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Proc Natl Acad Sci U S A. 2008 Jun 3; 105(22): 7869–7874.
Published online 2008 May 30. doi:  10.1073/pnas.0802805105
PMCID: PMC2408483

Functional significance of axonal Kv7 channels in hippocampal pyramidal neurons


Members of the Kv7 family (Kv7.2–Kv7.5) generate a subthreshold K+ current, the M− current. This regulates the excitability of many peripheral and central neurons. Recent evidence shows that Kv7.2 and Kv7.3 subunits are targeted to the axon initial segment of hippocampal neurons by association with ankyrin G. Further, spontaneous mutations in these subunits that impair axonal targeting cause human neonatal epilepsy. However, the precise functional significance of their axonal location is unknown. Using electrophysiological techniques together with a peptide that selectively disrupts axonal Kv7 targeting (ankyrin G-binding peptide, or ABP) and other pharmacological tools, we show that axonal Kv7 channels are critically and uniquely required for determining the inherent spontaneous firing of hippocampal CA1 pyramids, independently of alterations in synaptic activity. This action was primarily because of modulation of action potential threshold and resting membrane potential (RMP), amplified by control of intrinsic axosomatic membrane properties. Computer simulations verified these data when the axonal Kv7 density was three to five times that at the soma. The increased firing caused by axosomatic Kv7 channel block backpropagated into distal dendrites affecting their activity, despite these structures having fewer functional Kv7 channels. These results indicate that axonal Kv7 channels, by controlling axonal RMP and action potential threshold, are fundamental for regulating the inherent firing properties of CA1 hippocampal neurons.

Keywords: axon initial segment, CA1 pyramidal neurons, M-current, KCNQ channels

Neuronal Kv7 (KCNQ) channels form a noninactivating K+ current (also known as the M− current); this turns on at subthreshold potentials and regulates the excitability of a variety of peripheral and central neurons (13). Recent immunohistochemical evidence has shown that the principal subunits forming native M channels, Kv7.2 and Kv7.3 (3, 4), are concentrated at the axon initial segment (AIS) and nodes of Ranvier of central and peripheral principal neurons (59), where they colocalize with Na+ channels. Like Na+ channels, they contain an ankyrin G-binding motif that targets them to the AIS (5, 8). They are also expressed at lower densities at the soma and possibly dendrites and synaptic terminals (4, 6, 7, 10, 11).

Spontaneous mutations in Kv7 subunits cause epilepsy in humans (2) and mice (12). The hippocampus is strongly implicated in epilepsy (13) and accordingly, previous somatic recordings from these neurons have indicated that the Kv7 current is involved in determining several aspects of neuronal excitability, including the resting membrane potential (RMP), spike frequency adaptation, and burst suppression (e.g., refs. 1416). However, the specific contribution made by Kv7 channels in the AIS to these or other manifestations of excitability has not been determined. This is important, because some human epileptogenic mutations impair axonal Kv7 subunit expression (7).

We have used selective pharmacological and molecular tools coupled with computer modeling to understand the involvement of axonal Kv7 channels in shaping neuronal intrinsic activity in hippocampal CA1 neurons. We show that a high axonal Kv7 channel concentration is essential for regulating action potential threshold and RMP, thereby suppressing inherent spontaneous activity of individual neurons. Further, we show that, although there are few dendritic Kv7 channels, the enhanced numbers of action potentials caused by perisomatic Kv7 channel inhibition effectively back-propagated into distal dendrites, thereby increasing their activity. These results provide further insights into the underlying mechanisms responsible for the physiological and pathophysiological effects after Kv7 channel modulation.


Kv7 Channel Inhibition Induces Spontaneous Action Potential Firing by Modulating Intrinsic Membrane Properties.

