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Logo of jphysiolThe Journal of Physiology SiteMembershipSubmissionJ Physiol
J Physiol. Mar 15, 2008; 586(Pt 6): 1565–1579.
Published online Jan 10, 2008. doi:  10.1113/jphysiol.2007.146597
PMCID: PMC2375705

Electrical remodelling maintains firing properties in cortical pyramidal neurons lacking KCND2-encoded A-type K+ currents

Abstract

Considerable experimental evidence has accumulated demonstrating a role for voltage-gated K+ (Kv) channel pore-forming (α) subunits of the Kv4 subfamily in the generation of fast transient outward K+, IA, channels. Immunohistochemical data suggest that IA channels in hippocampal and cortical pyramidal neurons reflect the expression of homomeric Kv4.2 channels. The experiments here were designed to define directly the role of Kv4.2 in the generation of IA in cortical pyramidal neurons and to determine the functional consequences of the targeted deletion of Kv4.2 on the resting and active membrane properties of these cells. Whole-cell voltage-clamp recordings, obtained from visual cortical pyramidal neurons isolated from mice in which the KCND2 (Kv4.2) locus was disrupted (Kv4.2-/- mice), revealed that IA is indeed eliminated. In addition, the densities of other Kv current components, specifically IK and Iss, are increased significantly (P < 0.001) in most (~80%) Kv4.2-/- cells. The deletion of KCND2 (Kv4.2) and the elimination of IA is also accompanied by the loss of the Kv4 channel accessory protein KChIP3, suggesting that in the absence of Kv4.2, the KChIP3 protein is targeted for degradation. The expression levels of several Kv α subunits (Kv4.3, Kv1.4, Kv2.1, Kv2.2), however, are not measurably altered in Kv4.2-/- cortices. Although IA is eliminated in Kv4.2-/- pyramidal neurons, the mean ± s.e.m. current threshold for action potential generation and the waveforms of action potentials are indistinguishable from those recorded from wild-type cells. Repetitive firing is also maintained in Kv4.2-/- cortical pyramidal neurons, suggesting that the increased densities of IK and Iss compensate for the in vivo loss of IA.

Cortical computations are performed in circuits composed of multiple cortical cell types with distinct action potential dynamics, repetitive firing properties and dendritic structures (Douglas et al. 1996; Maass et al. 2004; Marino et al. 2005; London & Häusser, 2005). This electrophysiological heterogeneity largely reflects differences in the types, densities, properties and/or distributions of voltage-dependent ion channels (London & Häusser, 2005). Although the regulation of intrinsic membrane properties is clearly important for normal cortical neuron and circuit functioning, little is known about how distinction channel expression levels and distributions are achieved during normal cortical development and/or how these patterns are maintained and regulated in the adult brain (Davis, 2006).

Recent studies, however, suggest roles for shared clustering and targeting mechanisms, as well perhaps as local competition. In animals (medNav1.6−/−) harbouring a targeted disruption of Scn8a, which encodes the voltage-gated Na+ (Nav) channel pore-forming subunit Nav1.6 (Burgess et al. 1995), for example, Nav channel remodelling, that partially compensates for the loss of Nav1.6, is evident (Van Wart & Matthews, 2006). In addition, Nav channel remodelling appears to be cell-type specific; Nav1.1 expression is increased in cerebellar Purkinje neurons, whereas compensation in retinal ganglion cells reflects up-regulation of Nav1.2 (Van Wart & Matthews, 2006). Alternatively, the normal patterns and the ‘balance’ of ion channel expression levels in cortical neurons might be achieved by some form of homeostatic regulatory mechanism which would function to orchestrate (recruit) compensatory channels, constrain plasticity and maintain ‘normal’ excitability (Davis & Bezprozvanny, 2001; MacLean et al. 2003; Zhang & Linden, 2003; Turrigano & Nelson, 2004; Davis, 2006). Consistent with this hypothesis, experiments on isolated cortical neurons in vitro have demonstrated changes in the functional expression of voltage-gated Na+ and K+ channels in ‘electrically silenced’ cells, achieved by chronic treatment with the Na+ channel blocker, tetrodotoxin, or by over-expression of hyperpolarizing, inwardly rectifying K+ (Kir) channels (Desai et al. 1999; Burrone et al. 2002).

Voltage-gated K+ (Kv) currents are key regulators of excitability in mammalian cortical neurons, functioning to control resting membrane potentials, shape action potential waveforms, determine repetitive firing patterns, regulate the responses to synaptic inputs and modulate synaptic plasticity. Rapidly activating and inactivating (IA) Kv channels, for example, contribute to action potential repolarization and repetitive firing, and affecting neuronal outputs (Rogawski, 1985; Bekkers, 2000a,b; Kang et al. 2000; Frick & Johnston, 2005; Kim et al. 2005; Yuan et al. 2005). In addition, somatodendritic IA channels modulate the back-propagation of action potentials (into dendrites), and impact synaptic efficacy and plasticity (Hoffman et al. 1997; Stuart et al. 1997; Häusser et al. 2000; Johnston et al. 2003; Birnbaum et al. 2004). Exploiting dominant negative strategies in vitro has revealed that IA channels are encoded by Kv4 α subunits and that the acute loss of IA affects the input properties and alters the excitability of hippocampal and cortical pyramidal neurons (Kim et al. 2005; Yuan et al. 2005). It has also been reported that dendritic excitability is increased in temporal lobe epilepsy as a result of decreased IA channel availability (Bernard et al. 2004). Given these observations, the recent report that the phenotypic consequences of the in vivo elimination of KCND2 (Kv4.2) (Guo et al. 2005; Hu et al. 2006) on hippocampal pyramidal neurons are quite modest (Chen et al. 2006) was unexpected.

