Elimination of the Fast Transient in Superior Cervical Ganglion Neurons with Expression of KV4.2W362F: Molecular Dissection ofI_A

Multiple components of the outward K⁺ currents in SCG neurons

In the experiments here, neurons were isolated from the SCG of E21 or P1 rats, plated on glial monolayers, and maintained in vitro. Whole-cell voltage-gated K⁺currents were recorded in the presence of 1 μmTTX and 0.1 mm CdCl₂ to block voltage-gated Na⁺ and Ca²⁺ currents, respectively. Representative outward currents recorded from (three) isolated SCG neurons in response to brief (125 msec) and prolonged (6 sec) membrane depolarizations to varying test potentials from a holding potential of −90 mV are presented in Figure 1. The rates of rise and the amplitudes of the currents increase with increasing membrane depolarization; the largest and most rapidly activating current in each panel of Figure 1 was evoked at +50 mV. No voltage-gated K⁺ currents were recorded when the K⁺ in the recording pipettes was replaced with Cs⁺ (n = 9). The currents recorded (Fig. 1) and analyzed here, therefore, are assumed to reflect only the currents through Ca²⁺-independent voltage-gated K⁺ channels.

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Fig. 1.

Voltage-gated outward K⁺current waveforms in SCG neurons. Whole-cell outward K⁺ currents were recorded from isolated SCG neurons in response to 125 msec (left panels) and 6 sec (right panels) depolarizing voltage steps from a holding potential of −90 mV. Experiments were conducted as described in Materials and Methods with 1 μm TTX and 100 μm CdCl₂ in the bath solution to block voltage-gated inward Na⁺ and Ca²⁺currents, respectively. The left and right panels in A–C were recorded from the same cell. There are distinct and stereotyped differences in the waveforms of the currents in Type I, II, and III cells (see Results and Table 1). There is a prominent rapid component of current decay in Type I (A) and II (B) cells that is not evident in Type III (C) cells; current activation is also slower in Type III (C) cells (see Results).

As is clearly evident in Figure 1, the amplitudes and the waveforms of the outward K⁺ currents vary markedly among SCG neurons. Nevertheless, outward K⁺ current waveforms in SCG cells are stereotyped, and the records shown in Figure1A–C are representative of the three distinct phenotypes observed; these are referred to as Type I (A), II (B), or III (C). In the majority (>90%) of cells studied, currents similar to those in Figure 1, A and B, were recorded. There is a prominent rapid component of current decay in these (Type I and Type II) cells (right panels), consistent with the presence of a fast transient outward “A” K⁺ current (Belluzzi et al., 1985a; Storm, 1990; McFarlane and Cooper, 1992). This current is referred to here asI_A,fast, orI_Af, to distinguish it from another, more slowly inactivating, transient outward K⁺ currentI_A,slow(I_As) that is also seen in some SCG cells (see below). Analysis of the outward K⁺ currents evoked during long depolarizations in Type I SCG cells (Fig. 1A,right panel) revealed that current decay is well described by the sum of two exponentials, with decay time constants (τ_decay) that differ by more than an order of magnitude, and a noninactivating steady-state current (see below). The mean ± SEM τ_decay (n = 30) determined for the fast and slow components of decay in these (Type I) cells were 121 ± 14 and 2560 ± 187 msec, respectively (Table 1). Neither time constant displays any appreciable voltage dependence (data not shown). Two K⁺ currents with similar τ_decay values have been described previously in rat sympathetic neurons and were assumed to reflect the expression of distinct K⁺ channels (McFarlane and Cooper, 1992). The rapidly inactivating current has been referred to asI_A, orI_Af (Freshi, 1983; Galvan and Sedlmeir, 1984; Belluzzi et al., 1985a; Nerbonne et al., 1986;McFarlane and Cooper, 1992; Wang and McKinnon, 1995), and the slowly inactivating current has been given various names, includingI_K (Galvan and Sedlmeir, 1984;Belluzzi et al., 1985b; Nerbonne and Gurney, 1989) andI_A,slow (McFarlane and Cooper, 1992). The very slowly decaying current component (τ_decay = 2560 ± 187 msec) is referred to here as I_K to emphasize the slow inactivation kinetics and to distinguish this conductance pathway fromI_As (see below).