To assess the overall contribution made by Kv7 channels to hippocampal intrinsic excitability, we first suppressed all Kv7 currents in hippocampal CA1 neurons by bath-applying the inhibitors, XE991, and linopirdine onto hippocampal slices. Because we wanted to determine how alterations in Kv7 channel activity specifically affected intrinsic neuronal activity, we added glutamate and GABA receptor blockers to the external solution (see Methods). Under these conditions, the soma had a RMP of −67.0 ± 0.4 mV (n = 39). Consistent with many previous studies involving peripheral and central neuronal subtypes (e.g., refs. 1418), 3 μM XE991 and 10 μM linopirdine substantially reduced somatic RMP by 8.7 ± 0.9 mV (n = 9, P < 0.05, Fig. 1 A and B) and 9.2 ± 1.8 mV (n = 6, P < 0.05), respectively, and considerably increased the number of action potentials produced by depolarizing steps [Fig. 1A; supporting information (SI) Fig. S1]. These effects occurred over a period of 20 min and did not reverse up to 45 min after washout.

Fig. 1.
Effects of inhibiting Kv7 channel activity on cell excitability All records are somatic recordings from the same hippocampal CA1 pyramidal neuron. (A i and ii) Examples of membrane voltage responses to current injections before and after adding the Kv7 ...

Surprisingly, most neurons exhibited spontaneous action potentials in the presence of XE991 (13 of 15 neurons; Fig. 1Bii) or linopirdine (5 of 10 cells), respectively. The stronger effect of XE991 probably reflects its greater potency as a Kv7 channel inhibitor (IC50s for XE991 and linopirdine = 0.2–0.5 and 2.0–7.0 μM, respectively; refs. 4 and 19). In most cells, spontaneous firing was fairly regular, but in two neurons, bursting occurred. The initial frequency ranged from 0.2 to 10 Hz, often increasing to >30 Hz with time (Fig. 1). Further, the spiking was suppressed when the RMP was hyperpolarized to the original RMP, indicating that depolarization by Kv7 inhibitors contributed to the generation of unprompted firing. Importantly, spontaneous spiking persisted in the absence of GABA blockers (n = 3, data not shown), showing this was not because of reduced synaptic inhibition.

To further check that this spontaneous activity was generated postsynaptically, we abolished synaptic transmission within the slice by adding the nonspecific Ca2+ channel blocker, Cd2+ (200 μM), after XE991 application (Fig. 1Biii). XE991-induced spiking persisted in the Cd2+ solution (n = 5; Fig. 1Biii), with no further change in RMP (n = 5, Fig. 1Biii). The main effect of Cd2+ was to switch regular firing to bursting in all neurons tested (e.g., Fig. 1B), probably through block of postsynaptic Ca2+ channels and Ca2+-activated conductances (20, 21). The persistent Na+ current (22) did not contribute to the depolarization after Kv7 channel block, because the Na+ channel blocker, tetrodotoxin (1 μM), did not alter RMP after XE991 (n = 5, Fig. 1Biv). Altogether, the above results show that axosomatic Kv7 channels are vital for preventing spontaneous firing of CA1 pyramidal neurons, and that this property resides in their control of intrinsic membrane properties.

Ankyrin-Binding Inhibition Results in Spontaneous Action Potential Firing.

We next asked what the specific contribution of AIS Kv7 channels was to the suppression of spontaneous firing. For this, we designed a peptide (referred to ankyrin G-binding peptide, or ABP) that had an identical amino acid sequence (YIAEGESDTD) to the Kv7.2/Kv7.3 ankyrin G-binding motif, which plays a significant role in axonal targeting (5, 79). A BLAST (National Institutes of Health Database) search using Swiss-Prot also revealed that this binding motif is unique to Kv7.2/Kv7.3 subunits. Because expression of a construct containing this peptide sequence has been shown to bind to ankyrin G (7), we hypothesized that ABP might compete with Kv7.2/Kv7.3 subunits for binding to ankyrin G, thereby perhaps dissociating the channels from the axonal membrane.