The experiments here were designed to explore directly the role of Kv4.2 in the generation of IA in (mouse) cortical pyramidal neurons and to determine whether functional ionic remodelling occurs in (Kv4.2-/-) neurons as a result of the in vivo deletion of Kv4.2. Whole-cell voltage-clamp experiments revealed that IA is eliminated in Kv4.2-/- cortical pyramidal neurons, demonstrating directly a critical role for Kv4.2 in the generation of IA in these cells. The elimination of IA in most (~80%) Kv4.2-/- cells is accompanied by the marked up-regulation of two delayed rectifier Kv current components, IK and Iss. In addition, in contrast with the effects of the in vitro elimination of IA (Kim et al. 2005; Yuan et al. 2005), the waveforms of action potentials and the repetitive firing properties of Kv4.2-/- are indistinguishable from wild-type cells.

Methods

Animals used in the present study were handled in accordance with the guidelines published in the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health); all protocols were approved by the Washington University Animal Studies Committee.

Isolation and in vitro maintenance of mouse visual cortical neurons

Neurons were isolated from the primary visual cortices of postnatal day (P) 7–10 wild-type (WT) and Kv4.2-/- (Guo et al. 2005) FVB mice using methods previously described for the isolation of neonatal rat neocortical neurons (Locke & Nerbonne, 1997; Yuan et al. 2005). Briefly, animals were anaesthetized with 5% halothane, decapitated and the brains were removed. Primary visual cortices were isolated, minced, and incubated in papain-containing solution at 35°C for 60 min. Isolated cortical neurons were obtained by trituration and subsequent centrifugation (at 600 g for 10 min). Dissociated cells were resuspended in minimal Eagle's essential medium (MEM) containing 5% heat-inactivated horse serum and plated at a density of 10 × 103 cm−2 on monolayers of (rat) neocortical astrocytes (prepared as described in Locke & Nerbonne, 1997). Neuronal/glial cultures were maintained in a 95% O2–5% CO2 37°C incubator, and the medium was exchanged every other day.

Electrophysiological recordings

Whole-cell voltage- and current-clamp recordings were obtained from large, pyramidal-shaped visual cortical neurons isolated from WT and Kv4.2-/- (FVB) mice at room temperature (22–24°C); recordings were obtained 48–72 h following cell isolations. Recording pipettes were fabricated from borosilicate glass using a horizontal puller (model P-87; Sutter Instruments, Novato, CA, USA). When filled with the standard pipette solution (see below), electrode resistances were typically 2–3 MΩ. Data were collected using a Dagan Model 8900 patch-clamp amplifier (Dagan Instruments). Experimental parameters were controlled with a Gateway microcomputer, interfaced to the electrophysiological recording equipment through a Digidata 1332 interface, using pCLAMP software (Axon Instruments, Version 9). For all voltage-clamp recordings, the bath solution contained (mm): 140 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 Hepes, 10 glucose, 0.001 tetrodotoxin (TTX), and 0.1 CdCl2 (pH 7.4; 310 mosmol l−1). The pipette solution contained (mm): 135 KCl, 10 Hepes, 10 glucose, 5 mm BAPTA, 5 Mg-ATP, and 0.3 Tris-GTP (pH 7.3; 310 mosmol l−1). For current-clamp recordings, the TTX and CdCl2 were omitted from the bath solution, and the intracellular free Ca2+ concentration in the pipette solution was fixed at 10−7m by lowering the BAPTA concentration to 2.5 mm in the presence of 1 mm CaCl2.

In all experiments, tip potentials were zeroed before membrane–pipette seals were formed. Outward Kv currents were routinely recorded in response to (4 s) depolarizing voltage steps to test potentials between −40 mV and +40 mV from a holding potential of −70 mV. Sampling frequencies were 100 kHz to 10 kHz, and currents were low-pass filtered at 5 kHz prior to digitization and storage. To examine the kinetics of Kv current recovery from steady-state inactivation, a three-pulse protocol was used. After depolarizing to +40 mV for 4 s to activate and inactivate the currents, cells were hyperpolarized to −70 mV for varying times, ranging from 10 to 4000 ms; test depolarizations to +40 mV were then presented to assess the extent of recovery. Single action potentials and action potential trains were recorded in response to brief (2 ms) and prolonged (400 ms) depolarizing current injections of variable amplitudes.

Western blots

Whole-brain (minus the cerebellum and the brainstem) and cortical membrane preparations were prepared from the brains of anaesthetized (5% halothane) and decapitated adult (6–8 week) WT (n = 6) and Kv4.2-/- (n = 6) FVB mice using previously described methods (Guo et al. 2005). The protein content of each solubilized sample was determined using a Bio-Rad protein assay kit with bovine serum albumin as the standard, and immunoblots with anti-Kv subunit-specific antibodies were performed. Polyclonal and monoclonal antibodies useful for the detection of Kv4.2, Kv4.3, Kv1.4, Kv2.1, Kv2.2, KChIP1 and KChIP2 have been described (An et al. 2000; Brunet et al. 2004; Guo et al. 2005). The monoclonal anti-KChIP3 antibody was purchased from Antibodies, Inc. (Davis, CA, USA).

