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The Journal of Neuroscience, July 15, 2000, 20(14):5191-5199
Elimination of the Fast Transient in Superior Cervical Ganglion Neurons with Expression of KV4.2W362F: Molecular Dissection of IA
Sacha A. Malin and Jeanne M. Nerbonne Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Electrophysiological and molecular studies have revealed considerable heterogeneity in voltage-gated K+ currents and in the subunits that underlie these channels in mammalian neurons. At present, however, the relationship between native K+ currents and cloned subunits is poorly understood. In the experiments here, a molecular genetic approach was exploited to define the molecular correlate of the fast transient outward K+ current, IAf, in sympathetic neurons and to explore the functional role of IAf in shaping action potential waveforms and controlling repetitive firing patterns. Using the biolistic gene gun, cDNAs encoding a dominant negative mutant Kv4.2
-subunit (Kv4.2W362F) and enhanced green fluorescent protein (EGFP) were introduced into rat sympathetic neurons in vitro. Whole-cell voltage-clamp recordings obtained from EGFP-positive cells revealed that IAf is selectively eliminated in cells expressing Kv4.2W362F, demonstrating that Kv4
Key words: K+ channels; IA; Kv4
INTRODUCTION
Voltage-gated potassium (K+) currents are key regulators of excitability in mammalian neurons, and in most cell types, two broad classes of voltage-gated K+ currents have been distinguished: (1) rapidly activating and inactivating currents, IA, and (2) delayed rectifier K+ currents, IK (Rudy, 1988
; Storm, 1990
-subunits has revealed considerably more heterogeneity (Coetzee et al., 1999
At present, there is considerable interest in determining the molecular correlates of functional voltage-gated K+ channels in mammalian neurons and in defining the roles of these channels in shaping action potential waveforms, repetitive firing patterns, and responses to synaptic inputs (Coetzee et al., 1999
Previous studies have demonstrated that
MATERIALS AND METHODS
Culture of SCG neurons. Sympathetic neurons were isolated from the SCG of embryonic day 21 (E21) to postnatal day 1 (P1) Long-Evans rat pups using a procedure similar to that described by Chang et al. (1990)
Immunohistochemistry. The affinity-purified rabbit polyclonal anti-Kv4 pan antibody used in this study was raised against residues 484-502 in the C terminus of Kv4.2, and it has been shown to reliably detect Kv4.2 and Kv4.3 (Barry et al., 1995
Transfection of isolated SCG neurons with the gene gun. In preliminary experiments, 1.6 µm gold beads were coated with pCMV-EGFP (Clonetech), which encodes enhanced green fluorescent protein (EGFP), and propelled (450 psi; 2 mm carrier distance) into SCG neurons at 4 d in vitro using the gene gun (Bio-Rad), a biolistic projectile system. After transfections, the cultures appeared healthy, and expression of EGFP was readily detected 24 hr later (see Results). In experiments aimed at examining the effects of Kv4.2W362F or Kv4.2 expression in SCG neurons, the gold particles were coated with either pBK-CMV-Kv4.2 and pCMV-EGFP or pBK-CMV-Kv4.2W362F-FLAG and pCMV-EGFP in a 4:1 ratio (total of 10 µg of DNA). Because EGFP expression was used to identify transfected neurons for subsequent electrophysiological characterization, immunohistochemical experiments were performed to determine whether EGFP-expressing cells also expressed the Kv4.2W362F-FLAG construct (see Results).