Although the waveforms of the currents in individual (Type I) SCG cells are similar, the densities of I_Af andI_K vary considerably. For example,I_Af density at +50 mV ranged from 70 to 208 pA/pF, with a mean ± SEM of 81 ± 11 pA/pF (n = 30) (Table 1). Similarly,I_K densities at +50 mV in Type I cells varied considerably, ranging from 83 to 258 pA/pF with a mean ± SEM of 108 ± 12 pA/pF (Table 1). The density of the noninactivating K⁺ current component remaining at the end of 6 sec voltage steps (Fig. 1),I_SS (steady-state), is also variable; at +50 mV, for example, I_SS density ranged from 35 to 127 pA/pF, with a mean ± SEM of 74 ± 5 pA/pF (n = 30) (Table 1).

In some (9) of the 43 SCG cells studied, outward K⁺ current waveforms similar to those displayed in Figure 1B were observed. In these (Type II) cells, the decay phases of the outward K⁺ currents were not well described by biexponential functions, and three exponentials were required to adequately describe the data. The mean ± SEM τ_decay values derived from these fits were 95 ± 8, 480 ± 21, and 2800 ± 193 msec (Table 1). The fast τ_decay (95 ± 8 msec) and the very slow τ_decay (2800 ± 193 msec) are not significantly different from those determined forI_Af andI_K, respectively, in Type I cells. It is assumed, therefore, that these time constants also reflect expression of I_Af andI_K in Type II cells (see below). The component of current inactivation in Type II cells with a τ_decay = 480 ± 21 msec is referred to asI_A,slow, orI_As, and is assumed to reflect the expression of a novel conductance pathway, distinct fromI_A,fast,I_K, andI_SS (see below and Discussion). Although I_Af densities are similar in Type I and Type II cells, the densities ofI_K andI_SS are significantly (p < 0.005) lower in Type II than in Type I cells (Table 1).

In the other four (of 43 or ∼9%) SCG cells (Type III) studied, no rapidly inactivating K⁺ currents (similar to I_Af and/orI_As) were evident (Fig.1C). The decay phases of the outward currents in these cells were well described by a single exponential with a mean ± SEM τ_decay of 2200 ± 176 msec (Table 1). Peak outward K⁺ current densities in Type III cells, which lack both I_Af andI_As (Table 1), are lower than in Type I and II cells (Table 1). In addition, in Type III cells,I_SS contributes substantially more to the peak outward current than does I_SSin Type I or II cells (Table 1). In other respects, however, the properties of Type III cells are indistinguishable from Type I and II cells. The mean ± SEM whole-cell membrane capacitances and input resistances, for example, were 33 ± 3 pF and 0.52 ± 0.08 GΩ for the cells with I_Af(n = 39), and 25 ± 2 pF and 0.60 ± 0.04 GΩ for the cells lacking I_Af(n = 4).

I_Af is selectively attenuated in SCG neurons expressing Kv4.2W362F

On the basis of the kinetic properties ofI_Af and the sensitivity of this current to 4-AP, it seemed reasonable to suggest that this conductance pathway reflects the expression of Kv4 α-subunits. Consistent with this hypothesis, previous studies have documented the expression of Kv4.1, Kv4.2, and Kv4.3 mRNAs in SCG neurons (Dixon and McKinnon, 1996;Pankevych et al., 1999). In addition, immunohistochemical experiments with anti-Kv4 α-subunit-specific antibodies reveal the expression of these subunits in isolated SCG neurons (Fig.2). As evident in Figure2A, for example, Kv4.2 expression is readily detected in the cell bodies of isolated SCG neurons. The expression pattern for Kv4.3 is distinct (from that of Kv4.2): Kv4.3 staining is seen throughout the processes of isolated SCG neurons (Fig.2B). In addition, the anti-Kv4.3 labeling appears to be punctate (Fig. 2B), suggesting “clustering” of Kv4.3-encoding K⁺ channels. A similar staining pattern is seen with an anti-Kv4 pan antibody, which also strongly labels cell bodies and proximal processes (Fig.2C).

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Fig. 2.

Expression of Kv4 α-subunits in SCG neurons. Isolated wild-type SCG neurons were examined immunohistochemically 48 hr after plating, as described in Materials and Methods. Cultures were stained with anti-Kv4.2 (A), anti-Kv4.3 (B), and a pan-Kv4 antibody (C). Both Kv4.2 and Kv4.3 are readily detected in SCG neurons; Kv4.2 is localized to the cell bodies and proximal processes, whereas Kv4.3 is also detected in more distal processes. In addition, the anti-Kv4.3 appears more punctate. Scale bars, 50 μm.

