CD4 antibody (Caltag), rabbit anti-MAP2 antibody (Chemicon), Alexa660-conjugated strepavidin, Alexa350- and Alexa488-conjugated secondary antibodies (Amersham Pharmacia Life Science), and Cy2, Cy5, and biotin-conjugated secondary antibodies (The Jackson Laboratory).

DNA Constructs and Mutagenesis.

Plasmids pcDNA3-KCNQ2 and -KCNQ3 with and without extracellular HA tag (HA-KCNQ2 or HA-KCNQ3) were kind gifts of Thomas Jentsch, Hamburg University, Hamburg, Germany. CD4 fusion to KCNQ2 and KCNQ3 C-tails were constructed by inserting into the C-terminal domain of CD4 between engineered NotI and XhoI sites in pcDNA3-CD4 the following PCR fragments: full C-tail (amino acids 323–844), MP+A+Sid (amino acids 323–623), MP (amino acids 323–500), A domain (amino acids 501–579), and Sid (amino acids 580–623) of KCNQ2 and full C-tail (amino acids 362–872) of KCNQ3. Mutations in the ankyrin-G binding motifs of KCNQ2 (E810A/D812A) and KCNQ3 (E837A/D839A), and BFNC mutations were generated by the QuikChange Site-Directed Mutagenesis Kit (Stratagene) as previously described in refs. 7, 8, and 3135 and verified by sequencing the entire construct. The P681-FS and G838-FS mutations of KCNQ2 were introduced to pcDNA3-KCNQ2 containing the 3’ untranslated region of KCNQ2 subcloned into SacII and XbaI.

Immunocytochemistry.

Primary hippocampal cultures from 18-day embryonic rats were prepared and transfected at 7 days in vitro (DIV) as described in ref. 50. Surface immunostaining for HA-KCNQ channels at 10 DIV was performed as described in ref. 50 with the following modifications: Surface HA-KCNQ proteins were labeled with mouse anti-HA antibody (1:500 dilution) at 4°C overnight and visualized with biotin-conjugated secondary antibodies for 2 h, followed by Alexa 660-conjugated strepavidin. Neurons were then permeabilized and incubated with rat anti-HA antibody to label total HA-KCNQ proteins (1:1,000 dilution) and rabbit anti-MAP2 antibody (1:1,000 dilution), followed by Alexa488- and Alexa350-conjugated secondary antibodies, respectively. Immunostaining of surface CD4 fusion proteins or endogenous KCNQ2 and KCNQ3 proteins with rabbit anti-KCNQ2 and KCNQ3 antibodies (1:200 dilution) was performed as described in ref. 50.

Image Acquisition and Quantification.

Fluorescence images of pyramidal neurons were acquired and the fluorescence intensity profiles of the major dendritic and axonal processes were quantified as described in ref. 50 (see Fig. 8 legend). Grayscale-inverted images and camera lucida drawing were generated in photoshop (Adobe Systems, San Jose) as described in ref. 50 (see Fig. 7 legend). Background-subtracted mean fluorescence intensity of the axon within 30 μm of the soma (AIS) or at 50–80 μm from the soma (distal axon), or dendrites up to where MAP2 staining disappears (dendrite), was measured for determination of the AIS/distal axon and axon/dendrite ratios. All fluorescence intensity quantification was reported as mean ± SEM. ANOVA and post-ANOVA Tukey's multiple comparison tests were performed to identify the statistically significant difference between groups of three or more, whereas the Student t test was performed for groups of two by using prism4 (GraphPad, San Diego).

Supplementary Material

Supporting Figures:

Acknowledgments

We thank Melissa Ehlers for technical support and Bjorn Schroeder and Xiang Qian for valuable comments on the manuscript. This work was supported by a National Institutes of Health National Research Service Award postdoctoral fellowship and National Institute of Mental Health Grant MH65334. Y.N.J. and L.Y.J. are Howard Hughes Medical Institute investigators.

Abbreviations

AIS
axonal initial segment
BFNC
benign familial neonatal convulsions
DIV
days in vitro
HA
hemagglutinin

Footnotes

Conflict of interest statement: No conflicts declared.

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