To determine the effects of disrupting axonal Kv7–ankyrin binding in individual neurons without confounding changes in network activity, we initially made whole-cell somatic recordings in the presence of glutamatergic and GABAergic antagonists with ABP included in the internal pipette solution. [Because the AIS is <50 μm from the cell body (23), changes in AIS excitability can be monitored at the soma.] ABP delivered in this manner diffused throughout the cell (Fig. S2). With 10 mM ABP, the RMP initially remained steady at −66.8 ± 0.8 mV (n = 10) for at least 5 min, after which the neuron gradually depolarized to −61.9 ± 0.6 mV (n = 10, P < 0.01) >20 min (Fig. 2 A and C), reaching steady state thereafter. Following a parallel time course, action potential firing in response to depolarizing current steps also increased steadily (Fig. 2 A i and ii and D). This persisted even when the RMP was restored to that seen when the cell was first patched onto (Fig. 2D). Spontaneous spiking also occurred in 7 of 10 neurons tested (Fig. 2C i and ii), albeit at a much lower frequency (≈0.01 Hz) than that observed when all Kv7 channels were blocked with XE991 (Fig. 1Bii). Bath application of XE991 (3 μM) subsequent to ABP maximal effect further depolarized the cells (Fig. 2 A and C) and accelerated the spiking frequency to >10 Hz (Fig. 2Biii). Concentrations of ABP <10 mM also enhanced depolarization-induced action potential firing but did not induce spontaneous spiking, presumably because the amount was too low to disrupt enough binding of Kv7 channels to ankyrin G. It was difficult to patch cells with >10 mM peptide in the patch pipette.

Fig. 2.
Effects of selective disruption of axonal Kv7 channel function on intrinsic cell excitability. (A) Effects of including ABP (YIAEGESDTD) in the internal solution on RMP and action potential numbers measured by applying 400-ms current steps from −150 ...

The peptide targeted Kv7 channels, because ABP had no effect on cell excitability if neurons were pretreated with 3 μM XE991 (Fig. S3). It also appeared to have no effect on Na+ channels, because spike height and width were unchanged (see below). Further, inclusion of a scrambled version of ABP (sABP, 10 mM; Fig. S3) in the internal pipette solution had no effect on RMP or action potential firing.

Somatic Input Resistance Is Unaffected by ABP.

Although ABP depolarized the cell, this alone was not the cause of spontaneous action potential firing, because in control neurons, a comparable depolarization (≈5 mV) had no such effect. Hence, another membrane property must have changed. Because variations in membrane resistance can also affect cell excitability (e.g., ref. 24), we assessed the effect of ABP on input resistance, RN. No change in RN occurred with 10 mM ABP included in the pipette solution, whereas subsequent addition of 3 μM XE991 produced a substantial increase in RN (Fig. S4A), as in control cells with no intrapipette ABP (Fig. S4B). In support of this, whole-cell voltage–clamp recordings showed that the somatic Kv7 (M−) current was unaffected after 30-min dialysis with ABP (% inhibition = 9.2 ± 5.9%, n = 3), measured by applying a step potential from −20 mV to −50 mV (4). These results indicate that ABP did not affect somatic Kv7 channels.

Axonal Kv7 Channels Critically Regulate the Action Potential Threshold.

In central neurons, the AIS is the preferential site for action potential generation (23), raising the possibility that axonal Kv7 channels may play a significant role in controlling action potential production or kinetics. We tested this by evoking single spikes (Fig. 3A). With 10 mM ABP in the internal pipette solution, the spike threshold (ST) was lowered by 7.8 ± 0.8 mV (n = 10, P < 0.05, Fig. 3B) over a period of 25 min, remaining steady thereafter (Fig. 3C). Further, a smaller current injection was required to elicit a single action potential after ABP dialysis than at the beginning of the recording (data not shown). Subsequent application of XE991 then had no further effect (ST difference before and after XE991 = 0.7 ± 0.7 mV, n = 4; Fig. 3A). ABP had indeed targeted Kv7 channels, because it had no effect if neurons were pretreated with 3 μM XE991 (ST change = −2.0 ± 1.2 mV, n = 4). (Without ABP, XE991 reduced ST by 3.4 ± 0.7 mV, n = 6, P < 0.05.) The scrambled peptide, sABP (10 mM), had no effect on ST (change over 45 min = −0.2 ± 1.5 mV, n = 5). ABP, sABP, and XE991 had no effect on action potential height or half width or the overall rate of rise, indicating that Na+ and K+ channels involved in spike repolarization were unaffected. These results indicate that axonal Kv7 channels play a major role in determining action potential threshold.

