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Previous Article | Next Article
The Journal of Neuroscience, December 1, 2002, 22(23):10094-10105
Delayed Rectifier K+ Currents, IK, Are Encoded by Kv2
-Subunits and Regulate Tonic Firing in Mammalian Sympathetic Neurons
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
Previous studies have revealed the presence of four kinetically distinct voltage-gated K+ currents, IAf, IAs, IK, and ISS, in rat superior cervical ganglion (SCG) neurons and demonstrated that IK and ISS are expressed in all cells, whereas IAf and IAs are differentially distributed. Previous studies have also revealed the presence of distinct components of IAf encoded by
-subunits of the Kv1 and Kv4 subfamilies. In the experiments described here, pore mutants of Kv2.1 (Kv2.1W365C/Y380T) and Kv2.2 (Kv2.2W373C/Y388T) that function as Kv2 subfamily-specific dominant negatives (Kv2.1DN and Kv2.2DN) were generated to probe the functional role(s) of Kv2
Key words: K+ channels; IK; Kv2.1; Kv2.2; Kv2.1W365C/Y380T; Kv2.2W373C/Y388T; transgenics; Gene Gun; neuronal excitability; repetitive firing patterns
INTRODUCTION
Voltage-gated K+ channels are important determinants of neuronal membrane excitability, and differences in K+ channel expression patterns and densities contribute to the variations in action potential waveforms and repetitive firing patterns evident in different neuronal cell types (Pongs, 1999
). Electrophysiological studies have revealed that most mammalian neurons express multiple types of voltage-gated K+ currents with distinct time- and voltage-dependent properties (Rudy, 1988
We have shown previously that sympathetic neurons isolated from the rat superior cervical ganglion (SCG) express four kinetically distinct voltage-gated K+ currents: two transient A-type currents, IAf and IAs, a delayed rectifier current, IK, and a steady-state current, ISS (Malin and Nerbonne, 2000
Previous studies suggest that
MATERIALS AND METHODS
Construction of Kv2.1W365C/Y380T and Kv2.2W373C/Y388T. Rat Kv2.1 (obtained from R. Joho, University of Texas Southwestern, Dallas, TX) was subcloned into the pAlter vector (Promega, Madison, WI), and two pore mutations (W365C/Y380T) were introduced using the Altered Sites Mutagenesis II (pAlter) system (Promega). Codons TGG (W365) and TAC (Y380) were mutagenized to TGC (C) and ACC (T). To aid in the detection of the mutant construct, the FLAG epitope was added to the C terminus of Kv2.1W365C/Y380T by PCR. The corresponding mutations (W373C/Y388T) were also introduced into rat Kv2.2 (obtained from S. Trimmer, State University of New York, Stonybrook, NY) in pAlter using PCR. Codons TGG (W373) and TAC (Y388) were mutagenized to TGC (C373) and ACC (T388). The myc epitope tag was added on the C terminus of Kv2.2W373C/Y388T by PCR, and Kv2.2W373C/Y388T-myc was cloned into the pEF6/v5His vector (Promega). Both the Kv2.1W365C/Y380T and the Kv2.2W373C/Y388T constructs were sequenced in their entirety to ensure that no additional mutations were introduced.
Expression studies in human embryonic kidney-293 cells. The functional properties of Kv2.1W365C/Y380T and Kv2.2W373C/Y388T were examined in heterologous expression studies in human embryonic kidney (HEK)-293 cells. HEK-293 cells were maintained in growth medium [Eagle's Minimal Essential Medium (EMEM) (Invitrogen, San Diego, CA), containing 10% heat-inactivated fetal calf serum (FCS) and 100 U/ml penicillin-streptomycin], split, plated in 35 mm tissue culture dishes, and transfected with wild-type and mutant K+ channel
Isolation and in vitro maintenance of SCG neurons. Sympathetic neurons were isolated from the SCGs of embryonic day 21 to postnatal day 1 Long-Evans rat pups using a procedure similar to that described previously by Chang et al. (1990)
Antibodies. The monoclonal anti-Kv2.1 antibody used here was raised against residues 853-857 in the C terminus of Kv2.1 (Trimmer, 1991
Western blots. Extracts of HEK-293 cells transfected with wild-type and mutant Kv2.x constructs and rat SCG (and brain) membrane preparations were prepared using methods described in detail previously (Barry et al., 1995
Immunoprecipitations. For each immunoprecipitation experiment, the precipitating antibody (0.1 µg) and 50 µl of equilibrated protein-A Sepharose beads (Sigma) were added to 100 µl of the HEK-293 or SCG (or brain) protein preparation (prepared as described above) and mixed (by inversion) at 4°C overnight. Eluted proteins were fractionated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with the monoclonal anti-Kv2.1 antibody or the polyclonal anti-Kv2.1 antibody as described above. After exposure to the appropriate (anti-mouse or anti-rabbit) secondary antibody, protein bands were visualized by enhanced chemiluminescence as described above.