Electrophysiological recording. Whole-cell recordings were obtained from isolated SCG neurons at room temperature (22-25°C). Data were collected using an Axopatch-1B patch-clamp amplifier; experimental parameters were controlled with a P5-120 Gateway2000 computer through a TL-1 DMA Interface using the PClamp7 software package (Axon Instruments). Electrodes were fabricated from soda-lime glass (Chase 2502) with a two-stage puller, and the shanks were coated with a silicone elastomer (Sylgard, Dow Corning, Corning, NY). Pipette resistances were 1.5-3 M
after fire-polishing. For voltage-clamp recordings, the bath solution routinely contained (in mM): 140 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 5 glucose, 0.001 TTX, and 0.1 CdCl2 (pH 7.5, 300 mOsm). Recordings were also obtained from SCG neurons in the current-clamp mode, and the TTX and CdCl2 were omitted from the bath for these studies. The pipette solution for both current and voltage-clamp recordings contained (in mM): 135 KCl, 10 HEPES, 5 glucose, 3 MgATP, 0.5 NaATP, 2 EGTA, 1.1 CaCl2 (pH 7.5, 300 mOsm). Series resistances, estimated from the decays of the uncompensated capacitative transients, were 2-5 M
40 mV and +50 mV from a holding potential of
Data analysis. Data were compiled and analyzed using pCLAMP7 (Axon) and Excel (Microsoft) software and are presented as means ± SEM. To ensure adequate spatial control of the membrane voltage, the decay phase of the capacitative transients were analyzed, and only cells in which >90% of the amplitude of the capacitative transient decayed with a single exponential time course were included in this study. Only data obtained from cells with an input resistance >100 M
1 + A2e-t/
1,
< 30 pA). All current-clamp recordings were obtained from cells with overshooting action potentials and stable resting membrane potentials negative to
RESULTS
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 µM TTX 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
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Figure 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
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 Figure 1A-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
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Table 1. Outward K+ currents in wild-type and transfected SCG neuronsa Although the waveforms of the currents in individual (Type I) SCG cells are similar, the densities of IAf and IK vary considerably. For example, IAf 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, IK 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), ISS (steady-state), is also variable; at +50 mV, for example, ISS 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
In the other four (of 43 or ~9%) SCG cells (Type III) studied, no rapidly inactivating K+ currents (similar to IAf and/or IAs) 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
IAf is selectively attenuated in SCG neurons expressing Kv4.2W362F
On the basis of the kinetic properties of IAf and the sensitivity of this current to 4-AP, it seemed reasonable to suggest that this conductance pathway reflects the expression of Kv4
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Figure 2. Expression of Kv4
To test the hypothesis that Kv4
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Figure 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 in A and B). In addition, EGFP expression correlates with Kv4.2W362F (compare left and right 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 Figure 4. 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, IAf, 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 (
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Figure 4. IAf 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 and right 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 of IAf (see Results). In a small subset of Kv4.2W362F-expressing cells (C), a rapid component of decay with a mean ± SEM
In the remaining Kv4.2W362F-expressing SCG cells (n = 6), a rapid component of outward current decay is detected (Fig. 4C), suggesting that IAf is unaffected by Kv4.2W362F expression in a subset of SCG cells. Analysis of the currents in records such as those in Figure 4C revealed that the mean ± SEM density of this component is lower and the mean ± SEM
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Figure 5. Two components of IAf in 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
Action potential waveforms and repetitive firing in SCG neurons
Subsequent experiments were aimed at evaluating the role of IAf 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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Figure 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 of IAf 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.
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Table 2. Resting and active membrane properties of SCG neuronsa Elimination of IAf increases excitability (and percentage) of "adapting" SCG cells
Previous pharmacological studies suggest a primary role for IAf in the regulation of excitability and in action potential repolarization in SCG neurons (Galvan and Sedlmeir, 1984
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 of IAf: ~25% of wild-type (Fig. 6C, Table 2) and Kv4.2W362F-expressing (Fig. 7C, Table 3) cells fire tonically. These observations suggest that low IAf 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 (Table 2), 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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Figure 7. Elimination of IAf increases 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 in A-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).
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Table 3. Expression of Kv4.2W362F increases input resistance and the number of adapting SCG neuronsa In contrast to the marked effects on excitability, action potential durations are not affected measurably by elimination of IAf (Table 3). These observations suggest that IAf 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 (Table 2).
Increasing IAf reduces the number of adapting cells
The results of the experiments above suggest that low IAf density is an important determinant of the adapting phenotype. To further test this hypothesis, the effects of increasing IAf 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 (Table 4). 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 IAf 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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Figure 8. Increased IAf 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 in A-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.
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Table 4. Overexpression of Kv4.2 reduces the input resistance and the number of adapting neuronsa Analysis of voltage-clamp recordings from EGFP-positive cells in Kv4.2-transfected cultures revealed that the density of IAf 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 lack IAf and IAs (Table 1), were seen among the cells expressing Kv4.2. In the majority (69%) of the cells overexpressing Kv4.2, IAf, IK, and ISS are detected (Table 1). The waveforms of the currents (Fig. 9A) are similar to those in wild-type I cells (Fig. 3A), although IAf density is significantly (p < 0.04) higher and IK and ISS 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 IAf, IAs, IK, and ISS, although IAf density is significantly (p < 0.04) higher in the Kv4.2-expressing cells (Table 1).
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Figure 9. Overexpression of Kv4.2 increases IAf 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 in A and B were obtained from all cells (Table 1); no cells lacking IAf were evident. Analysis of the decay phases of the currents provided the mean ± SEM densities of IAf, IAs, IK, and ISS (Table 1), and mean ± SEM IAf 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).
DISCUSSION
Kv4
In the experiments here, four kinetically distinct voltage-gated K+ currents have been distinguished in SCG neurons: two transient currents, IAf and IAs, as well as a slowly inactivating current, IK, and a steady-state current, ISS (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 IAf, IK, and ISS, whereas Type II cells express IAf, IAs, IK, and ISS, and Type III cells express only IK and ISS. There is considerable variability in current densities within each grouping (Table 1), and interestingly, IK and ISS densities are significantly (p < 0.005) lower in Type II cells (the only cells that express IAs) than in either Type I or III cells (Table 1).