Equal amounts of proteins (20 μg) from WT and Kv4.2-/- mouse brains/cortices were fractionated on 8, 12 or 15% SDS-PAGE gels, transferred to PVDF membranes (Bio-Rad), and incubated in 0.2% I-Block (Tropix) in PBS containing 0.1% Tween 20 (blocking buffer) for 1 h at room temperature. This blocking step was followed by an overnight incubation at 4°C with one of the anti-Kvα or anti-Kvβ subunit-specific antibodies (at 0.25–5 μg ml−1) alone or together with a monoclonal anti-β-actin antibody. In each experiment, after washing, membrane strips were incubated with alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse IgG (Tropix) diluted 1: 5000 in blocking buffer, and bound antibodies were detected using CPSD, a chemiluminescent alkaline phosphate substrate (Tropix). After washing to remove excess substrate, membranes were exposed to X-ray film (Kodak). These films were scanned (Molecular Dynamics Densitometer) and the densities of Kv subunit and β-actin bands were determined by summing the pixel values (above background) in a defined (rectangular) area surrounding each protein band. The intensities of the bands corresponding to each Kv subunit protein were normalized to the intensity of the actin band in the same sample. Mean ± s.e.m. values from experiments completed on cortical proteins prepared from six WT and six Kv4.2-/- brains/cortices were determined.

Data analyses

Electrophysiological data were compiled and analysed using CLAMPFIT (Axon) and Excel (Microsoft) software. The spatial control of the membrane voltage in each cell was assessed by analyzing the decays of the capacitative transients evoked during brief (25 ms) ±10 mV voltage steps from the holding potential of −70 mV; only cells with capacitative transients well described by single exponentials were analyzed further. Whole-cell membrane capacitances, Cm, were determined by integration of the capacitative transients evoked during these brief (25 ms) subthreshold (±10 mV) voltage steps from −70 mV. Input resistances (Rin) were calculated from the steady-state currents recorded in response to the −10 mV hyperpolarizing voltage steps. Leak currents at −70 mV were always ≤ 20 pA and were not corrected. In each cell, the series resistance (Rs) was calculated by dividing the time constant of decay of the capacitive transient by the Cm (determined in the same cell). The mean ± s.e.m. (n = 109) Rs was 11.4 ± 0.9 MΩ and, in all cells, Rs was compensated electronically by ~90–95%. Because current amplitudes were ≤ 10 nA, voltage errors resulting from the uncompensated Rs were always ≤ 12 mV and were not corrected. Current amplitudes, Rin and Rs were monitored continuously during all recordings. Only cells in which these parameters remained constant were analyzed further.

Peak outward Kv currents in individual cells were defined as the maximum values of the outward K+ currents evoked during (4 s) depolarizing voltage steps. To determine the amplitudes of the individual Kv current components, the decay phases of the currents were fitted by the sum of two or three exponentials (as described in the text) with all parameters in the fits left free. As described in detail in previous studies on rat cortical pyramidal neurons (Yuan et al. 2005), the quality of the fits was assessed (and was evident) by visual inspection. In addition, correlation coefficients for all fits were determined. Current amplitudes, measured in individual cells, were normalized for differences in cell size (whole-cell Cm), and current densities (pA pF−1) are reported. The kinetic analyses also provided the time constants of inactivation of the individual Kv current components in WT and Kv4.2-/- neurons.

All current-clamp recordings were obtained from cells with overshooting action potentials and with stable resting membrane potentials ≤−50 mV. The (current) threshold for action potential generation in each cell was defined as the minimal current, applied for 2 ms, required to evoke a single action potential. The voltage threshold for action potential generation in each cell was determined from a phase plot of the first derivative of the membrane voltage with respect to time (dV/dt) as a function of the membrane voltage (Bean, 2007). The latency to firing on action potential in each cell was defined as the time after the onset of the depolarizing current injection required to bring the membrane to the (voltage) threshold for action potential generation. Action potential durations at 50% (and 90%) repolarization were determined by measuring the widths of the action potentials when the membrane voltage had returned halfway (or 90% of the way) back to the resting membrane potential. The amplitudes and durations of afterhyperpolarizations were determined by measuring the maximal deflections from, and the time required to return to, the resting membrane potential, respectively. To examine the effects of the targeted deletion of KCND2 on repetitive firing properties, the number of spikes evoked in response to varying amplitude current injections in individual cells were counted.

All averaged and normalized data are presented as mean ± s.e.m.; n values are presented in the text and/or in the figure legends. The statistical significance of apparent differences between different groups of cells was examined using Student's t test and, where appropriate, P values are presented in the text or in the figure legends.

Results

Multiple Kv currents expressed in mouse visual cortical pyramidal neurons

In initial experiments, neurons were isolated from the primary visual cortices of P7–P10 wild-type (WT) FVB mice, plated on glial monolayers, and maintained in vitro. Large, pyramidal-shaped neurons were selected for recordings (Desai et al. 1999; Yuan et al. 2005), and whole-cell voltage-gated K+ (Kv) currents were recorded in the presence of 1 μm TTX and 0.1 mm CdCl2 to block voltage-gated Na+ and Ca2+ currents, respectively. Representative Kv currents recorded from WT mouse visual cortical pyramidal neurons in response to (4 s) depolarizations to varying test potentials (−40 to +40 mV) from a holding potential of −70 mV are presented in Fig. 1A. Similar to previous studies on rat cortical neurons (Locke & Nerbonne, 1997; Yuan et al. 2005), Kv currents in mouse visual cortical pyramidal neurons activate rapidly and the rates of rise and the amplitudes of the currents increase with increasing membrane depolarization.