To test the hypothesis that Kv4 α-subunits underlieI_Af in SCG neurons, cells were transfected with a pore mutant of Kv4.2, Kv4.2W362F, that functions as a dominant negative (Barry et al., 1998). Previous studies have shown that coexpression of Kv4.2W362F with either Kv4.2 or Kv4.3 attenuates current amplitudes relative to cells expressing (wild-type) Kv4.2 or Kv4.3 alone (Barry et al., 1998). In the experiments here, beads were coated either with cDNA constructs encoding Kv4.2W362F and EGFP or with the EGFP cDNA alone, and cells were transfected using the biolistics gene gun (see Materials and Methods). Within ∼24 hr of transfection, EGFP expression was readily detected under epifluorescence illumination (Fig.3A); ∼10% of the cells in these cultures were EGFP positive. To determine whether EGFP-positive cells in cultures exposed to beads coated with Kv4.2W362F (and EGFP) also express the transgene, the cultures were fixed ∼48 hr after transfection and probed with the anti-FLAG M2 antibody. These experiments revealed that all EGFP-positive cells in these cultures (n = 112) also express Kv4.2W362F-FLAG. An example of an EGFP-positive, Kv4.2W362F-positive cell is illustrated in Figure3B. As is evident, the FLAG staining appears to be predominantly on the cell surface (arrow), whereas EGFP expression is detected in the cytosol (Fig. 3B). In addition, EGFP appears to fill the entire cell (Fig.3B).

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Fig. 3.

Immunohistochemical detection of Kv4.2W362F-FLAG in transfected SCG neurons. Isolated SCG neurons, transfected with EGFP alone (A) or with Kv4.2W362F-FLAG and EGFP (B) using the gene gun, were fixed and stained 24 hr later (see Materials and Methods). A,B, EGFP fluorescence (left panels) and Cy3 fluorescence (right panels) images of the same field. Anti-FLAG staining is only evident in cultures transfected with Kv4.2W362F-FLAG expression (compare right panels inA and B). In addition, EGFP expression correlates with Kv4.2W362F (compare left andright panels in B). Scale bar, 50 μm.

Representative whole-cell voltage-gated outward K⁺ current waveforms recorded from SCG neurons expressing Kv4.2W362F (and EGFP) are presented in Figure4. Control experiments revealed that the outward K⁺ currents in cells expressing EGFP alone are indistinguishable from those in wild-type cells (Fig.1). The waveforms of the outward K⁺currents in cells expressing Kv4.2W362F (Fig. 4), however, are very different from those recorded from wild-type cells. Specifically, the rapid component of current decay, I_Af, that is prominent in wild-type Type I (Fig. 1A) and Type II (Fig. 1B) SCG cells appears to be missing in (all) cells expressing Kv4.2W362F (Fig. 4). In contrast to the results obtained on wild-type and EGFP-expressing cells (Table 1), the decay phases of the outward currents in the majority (18 of 31, 58%) of Kv4.2W362F-expressing cells (Fig. 4A) were well described by a single exponential (τ_decay = 2205 ± 235 msec) and a steady-state outward current (Table 1). The waveforms of the currents in these cells (Fig.4A) are indistinguishable from wild-type Type III cells (Fig. 1C), suggesting expression of onlyI_K andI_SS (Table 1). In the remaining Kv4.2W362F-expressing cells (Fig.4B,C), two exponentials were required to fit the decay phases of the currents. In 7 of 31 cells, current waveforms similar to those in Figure 4B were recorded. Analysis of the decay phases of the outward K⁺ currents in these cells provided mean ± SEM τ_decay values of 490 ± 31 and 2473 ± 254 msec, consistent with the presence ofI_As andI_K;I_SS is also evident in these cells (Table 1). Although these (Fig. 4B) cells represent a novel (nonphysiological) class of SCG neurons, the percentage (22%) of cells with this phenotype and the distribution of current densities suggest that these are Type II cells that lack the fast transient current component, I_Af, attributable to the expression of Kv4.2W362F. Interestingly, the densities ofI_K andI_SS in these cells (Table 1) are significantly (p < .002) higher than in wild-type Type II cells (Table 1), suggesting thatI_K andI_SS are upregulated in Type II cells when I_Af is eliminated (see Discussion).

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Fig. 4.

I_Af is eliminated in SCG neurons expressing Kv4.2W362F-FLAG. Whole-cell depolarization-activated outward K⁺ currents were recorded from isolated SCG neurons transfected with Kv4.2W362F-FLAG as described in the legend to Figure 1. The left andright panels in A–C were recorded from the same cell. The waveforms of the currents recorded from cells expressing Kv4.2W362F are distinct from those recorded from wild-type cells or from cells expressing EGFP alone (Fig. 1). In the majority of cells (A, B), current activation and inactivation are slow, consistent with the absence ofI_Af (see Results). In a small subset of Kv4.2W362F-expressing cells (C), a rapid component of decay with a mean ± SEM τ_decay that is not significantly different from that determined forI_Af in wild-type Type I SCG cells (Table 1) is evident (see Results).