Fig. 3.
Effects of reducing axonal Kv7 channel function on action potential threshold. (A) Superimposed examples of single action potentials 5 (black trace) and 25 min (blue trace) postpatching with ABP included in the patch-pipette solution. Each action potential ...

Immunohistochemical Kv7 Subunit Distribution Appears Unaltered by ABP Treatment.

In CA1, intense immunolabeling for ankyrin-G, Kv7.2, and Kv7.3 subunits is detectable in pyramidal cell AIS (5, 6, 8). We assumed that the ABP disrupts ankyrin-G binding of Kv7.2/Kv7.3 channels and wondered whether this might cause subcellular redistribution of Kv7.2 and Kv7.3. We tested this using a membrane-targeted palmitoylated congener of the peptide (25). This replicated the electrophysiological effects of ABP delivered intracellularly, whereas a lipidated sABP (N-palmitoyl sABP) did not (Fig. S5). High densities of Kv7.2 and Kv7.3 were found in stained CA1 sections that were untreated or treated with N-palmitoyl sABP (as observed; see refs. 56 and 9). The density of these did not appear to be changed after treatment with the N-palmitoyl ABP (Fig. S6).

Computer Modeling Verifies That Axonal Kv7 Channels Regulate Spontaneous Firing.

The above observations provide an indication that the axonal location of Kv7 channels plays a crucial role in suppressing spontaneous activity. To confirm this, we used computer modeling (see Methods). Because the biophysical properties of the Kv7 (or M−) current in hippocampal neurons present in slices have not been clearly defined, we first obtained an activation curve using voltage–clamp recordings (SI Text and Fig. S7). These data were then input into the simulation program, NEURON, to obtain a “near-realistic” model of the CA1 pyramidal cell (see Methods). The basic conductance of Kv7 channels calculated from the voltage–clamp recordings was 0.02 S/cm2. Because there seem to be few functional Kv7 channels present in dendrites (see below and ref. 26), we restricted Kv7 channels to the soma and axon. Further, although immunohistochemistry has indicated that Kv7 protein is three times denser in the axon than the soma (7, 8), the relative number of functional axonal Kv7 channels is unknown. We therefore initially varied Kv7 channel density in axonal and somatic compartments. Then, to determine their effects on cell excitability, Kv7 channels were selectively removed from either all or selected compartments by setting their respective Kv conductances to zero. When doing this, we did not modify any other ionic conductances. However, because inserting axonal Kv7 channels hyperpolarized the model neuron by 1–2 mV (depending on Kv7 channel density), we adjusted the initial RMP to −65 mV to match the experimental data by slightly adjusting the passive leak current.

With an axon:soma Kv7 conductance ratio of 1:1 or 2:1, removal of all Kv7 conductance caused a long-lasting depolarization but not spontaneous action potential firing (Fig. 4Ai), although it did increase the number of action potentials in response to a step depolarization, as expected (Fig. S8). If, however, the axonal conductance was set between three and five times that at the soma, then taking away all Kv7 conductance generated unprompted firing (Fig. 4A ii and iii) in a similar manner to that caused by applying XE991 (Fig. 1). Interestingly, if axonal Kv7 conductance was six times or more at the soma, eliminating all Kv7 conductance produced limited firing (Fig. 4Aiv). This is because Kv7 channel deletion then depolarized the RMP to values above −50 mV, thereby inactivating Na+ channels and abbreviating spiking. Taken together with our experimental data, the model predicts that, for sustained spontaneous firing to occur after total Kv7 channel blockade (see Fig. 1), the axonal Kv7 channel density must be between three and five times greater than at the soma.

Fig. 4.
Relation between the subcellular distribution of Kv7 channels and spontaneous intrinsic excitability. Computer simulations with the Kv7 channel conductance in the axon and soma varied. The RMP in all cases was set to be −65 mV. A and B illustrate ...

To test how axonal Kv7 channels specifically contribute to spontaneous neuronal activity, we selectively removed the axonal Kv7 channel conductance, leaving the somatic Kv7 channels intact (thus imitating ABP). The results forecast that a minimum Kv7 axon:soma conductance ratio of 3:1 is required for spontaneous action potential firing to occur (Fig. 4B i–iii). Again, if the initial axonal density was six times (or more) than in the soma, then removal of the axonal channels produced limited action potential firing for reasons stated above (Fig. 4Biv).