Figure 1
Distinct Kv current waveforms in wild-type (WT) and Kv4.2-/- cortical pyramidal neurons

The decay phases of the Kv currents in WT cortical pyramidal neurons were well described by the sum of three exponentials with mean ± s.e.m. (n = 59) inactivation time constants (τdecay) (at +40 mV) of 27 ± 1 ms, 229 ± 15 ms and 2040 ± 64 ms (Table 1). The rapid component of current decay is attributed to the presence of the fast transient ‘A’ current, typically referred to as IA (Kang et al. 2000; Shibata et al. 2000; Song, 2002; Yuan et al. 2005) or ISA (Hoffman et al. 1997; Johnston et al. 2003; Birnbaum et al. 2004; Jerng et al. 2004). The more slowly decaying (τdecay~250 ms) transient Kv current is referred to as ID by analogy to previously described currents in rat hippocampal and cortical neurons (Storm, 1987, 1990; Locke & Nerbonne, 1997; Yuan et al. 2005). The slowly decaying (τdecay~2000 ms) and the non-inactivating Kv current components are referred to as IK and Iss, respectively.

Table 1
Kv current diversity and remodelling in Kv4.2-/- cortical pyramidal neurons

Selective elimination of IA in Kv4.2-/- cortical pyramidal neurons

Subsequent experiments were conducted on cells isolated from the visual cortices of mice (Kv4.2-/-) in which the KCND2 locus was disrupted by homologous recombination (Guo et al. 2005; Hu et al. 2006). The Kv current waveforms illustrated in Fig. 1B are representative of those recorded from the majority (39 of 50; ~80%) of Kv4.2-/- cortical pyramidal neurons. Although peak Kv current densities in these (Kv4.2-/-) and in WT cells are similar (Fig. 1C; Table 1), the waveforms of the currents are distinct. In particular, the rapidly decaying current component that is prominent in WT cells (Fig. 1A) is not evident (Fig. 1B).

Analyses of the Kv currents in recordings obtained from most (80%) Kv4.2-/- cells (Fig. 1B) revealed that, in contrast to WT cells, the decay phases of the currents were best described by the sum of two exponentials, with mean ± s.e.m. (n = 39) τdecay values of 174 ± 20 ms and 1761 ± 48 ms, and a non-inactivating current component (Table 1), consistent with the expression of ID, IK and Iss in these cells. No rapidly inactivating IA was detected in the majority of Kv4.2-/- cells (Fig. 1D). These analyses also revealed that the densities of IK and Iss were significantly (P < 0.001) higher in Kv4.2-/- cells (Fig. 1E), whereas ID densities in Kv4.2-/- cells were similar (Fig. 1D and Table 1) to those measured in WT cells.

In WT mouse cortical pyramidal neurons (n = 19), both IK and Iss were attenuated by application of 10 mm tetraethylammonium (TEA), whereas IA and ID are unaffected (Fig. 2). Both IK and Iss were also reduced by 10 mm TEA in Kv4.2-/- cells (n = 14), and analyses of the waveforms of the TEA-sensitive currents (Fig. 2C and F) confirmed that mean ± s.e.m.IK (Fig. 2G) and Iss (Fig. 2H) densities were significantly (P < 0.001) higher in Kv4.2-/-, than in WT, neurons (Table 1).

Figure 2
TEA-sensitive Kv current densities are higher in Kv4.2-/- cortical pyramidal neurons

Although IK and Iss densities were increased in most Kv4.2-/- cells, neither the time- nor the voltage-dependent properties of these currents were measurably affected by the targeted deletion of KCND2 (Table 1). Mean (± s.e.m.) whole-cell membrane capacitances (Cm) measured in Kv4.2-/- (19.3 ± 1.0 pF; n = 39) and WT (22.3 ± 1.2 pF; n = 59) neurons were also not significantly different. The increases in IK and Iss densities in Kv4.2-/- cells, therefore, cannot be attributed to differences in cell size, but rather suggest Kv current remodelling with the targeted disruption of KCND2 and the loss of IA.

A rapidly inactivating Kv current is evident in a subset of Kv4.2-/- pyramidal neurons

Although IA was clearly eliminated in most (39 of 50; ~80%) Kv4.2-/- neurons (Fig. 1), a rapidly inactivating Kv current was evident in the other 11 (~20% of the 50) Kv4.2-/- neurons examined. The waveforms of the Kv currents in these (Kv4.2-/-*) cells (Fig. 3B) were similar to the currents recorded from WT cells (Fig. 3A). The decay phases of the Kv currents in Kv4.2-/-* cells, for example, were well described by the sum of three exponentials with mean ± s.e.m. (n = 11) τdecay values (Table 1) that were not significantly different from those determined in WT cells. The densities of the peak (Fig. 3C) and the rapidly inactivating (Fig. 3D) Kv currents in Kv4.2-/-* cells, however, were significantly (P < 0.001) lower (Table 1) than in WT cells. In addition, the rapidly inactivating current in Kv4.2-/-* cells appears to activate at test potentials depolarized relative to wild-type IA (Fig. 3D). In contrast to the results obtained in the majority of Kv4.2-/- cells (Fig. 1), mean ± s.e.m.IK and Iss densities in Kv4.2-/-* and WT cells were not significantly different (Fig. 3 and Table 1).

Figure 3
A rapidly inactivating Kv current is present in a small subset of Kv4.2-/- cortical pyramidal neurons

The presence of a rapidly inactivating Kv current in this subset (Kv4.2-/-*) of Kv4.2-/- cells (with a τdecay value similar to the τdecay for wild-type IA) suggests either that Kv4.2 does not contribute to the formation of IA channels in a subset of cortical pyramidal neurons or, alternatively, that a novel transient (‘IA-type’) current was up-regulated in some cells with the elimination of IA. Light and electron microscopical studies have demonstrated that Kv4.3 is also expressed in the cell bodies and dendrites of some mouse cortical pyramidal neurons (Burkhalter et al. 2006), suggesting a possible role for Kv4.3 in the generation of the rapidly inactivating Kv current in Kv4.2-/-* cortical pyramidal neurons. Other Kv α subunits, such as Kv1.4 and Kv3.4, however, also produce rapidly activating and inactivating channels in heterologous expression systems, suggesting molecular heterogeneity in neuronal IA-type channels (Birnbaum et al. 2004). Subsequent experiments, therefore, were aimed at further characterization of the rapidly inactivating Kv current in Kv4.2-/-* neurons (Fig. 3B).