In the remaining Kv4.2W362F-expressing SCG cells (n = 6), a rapid component of outward current decay is detected (Fig.4C), suggesting that I_Af is unaffected by Kv4.2W362F expression in a subset of SCG cells. Analysis of the currents in records such as those in Figure 4Crevealed that the mean ± SEM density of this component is lower and the mean ± SEM τ_decay is longer than those determined for I_Af in wild-type Type I and II cells (Table 1). These observations suggest that there are actually two components of I_Af, only one of which is encoded by Kv4 α-subunits (and therefore affected by Kv4.2W362F expression) or, alternatively, that a novel current is upregulated in this subset of SCG cells (Fig. 4C) when I_Af is eliminated. The time constants of inactivation of currents in records such as those in Figure 4C varied over the range 138–249 msec. Although this range is similar to that (60–208 msec) observed in wild-type Type I and II cells, it is narrower, and the distributions of time constants are quite different (Fig. 5). In the Kv4.2W362F-expressing cells, τ_decay values fall between 138 and 249 msec, whereas in wild-type cells, there appear to be two distinct groups of cells, i.e., those with τ_decay values in the 50–130 msec range (26 of 39; 66%) and those with τ_decay values between 131 and 210 msec (13 of 39; 33%). These observations further suggest that only the faster current (τ_decay 50–130 msec) is encoded by Kv4 α-subunits (see Discussion).

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Fig. 5.

Two components of I_Afin SCG neurons. The time constants of inactivation of the rapid component of current decay were determined in wild-type, Kv4.2W362F-, and Kv4.2-expressing SCG cells, as described in Materials and Methods. The τ_decay values were binned in 40 msec increments for comparison purposes, and, as is evident, the distributions of τ_decay values are distinct (see Results).

Action potential waveforms and repetitive firing in SCG neurons

Subsequent experiments were aimed at evaluating the role ofI_Af in shaping the waveforms of individual action potentials and in determining the repetitive firing properties of SCG neurons. Although the waveforms of the action potentials recorded from wild-type SCG neurons are similar, the responses to prolonged (500 msec) depolarizing current injections are distinct (Fig.6A–C). In ∼45% of the SCG cells examined, phasic firing was observed, i.e., cells fire one or two action potentials in response to prolonged current injections (Fig. 6A, middle panel), and the number of action potentials elicited is unaffected by increasing the amplitude of the injected current (Fig.6A, right panel). Repetitive firing was observed, however, in the remaining ∼55% of the SCG cells studied (Fig. 6B,C). In approximately half of these cells, low amplitude current injections produce repetitive firing, and the frequency of firing decreases as a function of time after the onset of the current injection, i.e., these cells “adapt” (Fig. 6B, middle panel). When the amplitude of the injected current is increased, adapting cells become refractory and firing ceases, despite the maintained current injection (Fig. 6B,right panel). The remaining subset of cells displayed “tonic” activity, firing regularly spaced action potentials in response to prolonged depolarizing current injections (Fig.6C, middle panel). In addition, in tonic cells, the frequency of firing increases with the amplitude of the injected current (Fig. 6C, right panel). No refractoriness is evident in tonic cells during current injections ranging from 20 to 300 pA, clearly distinguishing these cells from both phasic and adapting neurons.

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Fig. 6.

Distinct repetitive firing patterns in SCG neurons. Action potentials and repetitive firing patterns were recorded from isolated EGFP-expressing SCG neurons in response to brief or prolonged depolarizing current injections, as described in Materials and Methods. Current-clamp recordings from three representative cells are shown in A–C. In each cell, single action potentials were elicited by 1.5 msec depolarizing current injections (left panels), and repetitive firing patterns were recorded in response to 500 msec depolarizing currents injections of 100 pA (middle panels) or 200 pA (right panels). Based on the response(s) to the 500 msec current injections, cells were classified as phasic (A), adapting (B), or tonic (C) (see Results and Table 2).