Irrespective of the ratio of axonal Kv7 channels to soma, selective removal of somatic Kv7 channels did not cause spontaneous firing (data not shown). Thus, the presence of axonal Kv7 channels is both necessary and sufficient to prevent spontaneous firing.

Computer Modeling Confirms That Axonal Kv7 Channels Control Action Potential Threshold.

Experimental results demonstrated that changes in action potential threshold (in addition to RMP depolarization) were required for spontaneous action potential firing to occur after disruption of axonal Kv7 ankyrin binding (see Figs. 2 and and3).3). We therefore used computer modeling to examine how axonal Kv7 channels affected the action potential threshold, simulating the experimental protocol in Fig. 3A. Starting with axonal Kv7 conductances two and three times those in the soma, selective removal of these conductances reduced action potential threshold by 6.8 and 12.7 mV, respectively. These results are in reasonable agreement with the change produced by disrupting axonal Kv7 channel activity with ABP.

Distal Dendrites Have Few Functional Kv7 Channels.

The above evidence points to a critical role for axonal Kv7 channels. However, Kv7 channels may also be present in dendrites (15, 27, 28). To determine whether this is so, we obtained recordings from distal dendrites (250–300 μm from the soma) and then bath-applied XE991 (3 μM) and linopirdine (10 μM). These two drugs reduced dendritic RMP by 4.7 ± 1.0 mV (n = 6, Fig. 5 A and B) and 5.0 ± 0.9 mV (n = 4), a lesser effect than at the soma (see above) and, in contrast to the soma (Figs. S1 and S4), did not affect dendritic RN (Fig. 5D). This implies that Kv7 channels were not likely to be present in distal dendrites (in keeping with recent data; see ref. 26).

Fig. 5.
Effects of XE991 on dendritic activity (A) Recordings from distal CA1 pyramidal dendrites (250–300 μm from the soma) under control conditions and in the presence of XE991 when the RMP was depolarized (DRMP) or restored artificially back ...

Nevertheless, the numbers of action potentials recorded in distal dendrites were significantly enhanced in the presence of XE991 and linopirdine, like at the soma (Fig. 5 A and C) and spontaneous spikes were noted in 50% of neurons (see Fig. 5A). In CA1 pyramidal neurons, most dendritic action potentials are those that have been initiated at the axon and back-propagated (29). Thus, the increased numbers of dendritic action potentials are likely to result from the enhanced axosomatic excitability. In support of this, the spontaneous activity and enhanced evoked action potential firing in the presence of XE991 and linopirdine persisted even when dendritic RMP was artificially restored to the predrug level (Fig. 5). These results indicate that, despite few functional dendritic Kv7 channels, alterations in firing after perisomatic Kv7 channels modulation are likely to affect dendritic excitability.


The principal conclusion arising from this study is that the function of axonal Kv7 channels in hippocampal CA1 pyramidal neurons is to suppress the inherent spontaneous activity of these neurons, and that they do this by regulating action potential threshold and RMP (Figs. 2 and and3).3). Further, we found that computer simulations could reproduce our experimental observations if the axonal Kv7 conductance was approximately three to five times greater than that at the soma (Fig. 4). This accords well with information from immunohistochemical data (7, 8).

Selectivity and Mechanism of Action of ABP.

Our principal experimental evidence for a specific role of axonal Kv7 channels stems from the use of a peptide (ABP) designed to selectively disrupt the association between Kv7 subunits and ankyrin G at the AIS. This peptide affected only Kv7 channels, because its actions were fully occluded by blocking all Kv7 channels with XE991; and it affected only axonal, not somatic, channels, because (unlike XE991) it did not affect somatic input resistance or M current amplitude. Interestingly, intracellular ABP did not affect Na+ channel function (at least within our experimental time frame), as judged from action potential properties, even though Na+ channels have a partly homologous ankyrin-binding motif (5, 9). This may reflect either the specificity of the peptide or tighter Na+-channel ankyrin binding [perhaps related to the different locations of the ankyrin-binding motif within the NaV1 and Kv7 channel proteins (9)].