Transfection of isolated SCG neurons. In control experiments, 1.6 µm gold beads were coated with pCMV-EGFP (Clontech, Palo Alto, CA) and propelled (450 psi; 2 mm carrier distance) into SCG neurons at 4 d in vitro using the Biolistics Gene Gun (Bio-Rad). After transfections, the cultures appeared healthy, and expression of EGFP was readily detected 24 hr later. In experiments aimed at examining the effects of Kv2.1, Kv2.1W365C/Y380T, Kv2.2W373C/Y388T, or Kv2.1W365C/Y380T plus Kv2.2W373C/Y388T expression in SCG neurons, the gold particles were coated with pBK-CMV-Kv2.1 and pCMV-EGFP (4:1 ratio); pAlter-CMV-Kv2.1W365C/Y380T and pCMV-EGFP (4:1 ratio); pEF6/v5His-EF6-Kv2.2W373C/Y388T and pCMV-EGFP (4:1 ratio); or pAlter-CMV-Kv2.1W365C/Y380T, pEF6/v5His-EF6-Kv2.2W373C/Y388T, and pCMV-EGFP (2:2:1 ratio). In all cases, a total of 10 µg of DNA was used in coating the beads. Because EGFP expression was used to identify transfected neurons for subsequent electrophysiological characterization, immunohistochemical experiments using the anti-FLAG and anti-MYC antibodies were also performed to determine whether EGFP-expressing cells also expressed the test constructs.
Electrophysiological recording. Whole-cell recordings were obtained from HEK-293 cells and from isolated SCG neurons 24-48 hr after transfection. Experiments were performed at room temperature (22-25°C), and data were collected using an Axopatch-1B patch-clamp amplifier interfaced to a P5-120 Gateway2000 computer through a Digidata 1200 and using the pClamp7 software package (Axon Instruments, Foster City, CA). 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. In current-clamp experiments on isolated SCG neurons, the TTX and CdCl2 were omitted from the bath. 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, and 1.1 CaCl2, pH 7.5, 300 mOsM. Series resistances, estimated from the decays of the uncompensated capacitative transients, were 2-5 M
In most experiments, voltage-gated K+ currents were evoked during 125 msec or 6 sec depolarizing voltage steps to test potentials between
40 mV and +50 mV from a holding potential of
Data analysis. Data were compiled and analyzed using pClamp7 (Axon Instruments) and Excel (Microsoft, Redmond, WA) software and are presented as means ± SEM. The decay phases of the capacitative transients were analyzed, and only cells in which >90% of the amplitude of the capacitative transient decayed over a single exponential time course were analyzed further. The mean ± SEM input resistance and capacitance of EGFP-expressing SCG neurons were 0.34 ± 0.03 G
t/
1 + A2e
1,
< 30 pA). All current-clamp recordings were obtained from cells with overshooting action potentials and stable resting membrane potentials negative to
RESULTS
Generation and characterization of dominant negative Kv2
In preliminary studies, a single point mutation was introduced into the pore region of Kv2.1, analogous to the approaches used to generate the dominant negative Kv4.2
To determine the functional properties of this construct, HEK-293 cells were transfected with Kv2.1DN (and EGFP) alone and in combination with Kv2.1, Kv2.2, or Kv1.4 (in a 1:1 ratio; see Materials and Methods), and outward K+ currents, evoked in response to 125 msec depolarizing voltage steps to potentials ranging from
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Figure 1. Kv2.1W365C/Y380T (Kv2.1DN) specifically attenuates Kv2.1-encoded currents in HEK cells. Whole-cell voltage-gated outward K+ currents were recorded from HEK-293 cells expressing Kv2.1DN alone (A), Kv2.1 or Kv2.1 plus Kv2.1DN (B), Kv2.2 or Kv2.2 plus Kv2.1DN (C), Kv1.4 or Kv1.4 plus Kv2.1DN (D), and EGFP. Cells were transfected with cDNA constructs (1:1) encoding these subunits, and currents were obtained from EGFP-positive cells as described in Materials and Methods. Schematic of Kv2.1DN is shown, with altered residues. A, In cells expressing Kv2.1DN alone, outward K+ currents are indistinguishable from those in wild-type HEK-293 cells. B, In HEK-293 cells coexpressing Kv2.1DN and Kv2.1, current densities are reduced markedly compared with those recorded from cells expressing Kv2.1 alone (compare left and right). C, In contrast, coexpression of Kv2.1DN with Kv2.2 reveals outward currents indistinguishable from those measured in cells expressing Kv2.2 alone. D, Outward K+ currents in cells expressing Kv2.1DN and Kv1.4 are indistinguishable from those expressing Kv1.4 alone.