The properties of phasic, adapting, and tonic cells are quite similar, despite the marked differences in repetitive firing patterns (Fig. 6). Resting membrane potentials and action potential amplitudes, for example, are indistinguishable in phasic, adapting, and tonic SCG cells (Table 2). Action potentials recorded from tonic cells, however, are significantly (p< 0.001) briefer than in either phasic or adapting cells (Table 2). At 90% repolarization, for example, the mean ± SEM action potential duration (APD₉₀) in tonic cells is 4.87 ± 0.20 msec, whereas mean ± SEM APD₉₀ values determined in phasic and tonic cells were 5.96 ± 0.20 and 6.55 ± 0.34 msec, respectively (Table 2). In addition, the input resistances of adapting cells are significantly (p < 0.001) higher than those of either phasic or tonic cells (Table 2). Consistent with this difference in input resistance, the mean ± SEM current threshold (24 ± 2 pA) for action potential generation is significantly (p < 0.003) lower in adapting cells than in phasic (67 ± 1 pA) or tonic (42 + 2 pA) cells (Table 2). Subsequent experiments were aimed at determining directly the role ofI_Af in mediating the phasic, tonic, and adapting firing patterns of SCG neurons by examining the effects of Kv4.2W362F expression on action potentials and repetitive firing.

Elimination of I_Af increases excitability (and percentage) of “adapting” SCG cells

Previous pharmacological studies suggest a primary role forI_Af in the regulation of excitability and in action potential repolarization in SCG neurons (Galvan and Sedlmeir, 1984; Belluzzi et al., 1985a; Nerbonne and Gurney, 1989). Application of 4-AP, often assumed to be a specific blocker ofI_Af, for example, reportedly increases the excitability of SCG neurons (Galvan and Sedlmeir, 1984). In the presence of 4-AP, the current threshold for action potential generation and the latency to firing are reduced, and action potentials are prolonged (Galvan and Sedlmeir, 1984; Belluzzi et al., 1985a; Nerbonne and Gurney, 1989). In some studies, the input resistances of SCG neurons were also reportedly increased after exposure to 4-AP (Galvan and Sedlmeir, 1984; Belluzzi et al., 1985a). If all of these effects are attributable to loss of I_Af, then similar changes in excitability and action potential durations would be expected to be observed in SCG neurons expressing Kv4.2W362F (and lacking I_Af).

Representative current-clamp recordings from SCG cells expressing Kv4.2W362F are presented in Figure 7. As in recordings from wild-type (or EGFP-expressing) SCG cells, the phasic, adapting, and tonic firing patterns were also observed in SCG neurons expressing Kv4.2W362F. In contrast to control cells, however, the majority (52%) of Kv4.2W362F-expressing cells are adapting (Fig.7B). This increase in the number of adapting cells reflects a decline in the fraction of neurons displaying the phasic phenotype from 43% of wild-type neurons (Fig. 6B, Table 2) to 24% of Kv4.2W362F-expressing cells (Fig. 7B, Table 3). The percentage of tonic cells, in contrast, is unaffected by loss ofI_Af: ∼25% of wild-type (Fig.6C, Table 2) and Kv4.2W362F-expressing (Fig. 7C, Table 3) cells fire tonically. These observations suggest that lowI_Af density correlates with the adapting phenotype (see Discussion). In addition to the redistribution of cells, the input resistances of phasic and tonic cells expressing Kv4.2W362F (Table 3) are significantly (p < 0.001) higher than in wild-type (phasic and tonic) cells (Table2), whereas the input resistances in adapting cells expressing Kv4.2W362F were not significantly different from wild-type adapting cells (Table 2). Indeed, the input resistances of Kv4.2W362F-expressing phasic, adapting, and tonic SCG cells are similar (Table 3). Consistent with this increase in input resistance, the current thresholds for firing single action potentials (and trains) in Kv4.2W362F-expressing phasic and tonic cells are significantly (p < 0.003) lower (Table 3) than in wild-type (phasic and tonic) cells (Table 2).

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Fig. 7.

Elimination of I_Afincreases the percentage of adapting cells. Action potentials and repetitive firing patterns were recorded, as described in the legend to Figure 6, from isolated SCG neurons expressing Kv4.2W362F. Records from three representative cells are shown inA–C. As in wild-type cells (Fig. 6), the phasic, adapting, and tonic firing patterns are observed in Kv4.2W362F-expressing cells. However, the percentage of adapting cells is increased markedly, and the percentage of phasic cells is decreased (Table 3) relative to the distribution of firing patterns in wild-type cells (Table 2).