How might ABP be working? Because mutant Kv7 channels that do not bind ankyrin G are not concentrated at the AIS (5, 8), one possibility is that, by disrupting ankyrin-binding, axonal Kv7 channels may redistribute after ABP treatment. However, immunohistochemistry did not reveal any obvious effect of a lipidated version of the ABP (which had the same functional effect as the intracellularly injected ABP) on Kv7.2 and Kv7.3 localization (Fig. S6). This may be because diffusion of all molecules in the AIS membrane is highly restricted, whether bound to ankyrin or not, because of constraints imposed by the entire actin-linked cytoskeletal apparatus (30). Alternatively, ABP might have induced a local reorganization such as redistribution of Kv7 channels from membrane to cytosol. We could not test this, because all available Kv7 antibodies are directed toward intracellular domains and so cannot distinguish between cytoplasmic and membrane protein [the small diameter of the AIS (<1 μm) also makes it very difficult to differentiate between cytoplasmic and membrane fluorescence even with confocal microscopy]. Nevertheless, the electrophysiological results clearly indicate that ABP affected axonal Kv7 channels.

Functional Significance of Axonal Kv7 Channels.

Kv7 channels are modulated by a variety of G protein-coupled receptors and intracellular signaling molecules (1). Our results indicate that modulating axonal Kv7 channel function can have a significant impact on neuronal processing under physiological conditions. For example, Kv7 channel activity is inhibited by acetylcholine (17), which, in the hippocampus, is probably mediated by the M3 receptor (31). Coincidentally, the M3 receptor is present at the CA1 neuropil (32). The increased action potential firing and enhanced back propagation of spikes into dendrites by transient Kv7 channel inhibition (Fig. 5) will lead to more NMDA receptor channel openings (because the Mg2+ block is removed by depolarization) and enhanced incoming excitatory synaptic inputs and, perhaps, synaptic integration (29). This may represent one way by which neurotransmitters such as acetylcholine contribute to processes such as learning and memory (33).

Our experiments also provide an explanation for why spontaneous mutations in Kv7 channels that disrupt axonal Kv7 targeting can result in epilepsy (7). In vivo, any spontaneous firing resulting from axonal channel disruption is likely to be enhanced by the more global Kv7 inhibition arising from activation of Gq-coupled receptors via afferent glutamatergic and cholinergic synaptic activity (3), similar to the amplifying effect of somatic Kv7 block by XE991 (Fig. 2), thereby leading to convulsions. Thus, a high density of AIS Kv7 channels is fundamental for regulating hippocampal pyramidal inherent activity and maintaining neural network homeostasis.


Electrophysiological Studies.

Methods were similar to those described in ref. 24. Briefly, 5- to 6-wk-old rats were anesthetized and perfused intracardially with ice-cold modified artificial cerebrospinal fluid containing 110 mM choline chloride, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 0.5 mM CaCl2, 7 mM MgCl2, and 7 mM glucose, and bubbled with 95% O2/5% CO2 (pH 7.2). Four hundred-micrometer-thick hippocampal–entorhinal slices were then prepared by using a vibratome (Leica VT1000S) and incubated for 1 h at room temperature in a holding chamber containing an external recording solution: 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 2 mM CaCl2, 2 mM MgCl2, and 10 mM glucose and bubbled with 95% O2/5% CO2 (pH 7.2). For recording purposes, slices were placed in a chamber with external recording solution supplemented with 0.05 mM 2-amino-5-phosphonovaleric acid (APV), 0.01 mM 6-cyano-7-nitroquinoxaline-2,3-dione, 0.01 mM bicuculline, and 0.001 mM CGP 55845 and maintained at 34–36°C. For some recordings, the external solution also had 0.01 mM tetrodotoxin and 0.2 mM Cd2+. The internal pipette solution was 120 mM KMeSO4, 20 mM KCl, 10 mM Hepes, 2 mM MgCl2, 0.2 mM EGTA, 4 mM Na2-ATP, 0.3 mM Tris·GTP, and 14 mM Tris-phosphocreatine, pH 7.3 (adjusted with KOH; pipette resistance, 8–9 MΩ). Whole-cell current–clamp recordings were obtained by using a bridge-mode amplifier (Axoclamp 2A), filtered at 10 kHz and sampled at 30 kHz. Series resistances were in the order of 10–20 MΩ. Voltage–clamp recordings were obtained by using an Axopatch 2A amplifier, filtered at 1 kHz and sampled at 5 kHz. All recordings were acquired by using pClamp (Axon Instruments) 8.0 and stored on a computer for further analysis.