No rapidly recovering Kv currents in Kv4.2-/-* cortical pyramidal neurons

In addition to rapid inactivation, IA is typically characterized by rapid recovery from inactivation (Birnbaum et al. 2004; Jerng et al. 2004). Subsequent experiments, therefore, were aimed at determining the time courses of recovery (from steady-state inactivation) of the rapidly inactivating Kv currents in WT and Kv4.2-/-* cortical pyramidal neurons (Fig. 3). Because the rapidly inactivating Kv current is only evident in ~20% of the cells, the Kv4.2-/-* cells had to be identified for these experiments based on the presence of this component in initial recordings using the standard voltage-clamp protocol (as in Figs 1 and and2).2). Once identified, the kinetics of recovery from inactivation of the rapidly inactivating current were examined. To determine recovery kinetics, Kv4.2-/-* and WT cells were first depolarized to +40 mV for 4 s to inactivate the currents and subsequently hyperpolarized to −70 mV for varying times ranging from 10 ms to 4000 ms. Test depolarizations to +40 mV were then presented to assess the extent of recovery. Typical Kv current waveforms recorded from WT and Kv4.2-/-* neurons using this protocol are illustrated in Fig. 4 (panels A and B). The amplitudes of the inactivating currents evoked at +40 mV following each recovery period in each cell were determined from exponential fits to the decay phases of the currents evoked during the test depolarizations. These values were subsequently normalized to the current amplitudes determined from fits to the Kv currents recorded (in the same cell) during the prepulse or during the final test pulse. As would be expected if recovery is complete following the 4000 ms recovery period, these two methods of analyses gave indistinguishable results.

Figure 4
The rapidly inactivating Kv current in Kv4.2-/-* cortical pyramidal neurons recovers slowly from inactivation (and is distinct, therefore, from WT IA)

The mean ± s.e.m. normalized amplitudes of the rapidly inactivating currents in WT and Kv4.2-/-* cells are plotted as a function of recovery time in Fig. 4C. As is evident, the rates of recovery of the rapidly inactivating currents in WT and Kv4.2-/-* cells are markedly different. The recovery data for IA in WT cells were well described by a single exponential with a mean ± s.e.m. (n = 7) time constant of 110 ± 17 ms. Although recovery of the rapidly inactivating current component in Kv4.2-/-* cells was also well described by a single exponential, recovery in these cells was slow, characterized by a mean ± s.e.m. (n = 9) time constant of 892 ± 159 ms (Fig. 4C). There was no fast component of recovery in Kv4.2-/-* cells, indicating that the rapidly inactivating current in Kv4.2-/-* cells is distinct from WT IA.

No heteropodatoxin (HpTx)-sensitive currents are evident in Kv4.2-/-* cortical neurons

The results presented above suggest the presence of a novel rapidly inactivating, slowly recovering Kv current in Kv4.2-/-* cortical pyramidal neurons. This current could reflect the presence of homomeric Kv4.3 channels or, alternatively, rapidly inactivating Kv channels encoded by non-Kv4 α subunits. Previous studies have demonstrated that the heteropodatoxins (HpTx-2 and -3) are selective for Kv4-encoded channels (Sanguinetti et al. 1997; Guo et al. 2005; Wang & Schreurs, 2006). Subsequent experiments, therefore, were focused on determining if there was an HpTx-sensitive Kv4-encoded current in Kv4.2-/-* neurons. As in the inactivation recovery experiments just described, Kv4.2-/-* cells had to be pre-selected for these analyses based on the presence of a fast component of inactivation. In WT cortical pyramidal neurons (n = 12), exposure to 500 nm HpTx-3 markedly attenuated the rapidly inactivating (IA) current (Fig. 5B). The waveforms of the HpTx-3-sensitive currents in WT cells (Fig. 5C) are consistent with the selective attenuation of IA.

Figure 5
No heteropodatoxin (HpTx)-sensitive Kv currents are evident in Kv4.2-/-* cortical pyramidal neurons

In contrast, Kv currents in Kv4.2-/-* neurons (n = 21) (Fig. 5D) were not measurably affected by 500 nm HpTx-3 (Fig. 5E), and no rapidly inactivating HpTx-sensitive currents were evident (Fig. 5F). The simplest interpretation of these results is that there are no Kv4-encoded currents in Kv4.2-/-* cells, i.e., that the rapidly inactivating, slowly recovering Kv current in Kv4.2-/-* neurons is not encoded by Kv4.3 α subunits. Analyses of the Kv currents remaining in the presence of HpTx-3 in WT cells (in records such as those illustrated in Fig. 5B) did not reveal the presence of a rapidly inactivating, slowly recovering Kv current similar to the current seen in Kv4.2-/-* cells (Fig. 5E), suggesting that this Kv current is not normally expressed in WT cortical pyramidal neurons (see Discussion).