In contrast to the marked effects on excitability, action potential durations are not affected measurably by elimination ofI_Af (Table 3). These observations suggest that I_Af does not play a prominent role in determining action potential durations in SCG neurons (see Discussion). Tonic cells expressing Kv4.2W362F, however, fire at higher frequencies (Fig. 7) than their wild-type counterparts (Fig.6) in response to current injections of the same amplitude. In response to a 200 pA current injection, for example, wild-type tonic cells fire at mean ± SEM frequencies of 15 ± 1 Hz, whereas Kv4.2W362F-expressing cells fire at 29 ± 2 Hz. The likely interpretation of this observation is the fact that the current thresholds for action potential generation are decreased in cells expressing Kv4.2W362F (Table 3) as compared with wild-type cells (Table2).

Increasing I_Af reduces the number of adapting cells

The results of the experiments above suggest that lowI_Af density is an important determinant of the adapting phenotype. To further test this hypothesis, the effects of increasing I_Af density on the firing properties of SCG neurons were examined. In these experiments, SCG neurons were transfected with a construct encoding wild-type Kv4.2 (as well as EGFP), and current-clamp recordings were obtained from EGFP-positive cells. These experiments revealed firing patterns (Fig. 8) that are indistinguishable from those of wild-type (and EGFP-expressing) SCG neurons (Fig. 6), although the distribution of cells displaying the various firing patterns is quite distinct (Table4). Half of the cells transfected with the Kv4.2 construct are tonic (Fig. 8C), only 14% of the cells are adapting (Fig. 8B), and the remaining 36% of the cells are phasic (Fig. 8A). There is a marked increase in tonic cells and a corresponding decrease in the number of adapting cells with overexpression of Kv4.2 (Fig. 8B, Table 4). The properties of the Kv4.2-expressing cells that are adapting (Fig. 8B) are also distinct from those of wild-type adapting cells (Table 2) in that the current threshold for action potential generation is increased significantly (p < 0.003) by Kv4.2 expression (Table 4), consistent with a decrease in input resistance (Table 4). These results suggest that all adapting cells are converted to phasic or tonic cells as a function of I_Af density (see Discussion). Analysis of the waveforms of evoked action potentials also revealed that APD₅₀ values (Table 4) in Kv4.2-expressing phasic cells are significantly (p < 0.001) shorter than in wild-type phasic cells (Table 2). In contrast, mean action potential durations in adapting and tonic SCG cells overexpressing Kv4.2 are not significantly different from those in wild-type (adapting and tonic) SCG cells (compare Table 2 and Table 4).

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Fig. 8.

Increased I_Af density decreases action potential durations and increases the percentage of tonic SCG neurons. Action potentials and repetitive firing patterns, recorded as described in the legend to Figure 6, were obtained from isolated SCG neurons 24 hr after transfection with wild-type Kv4.2. Records from three representative cells are shown inA–C. As in wild-type cells, the phasic (A), adapting (B), and tonic (C) firing patterns were seen in Kv4.2 overexpressing cells. In cultures overexpressing Kv4.2, however, the percentage of adapting cells is lower and the percentage of tonic cells is higher than seen in wild-type (Table 2) or Kv4.2W362F-expressing (Table 3) SCG cells.

Analysis of voltage-clamp recordings from EGFP-positive cells in Kv4.2-transfected cultures revealed that the density ofI_Af is increased relative to the currents in wild-type (and EGFP-expressing) SCG neurons. In all cells expressing wild-type Kv4.2, a prominent fast transient current is evident (Fig. 9). Analysis of the decay phases of the outward currents revealed that inactivation is best described by the sum of two (Fig. 9A) or three (Fig.9B) exponentials. The mean ± SEM decay time constants derived from these fits are similar to those determined in wild-type I and II SCG cells (Table 1). No Type III neurons, which lackI_Af andI_As (Table 1), were seen among the cells expressing Kv4.2. In the majority (69%) of the cells overexpressing Kv4.2, I_Af,I_K, andI_SS are detected (Table 1). The waveforms of the currents (Fig. 9A) are similar to those in wild-type I cells (Fig. 3A), althoughI_Af density is significantly (p < 0.04) higher andI_K andI_SS densities are significantly (p < 0.005) lower than in wild-type Type I cells (Table 1). In the remaining Kv4.2-expressing cells, three exponentials were required to fit the decay phases of the outward currents. These cells (Fig. 9B) are similar to wild-type Type II cells, expressing I_Af,I_As,I_K, andI_SS, althoughI_Af density is significantly (p < 0.04) higher in the Kv4.2-expressing cells (Table 1).

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Fig. 9.