In experiments aimed at testing the hypothesis that
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 than IAf 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 IAf channels, one generated by Kv4
IAf 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) IAf channels are open at rest, an observation supported by voltage-clamp data documenting an IAf "window current" in the
The results presented here also reveal that elimination of IAf (by Kv4.2W362F expression) does not measurably affect action potential duration in SCG neurons. This finding seems in conflict with several previous studies suggesting that IAf (IA) 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 IAf density (Nerbonne and Gurney, 1989
The role of IAf in determination of firing patterns
The experiments completed here reveal that IAf does play a prominent role in determining the repetitive firing properties of SCG neurons. Elimination of IAf resulted in an increase in the input resistances of phasic and tonic (but not adapting) cells (Table 3). The loss of IAf also resulted in an increased number (and percentage) of adapting cells (Table 3), whereas increasing the density of IAf decreased the number of adapting cells (Table 4). Taken together, these results suggest that low IAf density is correlated with the adapting firing pattern. The changes in the distributions of cells also suggest that reductions in IAf density can convert phasic cells to the adapting phenotype. Adapting cells, in contrast, can be made to fire tonically when IAf density is increased. Thus, although IAf 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 IAf 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 IAf play an important role in defining this firing class. One likely candidate is the M-current, IM, which has been linked previously to phasic firing in SCG and other neurons (Brown and Adams, 1980
FOOTNOTES Received March 14, 2000; revised April 24, 2000; accepted April 25, 2000.
Correspondence should be addressed to Dr. J. M. Nerbonne, Washington University Medical School, 660 S. Euclid, Box 8130, St. Louis, MO 63110. E-mail: jnerbonn{at}pcg.wustl.edu.
This work was supported by the National Science Foundation (predoctoral fellowship to S.A.M.) and National Institutes of Health (NS-30676). We thank Dr. Dianne Barry for the wild-type Kv4.2 and mutant Kv4.2W362F constructs and Pat Lampe for providing SCG cultures for some of the preliminary experiments. The technical assistance provided by Rebecca Hood and Amy Coleman in the preparation and maintenance of the glial monolayer cultures and by Andrew Benedict with the confocal microscopy is gratefully acknowledged.
REFERENCES
- Baro DJ, Levini RM, Kim MT, Willms AR, Lanning CC, Rodriguez HE, Harris-Warrick RM (1997) Quantitative single-cell-reverse transcription-PCR demonstrates that A-current magnitude varies as a linear function of shal gene expression in identified stomatogastric neurons. J Neurosci 17:6597-6610
[Abstract/Free Full Text] .- Barry DM, Trimmer JS, Merlie JP, Nerbonne JM (1995) Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels? Circ Res 77:361-369
[Abstract/Free Full Text] .- Barry DM, Xu H, Schuessler RB, Nerbonne JM (1998) Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4
[Abstract/Free Full Text] .- Belluzzi O, Sacchi O, Wanke E (1985a) Identification of delayed potassium and calcium currents in the rat sympathetic neurone under voltage clamp. J Physiol (Lond) 358:109-129
[Abstract/Free Full Text] .- Belluzzi O, Sacchi O, Wanke E (1985b) A fast transient outward current in the rat sympathetic neurone studied under voltage-clamp conditions. J Physiol (Lond) 358:91-109
[Abstract/Free Full Text] .- Brahmajothi MV, Campbell DL, Rasmusson RL, Morales MJ, Trimmer JS, Nerbonne JM, Strauss HC (1999) Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes. J Gen Physiol 113:581-600
[Abstract/Free Full Text] .- Brown DA, Adams PR (1980) Muscarinic suppression of a novel voltage-sensitive K+ current in a vertebrate neurone. Nature 283:673-676[Medline].
- Cassell JF, Clark AL, McLachlan EM (1986) Characteristics of phasic and tonic sympathetic ganglion cells of the guinea-pig. J Physiol (Lond) 372:457-483
[Abstract/Free Full Text] .- Chang JY, Martin DP, Johnson EM (1990) Interferon suppresses sympathetic neuronal cell death caused by nerve growth factor deprivation. J Neurochem 55:436-445[ISI][Medline].
- Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Meira E, Rudy B (1999) Molecular diversity of K+ channels. Ann NY Aca Sci 868:233-285.
- Dixon JE, McKinnon D (1996) Potassium channel mRNA expression in prevertebral and paravertebral sympathetic neurons. Eur J Neurosci 8:183-191[ISI][Medline].
- Fiset C, Clark RB, Larsen T, Giles WR (1997) A rapidly activating sustained current modulates repolarization and excitation-contraction coupling in adult mouse ventricle. J Physiol (Lond) 504:557-563[ISI][Medline].
- Freshi JE (1983) Membrane currents of cultured rat sympathetic neurons under voltage clamp. J Neurophysiol 50:1460-1478