Functional effects of IA and Kv current remodelling in Kv4.2-/- neurons

Previous studies suggest that IA channels function to control action potential waveforms, interspike intervals and repetitive firing in central neurons (Birnbaum et al. 2004; Jerng et al. 2004). Combined current-voltage-clamp recordings, for example, revealed that IA channels contribute to action potential repolarization in cortical neurons (Kang et al. 2000). In addition, action potentials were prolonged in isolated (rat) cortical pyramidal neurons in which IA was eliminated following in vitro transfection with a dominant negative Kv4.2 construct, Kv4.2 DN (Yuan et al. 2005). Input resistances were increased and the (current) thresholds for action potential generation were reduced in Kv4.2 DN-expressing cells (Yuan et al. 2005). In contrast to Kv4.2-/- neurons, however, there were no measurable changes in other Kv currents in isolated (rat) cortical pyramidal neurons expressing Kv4.2 DN and lacking IA (Yuan et al. 2005). Subsequent experiments, therefore, were focused on determining the functional consequences of the in vivo loss of IA and the Kv current remodelling evident in Kv4.2-/- neurons.

Action potentials, evoked in response to brief depolarizing current injections in isolated WT mouse visual cortical pyramidal neurons, rise rapidly to a maximal potential of ~+50 mV, and repolarization is rapid (Fig. 6A). The waveforms of evoked action potentials in Kv4.2-/- (Fig. 6B) cells were quite similar to those in WT cells. Further analyses revealed that mean ± s.e.m. resting membrane potentials, current thresholds for action potential generation, action potential amplitudes, action potential durations and the amplitudes and durations of afterhyperpolarizations in Kv4.2-/- and WT neurons were not significantly different (Table 2). In addition, as illustrated in the voltage trajectory (dV/dt versus time) and the phase (dV/dt versus voltage) plots in Fig. 6, the waveforms and the properties of the action potentials in WT (Fig. 6A) and Kv4.2-/- (Fig. 6B) cortical pyramidal neurons were indistinguishable.

Figure 6
Action potential waveforms in WT and Kv4.2-/- cortical pyramidal neurons are indistinguishable
Table 2
Resting and active membrane properties of WT and Kv4.2-/- cortical pyramidal neurons

Analyses of the phase plots (Fig. 6) further revealed that there were no significant differences in the voltage thresholds for action potential generation or in the slopes of the rising or falling phases of the action potentials in WT and Kv4.2-/- cells (see Discussion). Taken together, the results above suggested that Kv current remodelling (increased IK and Iss) negates the effects of the loss of IA on single action potentials. To explore directly the role of the up-regulation of IK and Iss in the maintenance of action potential waveforms in Kv4.2-/- neurons, outward Kv currents activated during single action potentials were measured. Kv currents, evoked in response to voltage-clamp protocols mimicking typical action potential waveforms, were examined in WT (Fig. 7A) and Kv4.2-/- (Fig. 7B) cells in the absence and presence of TEA. These experiments revealed that the densities and the waveforms of the Kv currents evoked during single action potentials under control conditions in WT (Fig. 7A) and Kv4.2-/- (Fig. 7D) neurons were indistinguishable. As also illustrated in Fig. 7, however, the amplitudes/densities of the TEA-sensitive currents evoked during single action potentials were higher in Kv4.2-/- (n = 14) than in WT (n = 15) cells.

Figure 7
The amplitudes of the TEA-sensitive Kv currents evoked during action potentials are larger in Kv4.2-/- than in WT cells

In response to prolonged (400 ms) low amplitude (10–100 pA) depolarizing current injections, WT cortical pyramidal neurons fire repetitively (Fig. 8A) at frequencies that vary with stimulus strength (Fig. 8C). Repetitive firing was also readily evoked in Kv4.2-/- cortical neurons (Fig. 8B), and the responses to prolonged current injections were indistinguishable from those observed in WT cells (Fig. 8C).

Figure 8
Repetitive firing is maintained in Kv4.2-/- neurons

Molecular basis of Kv current remodelling in Kv4.2-/- cortical pyramidal neurons

The electrophysiological finding that IA was eliminated in all Kv4.2-/- cortical pyramidal neurons and that there was no evidence for the presence of residual HpTx-sensitive (Kv4-encoded) channels was surprising given that Kv4.3 can form functional Kv channels in heterologous systems (Kim et al. 2007). These observations suggested the interesting possibility that the expression of Kv4.3 might be affected directly by the loss of Kv4.2. In addition, the voltage- and current-clamp data demonstrated the functional up-regulation of the delayed rectifier Kv currents, IK and Iss, in (most) Kv4.2-/- neurons, suggesting that the Kv α subunits encoding these channels might be up-regulated in Kv4.2-/- neurons/cortices. Previous studies suggest that, similar to other cell types, Kv2 α subunits encode IK in mouse cortical pyramidal neurons (Murakoshi & Trimmer, 1999; Du et al. 2000; Malin & Nerbonne, 2002; Pal et al. 2003; Guan et al. 2007), although the specific roles of the two Kv2.x pore-forming α subunits in this subfamily, Kv2.1 and Kv2.2, have not been defined.

Subsequent experiments were focused on testing the hypothesis that molecular remodelling occurs in Kv4.2-/- cortices. To determine the effects of the targeted deletion of Kv4.2 on the expression levels of Kv subunit proteins, Western blot analyses of tissue lysates and membrane proteins were completed. Representative Western blots of total protein isolates from WT and Kv4.2-/- cortices probed with several Kv pore-forming and accessory subunit-specific antibodies are presented in Fig. 9A and B. As illustrated in Fig. 9A, Kv4.3 expression levels were similar in WT and Kv4.2-/- cortices. The expression levels of several other Kv α subunits, specifically Kv1.4, Kv1.2, Kv2.1 and Kv2.2, were also indistinguishable in Kv4.2-/- and WT cortices (Fig. 9C), suggesting that these proteins are unaffected by the elimination of Kv4.2. Interestingly, however, the expression of the Kv4 channel accessory subunit, KChIP3 (An et al. 2000), was markedly reduced in Kv4.2-/- cortices (Fig. 9C), whereas the expression levels of two other KChIP proteins, KChIP1 and KChIP2, in WT and Kv4.2-/- cortices were not significantly different (Fig. 9C).