Overexpression of Kv4.2 increasesI_Af density in SCG neurons. Isolated SCG neurons were transfected with Kv4.2 (and EGFP), and outward K⁺ currents were recorded from EGFP-expressing cells as described in the legend to Figure 1. Records similar to those inA and B were obtained from all cells (Table 1); no cells lacking I_Af were evident. Analysis of the decay phases of the currents provided the mean ± SEM densities of I_Af,I_As,I_K, andI_SS (Table 1), and mean ± SEMI_Af densities were increased significantly in both Type I (A) and Type II (B) SCG cells as compared with wild-type Type I and II cells (Table 1).

Kv4 α-subunits underlie I_Af in SCG neurons

In the experiments here, four kinetically distinct voltage-gated K⁺ currents have been distinguished in SCG neurons: two transient currents, I_Afand I_As, as well as a slowly inactivating current, I_K, and a steady-state current, I_SS (Table 1). In addition, these currents are differentially distributed, and SCG cells have been classified as Type I, II, or III based on expression differences. Type I cells express I_Af,I_K, andI_SS, whereas Type II cells expressI_Af,I_As,I_K, andI_SS, and Type III cells express onlyI_K andI_SS. There is considerable variability in current densities within each grouping (Table 1), and interestingly,I_K andI_SS densities are significantly (p < 0.005) lower in Type II cells (the only cells that express I_As) than in either Type I or III cells (Table 1).

In experiments aimed at testing the hypothesis that α-subunits of the Kv4 subfamily of voltage-gated K⁺ channels underlie I_Af in SCG neurons, the biolistics method (gene gun) was used to introduce a mutant Kv4.2 α-subunit, Kv4.2W362F, that functions as a dominant negative (Barry et al., 1998), into these cells in vitro. Voltage-clamp recordings from Kv4.2W362F-expressing cells revealed that in the vast majority (∼80%) of cells no fast transient outward currents (I_Af) were detected (Fig. 3, Table 1). The densities of the other currents,I_As,I_K, andI_SS, are largely unaffected by Kv4.2W362F expression, although I_Kdensity is significantly higher in theI_As-expressing subset of these cells (Table 1, column B) than in wild-type Type II cells (Table 1). The simplest interpretation of these findings is thatI_Af is encoded by Kv4 α-subunits in most SCG neurons. Consistent with this hypothesis,I_Af was evident in all cells expressing wild-type Kv4.2 (Fig. 9), and meanI_Af density was significantly (p < 0.04) higher than in wild-type cells (Table 1).

Nevertheless, a rapidly inactivating current remains in ∼20% (6 of 31) of the SCG cells expressing Kv4.2W362F (Table 1). The density of this fast transient current is somewhat lower thanI_Af density in wild-type Type I or II cells (Table 1), which could reflect incomplete removal of Kv4-induced currents. Alternatively, it is possible that there are two populations of I_Af channels, one generated by Kv4 α-subunits and the other by another Kv subfamily. Analysis of the decay phases of the currents revealed that τ_decay values forI_Af in wild-type Type I and II cells are quite variable, ranging from 53 to 209 msec, and that there appear to be two populations of cells (Fig. 5). In cells expressing Kv4.2W362F, in contrast, the τ_decay for the rapid component of current decay ranged from 139 to 254 msec; there were no cells with τ_decay values in the 50–130 msec range (Fig. 5). In Kv4.2-expressing SCG cells, the τ_decay values forI_Af are all < 130 msec (Fig. 5). Because the mean ± SEM τ_decay values forI_Af recorded from Kv4.2- and Kv4.2W362F-expressing cells are significantly different (p < 0.001) and the distribution of τ values is nonoverlapping (Fig. 5), it seems reasonable to suggest that there are two distinct types of I_Af channels in SCG neurons that reflect the functional expression of distinct gene products. Interestingly, the identification of two molecularly distinct components of a “single” electrophysiologically defined current has been reported previously for I_Kr inXenopus spinal neurons (Ribera, 1996) and forI_{K, slow} in mouse ventricular myocytes (Xu et al., 1999b). Recent studies have also revealed two components of recovery of I_Af from steady-state inactivation with time constants of ∼100 and ∼1000 msec (n = 2), observations consistent with the suggestion that there are two components I_Af. The more rapidly inactivating currents (mean τ_decay∼ 80 msec) present in the majority (80%) of (Type I and II) SCG neurons reflect the expression of Kv4 α-subunits. Although the molecular identity of the fast transient currentI_Af remaining in (∼20%) Kv4.2W362F-expressing cells has not been defined, one potential candidate is Kv1.4, a Kv α-subunit that also produces A-type K⁺ currents when expressed in heterologous systems (Rudy, 1988). Importantly, Kv1.4 mRNA expression has been documented in SCG neurons (Dixon and McKinnon, 1996). Further experiments aimed at testing directly the hypothesis that Kv1.4 underlies the more slowly inactivating component ofI_Af are clearly warranted.