Data Analysis.

All measurements were made by using Clampfit (v8.0). Because Kv7 channels are most active at voltages positive to −70 mV (Fig. S7), we measured RN by applying a 400-ms 100-pA step from −70 mV. To calculate RN, the steady-state voltage in the last 25 ms of the step was subtracted from −70 mV and divided by the magnitude of current applied. The action potential threshold was measured by differentiating the action potential voltage with respect to time (dV/dt). Threshold was defined as the point where dV/dt was >0. Action potential height was measured from dV/dt to the peak whereas spike width was defined as half the height. Group data are expressed as mean ± SEM. Statistical significance was determined by using either paired or unpaired Student's t tests, as appropriate.


All chemicals were obtained from Sigma, except for XE991, linopirdine, CNQX, CGP 55848, bicuculline, and APV, which were purchased from Tocris. ABP, sABP, and analogues were custom-made either by the Advanced Biotechnology Center (Imperial College London) or Genscript and dissolved at the relevant concentration in either the internal or external solution as appropriate. Stock solutions of bicuculline and CGP 55848 were made in DMSO (final dilution, <0.1%) and stored at −20°C until use. Aqueous stock solutions of XE991, linopirdine, CNQX, and APV were also kept at −20°C until use.


Three hundred fifty- to four hundred-micrometer slices were treated with 50 μM palmitoylated ABP or palmitoylated sABP and then fixed by using 4% paraformaldehyde for 30–45 min. The slices were then equilibrated in 30% sucrose, embedded (OTC Tissue Tek Compound, Sakura Finetek), cryostat sectioned at 20 μm, and mounted onto Superfrost Plus (Fisher) slides. Because antigen retrieval was necessary, the sections were microwave-irradiated in 10 mM Na-Citrate, pH 6.0. Immunoreactions were then performed by using rabbit Kv7.2-N and guinea pig Kv7.3-N antibodies, as described (5, 8).

Computational Modeling Methods.

All simulations were implemented and run with NEURON (v5.8) by using the variable time step feature. The 3D reconstruction of the CA1 pyramidal neuron was one of those used previously (neuron 9068802; see ref. 34). Unless otherwise noted, the same standard uniform passive properties were used for control conditions (τm = 28 ms, Rm = 28kΩ·cm2, Ra = 150Ω·cm). RMP was set at −65 mV and temperature at 35°C. Active somatic and dendritic properties included Na+, DR-, A-, and Kv7-type potassium conductances, and Ih current. To take into account Ca2+ channels opened at rest, a low-threshold Ca2+ conductance, a Ca-dependent K+ conductance, and a simple Ca2+ extrusion mechanism were included at uniform density and distribution in all compartments. Channel kinetics and distribution were based on the available experimental data for CA1 pyramidal neurons (35). The model and simulation files are available for public download under the ModelDB section (36) of the SenseLab database (http://senselab.med.yale.edu). The Kv7 activation was modeled with a Boltzmann curve (v1/2 = −40, k = 10). The voltage dependence of the time constant was obtained from the experimental data (SI Text and Fig. S7) by using identical activation and deactivation rate constants at V0.

Supplementary Material

Supporting Information:


We thank Dr. P. Thomas and Prof. M. Duchen for help in acquiring the ABP-FITC image (Fig. S2) and Mr. T. Kao for aiding with the immunohistochemistry. This work was supported by a Wellcome International Prize Travel Research Fellowship [work plan approved in January 2003 (M.M.S.)] and grants from the Epilepsy Research Foundation (M.M.S.), European Union (D.A.B.), and National Institutes of Health (E.C.C.).


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0802805105/DCSupplemental.


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