Figure 9
Marked reduction in KChIP3 expression in Kv4.2-/- cortices

Discussion

Targeted deletion of Kv4.2 eliminates IA in cortical pyramidal neurons

The experiments here were focused on determining directly the role of Kv4.2 α subunits in the generation of IA channels in cortical pyramidal neurons and on exploring the functional consequences of the in vivo elimination of IA on action potential waveforms and repetitive firing. These experiments revealed that there were no rapidly inactivating Kv currents in the vast majority (39 of 50; 80%) of Kv4.2-/- neurons and, in addition, that there were no rapidly recovering Kv currents in any (n = 50) Kv4.2-/- cortical pyramidal neurons. Interestingly, it was recently reported that IA channels were eliminated in the apical dendrites of Kv4.2-/- hippocampal CA1 pyramidal neurons (Chen et al. 2006). Taken together, these observations are consistent with previous suggestions that Kv4.2 encodes IA channels in both hippocampal and cortical pyramidal neurons (Sheng et al. 1992; Serodio & Rudy, 1998; Johnston et al. 2003; Jerng et al. 2004; Trimmer & Rhodes, 2004).

Although the elimination of IA with the deletion of Kv4.2 suggests that functional IA channels in mouse cortical pyramidal neurons are Kv4.2 homomers, it is possible that there are also heteromeric Kv4.2/Kv4.3 IA channels. It has been demonstrated that Kv4.3 is expressed in pyramidal neurons in mouse primary visual cortex (Burkhalter et al. 2006), and this subunit could contribute to the formation of functional homomeric or heteromeric IA channels. Because there were no residual rapidly recovering, heteropodatoxin-sensitive (i.e. Kv4.x-encoded) Kv currents in Kv4.2-/- cortical pyramidal neurons, however, the results presented here do suggest that Kv4.3 cannot produce functional IA channels in cortical pyramidal neurons in the absence of Kv4.2. Additional experiments will be necessary to test this hypothesis and to explore the functional role of Kv4.3 in the generation of IA channels directly.

Electrical remodelling in Kv4.2-/- cortical pyramidal neurons

In (most) Kv4.2-/- cortical pyramidal neurons, the densities of the delayed rectifier Kv currents, IK and Iss, were significantly higher than in WT cells, suggesting substantial ‘electrical’ remodelling owing to the elimination of IA. These observations clearly demonstrate that ID, IK and Iss reflect the expression of molecular entities distinct from Kv4.2. In addition, the finding that ID (unlike IK or Iss) is unaffected by the deletion of Kv4.2 and the elimination of IA suggests that the in vivo‘electrical’ remodelling in Kv4.2-/- cortical pyramidal neurons does not reflect a general, ‘non-specific’ increase in Kv currents, but rather is specific for IK and Iss. It is certainly possible, however, that other (non-Kv) currents, such as Ca2+-dependent K+ currents, or even inward Na+ and/or Ca2+ currents, are altered in Kv4.2-/- neurons. These possibilities were not examined directly in the experiments completed here because the recording conditions employed in the voltage-clamp experiments eliminated inward currents and Ca2+-dependent currents. In addition, because the experiments here were performed on pyramidal neurons isolated from young (P7–P10) neurons, it is certainly possible that remodelling in mature neurons could be distinct (more pronounced or possibly reversed) in neurons from mature animals. Similarly, it is possible that remodelling might be distinct in the dendrites of intact cortical pyramidal neurons.

The marked Kv current remodelling in Kv4.2-/- cortical neurons probably contributed to the fact that no overt phenotype was evident in these (Kv4.2-/-) mice, an observation that was initially surprising given the dramatic cellular phenotypes evident in isolated (hippocampal and cortical) neurons expressing a mutant Kv4 (dominant negative) construct that also resulted in the elimination of IA (Kim et al. 2005; Yuan et al. 2005). In addition, attenuation of IA has been linked to acquired experimental temporal lobe epilepsy (Bernard et al. 2004) and a Kv4.2 truncation mutant has been identified in a patient with temporal lobe epilepsy (Singh et al. 2006). Although effects on the back propagation of action potentials into the dendrites of Kv4.2-/- CA1 hippocampal neurons and on the inducibility of hippocampal long-term potentiation have been reported, the effects were rather modest (Chen et al. 2006). These observations, in light of the findings here, may suggest that electrical remodelling also occurs in Kv4.2-/- hippocampus and compensates for the elimination of IA. Further experiments will be necessary to explore this hypothesis directly.

In a subset (11 of 50 or ~20%) of cells from Kv4.2-/- visual cortices, a rapidly inactivating Kv current that appeared initially to be similar to IA in WT cells was evident. Further analyses, however, revealed that the rapidly inactivating Kv currents in these (Kv4.2-/-*) cells recovered slowly from inactivation and were insensitive to the Kv4 channel-specific toxin, HpTx-3. Taken together, therefore, the results here demonstrate that IA was indeed eliminated in all Kv4.2-/- cortical pyramidal neurons. The results of the HpTx-3 experiments suggest that the presence of the rapidly inactivating, slowly recovering Kv current in this subset (~20%) of Kv4.2-/- pyramidal cells also reflects electrical remodelling and further that the molecular basis of this remodelling is distinct from that observed in the vast majority (80%) of Kv4.2-/- cells.