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Table 1. Subunit-specific assembly of Kv2.1DN and Kv2.2DN in HEK-293 cellsa A similar strategy was exploited to generate a double pore mutant of Kv2.2, Kv2.2W373C/Y388T, that also functions as a dominant negative (Kv2.2DN); this construct was myc epitope-tagged at the C terminus (see Materials and Methods). Electrophysiological experiments revealed that mean ± SEM peak outward K+ current densities (26 ± 6 pA/pF; n = 10) recorded from HEK-293 cells expressing Kv2.2DN (Fig. 2A) are not significantly different from those recorded from wild-type or mock-transfected HEK-293 cells (15 ± 6 pA/pF; n = 11). However, coexpression of Kv2.2DN with wild-type Kv2.2 (1:1) markedly reduced peak outward K+ current densities compared with the currents recorded from cells expressing Kv2.2 (and EGFP) alone (Fig. 2B, Table 1). The time- and voltage-dependent properties of the residual currents are indistinguishable from those determined in HEK-293 cells expressing Kv2.2 alone. Increasing the relative amount of the Kv2.2DN to wild-type Kv2.2 cDNA to 5:1 eliminated the Kv2.2-encoded K+ currents. Similar to the findings with Kv2.1DN and Kv2.2 (Fig. 1C), coexpression of Kv2.2DN with wild-type Kv2.1 at a 1:1 (Fig. 2C, Table 1) or 5:1 ratio does not measurably affect Kv2.1-encoded currents. Immunohistochemical experiments with the anti-myc antibody revealed that the expression levels of the Kv2.2DN-myc, however, are similar in Kv2.2- and Kv2.1-expressing HEK-293 cells (data not shown). Therefore, these combined observations are consistent with the hypothesis advanced above that Kv2.1 and Kv2.2 do not coassemble in HEK-293 cells (see also below).
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Figure 2. Kv2.2W373C/Y388T (Kv2.2DN) specifically attenuates Kv2.2-encoded currents in HEK cells. Whole-cell voltage-gated outward K+ currents were recorded from HEK-293 cells expressing Kv2.2DN (A), Kv2.2 or Kv2.2 plus Kv2.2DN (B), Kv2.1 or Kv2.1 plus Kv2.2DN (C), and EGFP, as described in the legend to Figure 1. A, Schematic of Kv2.2DN is shown, with the altered residues indicated. In cells expressing Kv2.2DN alone, outward K+ currents are similar to those in wild-type cells. B, When Kv2.2DN is coexpressed with Kv2.2, however, Kv2.2-encoded outward K+ currents are reduced compared with cells expressing Kv2.2 alone (compare left and right). C, In contrast, Kv2.2DN does not affect Kv2.1-encoded currents.