I_Af density and neuronal excitability

Whole-cell current-clamp recordings from SCG neurons revealed distinct repetitive firing patterns, and cells were classified as phasic, adapting, or tonic based on stereotyped differences in the response to prolonged current injections (Fig. 6). The waveforms of individual action potentials recorded in phasic, tonic, and adapting cells are similar (Fig. 6), although action potential durations are briefer in tonic than in phasic or adapting cells (Table 2). There are also significant differences in the input resistances and current thresholds for action potential generation in adapting compared with phasic or tonic cells (Table 2). In SCG cells expressing Kv4.2W362F, the mean ± SEM input resistances were increased compared with wild-type cells (Table 3). This finding suggests that (some) I_Af channels are open at rest, an observation supported by voltage-clamp data documenting anI_Af “window current” in the −70 to −50 mV range (Belluzzi et al., 1985a). Preliminary experiments have revealed that perfusion of wild-type SCG neurons with 300 nm Heteropoda toxin-3 (HpTx), a spider toxin (Sanguinetti et al., 1997) that blocksI_Af selectively (n = 2), increases the input resistances of SCG neurons to a mean ± SEM of 0.74 ± 0.27 GΩ (n = 17) from the control values of 0.45 ± 0.11 GΩ (n = 16). Importantly, neither the expression of Kv4.2W362F nor the application of HpTx-3 affects I_K1 density. Thus, the removal ofI_Af specifically increases input resistance. The concomitant decrease in the current threshold for action potential generation (compare Table 2 and Table 3) reveals thatI_Af regulates excitability in SCG neurons by opposing depolarizing inputs and influencing the current required to reach threshold.

The results presented here also reveal that elimination ofI_Af (by Kv4.2W362F expression) does not measurably affect action potential duration in SCG neurons. This finding seems in conflict with several previous studies suggesting thatI_Af(I_A) plays a role in action potential repolarization in SCG cells. Action potential durations, for example, reportedly decrease during late embryonic development in parallel with an increase in I_Af density (Nerbonne and Gurney, 1989). In addition, in SCG neurons, action potential durations follow the steady-state inactivation curve forI_Af and are prolonged by 4-AP (Belluzzi et al., 1985a). These seemingly disparate findings could reflect the fact that additional currents (toI_A) are changing during development and/or are affected by 4-AP. The molecular genetic approach to manipulating functional K⁺ channel expression used here, however, should not be confounded by these uncertainties. Importantly, action potential durations are decreased when Kv4.2 is overexpressed and I_Af is increased.

The role of I_Af in determination of firing patterns

The experiments completed here reveal thatI_Af does play a prominent role in determining the repetitive firing properties of SCG neurons. Elimination of I_Af resulted in an increase in the input resistances of phasic and tonic (but not adapting) cells (Table 3). The loss ofI_Af also resulted in an increased number (and percentage) of adapting cells (Table 3), whereas increasing the density of I_Af decreased the number of adapting cells (Table 4). Taken together, these results suggest that low I_Af density is correlated with the adapting firing pattern. The changes in the distributions of cells also suggest that reductions inI_Af density can convert phasic cells to the adapting phenotype. Adapting cells, in contrast, can be made to fire tonically when I_Af density is increased. Thus, although I_Af is not the sole determinant of repetitive firing patterns in SCG neurons, this conductance does play a prominent role.

Tonic cells are characterized by briefer action potentials than phasic or adapting cells (Table 2), and shortening action potential duration by increasing I_Af expression increases the percentage of tonic cells (Table 4). Nevertheless, ∼30% of Kv4.2-expressing cells are phasic (Table 4), suggesting that currents other than I_Af play an important role in defining this firing class. One likely candidate is the M-current,I_M, which has been linked previously to phasic firing in SCG and other neurons (Brown and Adams, 1980;Freshi, 1983; Galvan and Sedlmeir, 1984; Cassell et al., 1986; Wang and McKinnon, 1995). Blockade of the M-current has previously been shown to be sufficient to convert phasic SCG neurons to tonic firing (Cassell et al., 1986). Interestingly, the results here show that the same conversion may be accomplished with a decrease in action potential duration, which suggests that action potential duration is a key component of I_M activation and regulation of repetitive firing properties in SCG neurons. Further experiments aimed at testing this hypothesis directly are clearly warranted.