Functional consequences of the elimination of IA in Kv4.2-/- neurons

Action potential waveforms in Kv4.2-/- visual cortical pyramidal neurons were not significantly different from those measured in WT cells. The currents required to evoke action potentials, as well as action potential durations and the amplitudes and durations of afterhyperpolarizations in Kv4.2-/- and WT mouse visual cortical pyramidal neurons, were indistinguishable. These observations are distinct from those obtained in previous in vitro studies on isolated (rat) cortical and hippocampal (CA1) pyramidal neurons, transfected with the dominant negative Kv4.2 construct, Kv4.2 DN (Kim et al. 2005; Yuan et al. 2005). In isolated Kv4.2 DN-expressing neurons examined 24–48 h after transfection, IA was eliminated, cell input resistances were increased, and the currents required to evoke action potentials were reduced (Yuan et al. 2005). The functional ‘knockout’ of IAin vitro also resulted in markedly increased action potential durations, and the amplitudes and durations of afterhyperpolarizations were reduced (Kim et al. 2005; Yuan et al. 2005). Repetitive firing was also affected in Kv4.2 DN-expressing neurons: in response to low amplitude current injections, Kv4.2 DN-expressing neurons fired at higher frequencies than WT cells, whereas at high stimulus intensities, the excitability of these cells was reduced (Yuan et al. 2005). In hippocampal pyramidal neurons expressing Kv4.2 DN, frequency-dependent action potential broadening during repetitive firing was increased and back-propagation of action potentials into dendrites was enhanced relative to WT cells (Kim et al. 2005). Interestingly, overexpression of wild-type Kv4.2 in hippocampal pyramidal neurons also reportedly reduced frequency-dependent action potential broadening and inhibited dendritic back-propagation (Kim et al. 2005).

The finding here that action potential waveforms in Kv4.2-/- and WT cells are indistinguishable suggests that the Kv current remodelling observed in Kv4.2-/- cells negates the effects of the loss of IA. Action potential clamp experiments, focused on testing this hypothesis directly, indeed revealed that the densities of the total outward Kv currents evoked during single action potentials in WT and Kv4.2-/- neurons were not significantly different. In addition, the densities of the TEA-sensitive Kv currents evoked during single action potentials were much larger in Kv4.2-/-, compared with WT, cells. Taken together, these results suggest that the up-regulation of the TEA-sensitive Kv currents, IK and Iss, underlies the observed maintenance of action potential waveforms in Kv4.2-/- neurons.

Repetitive firing was also maintained in Kv4.2-/- neurons. These observations are also distinct from those observed in Kv4.2 DN cells (Kim et al. 2005; Yuan et al. 2005), clearly suggesting that the in vivo Kv current remodelling in Kv4.2-/- cells results in the maintained ability to fire repetitively (and at high frequencies) in spite of the loss of IA.

Molecular basis of electrical remodelling in Kv4.2-/- cortical neurons

Biochemical experiments completed here revealed that the expression levels of several Kv α subunit proteins, including Kv2.1 and Kv2.2, were similar in WT and Kv4.2-/- cortices. If the hypothesis is correct that Kv2 α subunits encode endogenous IK channels in WT mouse cortical pyramidal neurons and the up-regulated IK in Kv4.2-/- neurons, the findings that Kv2.1 and Kv2.2 protein expression levels were unaffected in Kv4.2-/- cortices suggests that post-translational mechanisms probably underlie the increased IK densities in Kv4.2-/- cells. The biochemical experiments here, however, were performed on protein preparations from whole mouse cortices, and it is possible, therefore, that changes in Kv2.x α subunit expression in pyramidal neurons might have gone undetected. If remodelling is different in the dendrites of cortical pyramidal neurons, this would also complicate the interpretation of the biochemical data presented, particularly in the context of the electrophysiological remodelling, described in P7–P10 cortical pyramidal neurons examined in vitro 24–48 h after isolation. Clearly, further experiments focused on determining the molecular basis of Kv current remodelling at the cellular level will be necessary to test these hypotheses directly. Further studies focused on defining the molecular basis of Iss channels in WT neurons and the up-regulated Iss in Kv4.2-/- neurons are also needed.

The biochemical experiments completed here also revealed that the expression of the KChIP3 protein is reduced markedly in Kv4.2-/- cortices, whereas the expression levels of the KChIP1 and KChIP2 proteins in WT and Kv4.2-/- cortices are not significantly different. These observations are particularly interesting in light of the recent report that the expression levels of the various KChIP isoforms are down-regulated in Kv4.2-/- (compared with WT) brains in a region- and cell type-specific manner (Menegola & Trimmer, 2006). The marked and selective reduction in KChIP3 expression seen here, therefore, was unexpected and, in addition, suggests that, in the absence of Kv4.2, the KChIP3 protein is targeted for degradation. Contrary to previous suggestions (based on immunohistochemical data) that KChIP2 and KChIP4 associate with Kv4.2 (Rhodes et al. 2004; Trimmer & Rhodes, 2004), the results here suggest that KChIP3 is probably the accessory subunit of Kv4.2-encoded IA channels in cortical pyramidal neurons. Interestingly, the loss of the KChIP3 protein is also correlated observationally with the increase in IK and Iss densities, a finding that suggests the interesting possibility that there is also a mechanistic link.

Acknowledgments

The authors thank Ms. Amy Coleman for expert technical assistance in the preparation and maintenance of glial and neuronal cultures, and Mr. Rick Wilson for maintaining and genotyping mice, and for his assistance with the figures. The financial support provided by the National Institutes of Health and Washington University Medical School is gratefully acknowledged.

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