Expression of Kv2.1DN or Kv2.2DN attenuates IK in SCG neurons
To assess the functional consequences of expression of the Kv2.x dominant negative constructs on outward K+ currents in SCG neurons, cells were transfected with Kv2.1DN-FLAG or Kv2.2DN-myc (and EGFP) using the Biolistics Gene Gun (see Materials and Methods). As reported previously (Malin and Nerbonne, 2000
Representative whole-cell voltage-gated outward K+ currents recorded from three EGFP-positive Kv2.1DN-expressing SCG cells are presented in Figure 3 (middle). As in wild-type cells (Fig. 3, left), type I, type II, and type III Kv2.1DN-expressing cells were readily distinguished (Table 2). Most (~70%) cells express IAf, IK, and ISS, and are therefore classified as type I cells (Malin and Nerbonne, 2000
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Figure 3. Expression of Kv2.1DN or Kv2.2DN reduces IK density in SCG neurons. Whole-cell voltage-gated outward K+ currents were recorded from isolated SCG neurons in response to 6 sec depolarizing voltage steps to test potentials between
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Table 2. Expression of Kv2.1DN or Kv2.2DN attentuates IK in SCG neuronsa Similar experiments were conducted on cells expressing Kv2.2DN, and representative outward K+ currents recorded from (3) Kv2.2DN- (and EGFP) expressing SCG neurons are shown in Figure 3 (right). As in wild-type and Kv2.1DN-expressing SCG cells, most (~70%) Kv2.2DN-expressing cells can be classified as type I, expressing IAf, IK, and ISS. In Kv2.2DN-expressing type I cells (Fig. 3), IK density is significantly (p < 0.005) lower than in wild-type I SCG neurons (Table 2). The densities of IAf and ISS and the kinetics of IAf and IK decay, in contrast, are unaffected by Kv2.2DN expression (Table 2). In ~20% of the Kv2.2DN-expressing SCG cells, IAf, IAs, IK, and ISS were evident, and these cells were classified as type II (Malin and Nerbonne, 2000
Coexpression of Kv2.1DN and Kv2.2DN eliminates most of IK in SCG neurons
The results of the experiments described above suggested that the Kv2.1 and Kv2.2
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Figure 4. Coexpression of Kv2.1DN and Kv2.2DN eliminates IK in most SCG neurons. Isolated SCG neurons were transfected with both Kv2.1DN and Kv2.2DN (and EGFP) using the Biolistics Gene Gun (as described in Materials and Methods), and outward K+ currents were recorded from EGFP-positive cells as described in the legend to Figure 3. Two distinct current waveforms were evident in these recordings: most (75%) cells were found to express only IAf and ISS and therefore are type I cells lacking IK; the remaining cells (25%) express IAf, IAs, IK, and ISS and are classified as type II cells with reduced IK density (Table 2). The densities of IAf, IAs, and ISS in Kv2.1DN plus Kv2.2DN-expressing type I and type II cells are indistinguishable from those measured in wild-type I and II cells (Table 2).
Expression of Kv2
Together, the simplest interpretation of the results presented above is that Kv2.1- and Kv2.2-encoded K+ channels underlie IK in type I cells, whereas only Kv2.1 (and not Kv2.2) channels contribute to IK in type II cells. These findings suggest that there is an additional component of IK in type II SCG neurons that is not encoded by Kv2
Biochemical experiments were performed on extracts of Kv2.1 and Kv2.2-transfected HEK-293 cells and on fractionated SCG neuronal membranes using specific anti-Kv2.1 and anti-Kv2.2 antibodies (see Materials and Methods). As illustrated in Figure 5A, the anti-Kv2.1 antibody detects a single band at ~114 kDa in HEK-293 cells transfected with Kv2.1 and Kv2.2. Similarly, in blots probed using the anti-Kv2.2 antibody, a single protein band at ~90 kDa was identified (Fig. 5A). Western blot analysis also revealed robust expression of Kv2.1 and Kv2.2 in lysates of rat SCG (Fig. 5A). Importantly, both the anti-Kv2.1 and the anti-Kv2.2 antibodies can be used to immunoprecipitate proteins (against which each of these antibodies was targeted) from homogenates of HEK-293 cells transfected with both the Kv2.1 and Kv2.2 cDNAs (Fig. 5B). In contrast, however, Kv2.2 does not coimmunoprecipitate with the anti-Kv2.1 antibody, and Kv2.1 does not precipitate with the anti Kv2.2 antibody (Fig. 5B). When the immunoprecipitation was performed with the anti-Kv2.1 antibody, the Kv2.2 (nonprecipitating) protein was identified in the supernatant (Fig. 5B, lanes S). In addition, when the anti Kv2.2 antibody was used in the immunoprecipitation, the Kv2.1 (nonprecipitating) protein was readily detected in the supernatants (Fig. 5B, lanes S). Similar results were obtained in experiments performed on lysates of rat SCG. The anti-Kv2.1 antibody precipitates the Kv2.1 protein but not the Kv2.2 protein expressed in SCG neurons (Fig. 5C). Similarly, after immunoprecipitations with the anti-Kv2.2 antibody, the Kv2.2 protein is found in the pellet (Fig. 5C, lane P), whereas the Kv2.1 protein is in the supernatant (Fig. 5C, lane S). Together, these data suggest that Kv2.1 and Kv2.2 do not associate either in HEK-293 cells or in SCG neurons but rather preferentially form monomeric (Kv2.1 or Kv2.2) voltage-gated K+ channels.
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Figure 5. Kv2.1 and Kv2.2 are expressed in rat SCGs but do not appear to associate. A, Lysates of control and transfected HEK-293 cells and of isolated rat SCGs were fractionated in SDS-PAGE gels and immunoblotted (IB) with the monoclonal anti-Kv2.1 (left) and the polyclonal anti-Kv2.2 (right) antibodies. B, C, Lysates prepared from transfected HEK-293 cells (B) and rat SCG neurons (C) were immunoprecipitated (IP) with either the monoclonal anti-Kv2.1 or the polyclonal anti-Kv2.2 antibody, fractionated, and immunoblotted with the same antibodies. Although both the anti-Kv2.1 and anti-Kv2.2 antibodies reliably immunoprecipitate the proteins against which each of these antibodies were generated, the Kv2.1 and Kv2.2
Kv2.1-encoded IK channels regulate action potential durations in tonic SCG cells
In wild-type rat SCG neurons, three distinct repetitive firing patterns, phasic, adapting, and tonic, are observed in response to prolonged (6 sec) depolarizing current injections (Malin and Nerbonne, 2000
In initial experiments focused on exploring the hypothesis that Kv2.x-encoded IK channels might play a role in shaping action potential waveforms in SCG neurons, Kv2.x-expressing HEK-293 cells and isolated SCG neurons were held at
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Figure 6. Activation of Kv2-encoded K+ currents during action potentials in SCG neurons. To explore directly the activation of Kv2.x-encoded K+ currents during action potential waveforms in SCG neurons, cells were held at the typical resting membrane potential of SCG neurons (~48 mV; see Table 3), and outward K+ currents activated by typical phasic, adapting, and tonic action potential waveforms were recorded. The voltage-clamp paradigms are illustrated at the bottom. Representative outward current waveforms in Kv2.1-expressing HEK-293 cells driven by the phasic adapting and tonic action potential waveforms are illustrated at the top. Representative outward K+ current waveforms in isolated SCG neurons (activated using the action potential voltage-clamp paradigms shown below the record) are presented at the bottom. Action potential clamp recordings from wild-type (solid line), Kv2.1DN-expressing (short dashed line), and Kv2.2DN-expressing (long dashed line) SCG cells are superimposed for comparison purposes.
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Table 3. Contribution of Kv2.1- and Kv2.2-encoded IK to action potential repolarizationa To probe directly the activation of Kv2.x channels in SCG cells, similar experiments were performed in SCG cells using the action potential clamp paradigms (Fig. 6). Peak outward K+ current densities measured in SCG cells with the action potential clamp paradigm are 50-60 pA/pF (Table 3). Comparison of these values with current densities measured using the conventional step depolarization paradigm (Fig. 3) reveals that peak current densities activated during the action potential clamp are ~25% of the mean peak current densities measured at +50 mV in response to step depolarizations from
The phasic, adapting, and tonic firing patterns seen in wild-type SCG cells are also evident in recordings from cells expressing Kv2.1DN, and the selective attenuation of the Kv2.1-encoded IK does not affect the distribution of firing patterns (Fig. 7). The expression of Kv2.1DN also does not affect the mean ± SEM input resistances, resting potentials, action potential amplitudes, or the current thresholds for action potential generation in phasic, adapting, or tonic SCG cells (Table 4). As in wild-type SCG cells, Kv2.1DN-expressing adapting cells are readily distinguished from phasic and tonic cells by increased input resistances and lower current thresholds for action potential generation (Table 4). In tonic cells expressing the Kv2.1DN, however, the mean ± SEM action potential duration at 90% repolarization is significantly (p < 0.02) longer than in wild-type tonic cells (Fig. 7B, Table 4). Interestingly, although action potential durations in Kv2.1DN-expressing tonic cells are prolonged, tonic firing is not eliminated, and the firing frequencies of tonic cells are unchanged. Therefore, brief action potentials, regulated by Kv2.1-encoded IK, are not the sole determinant of tonic firing.
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Figure 7. Phasic, adapting, and tonic firing patterns in SCG neurons expressing Kv2.1DN. SCG neurons were transfected with Kv2.1DN (and EGFP), and action potentials and repetitive firing patterns were recorded in response to brief or prolonged depolarizing current injections, as described in Materials and Methods. A, Current-clamp recordings from three representative Kv2.1DN-expressing cells are shown. In each cell, single action potentials were elicited by 1.5 msec current injections (left), and repetitive firing patterns were recorded in response to 100 pA (middle) or 200 pA (right) 500 msec current injections. Based on the response(s) to the 500 msec current injections, cells were classified as phasic (top), adapting (middle), or tonic (bottom) (Table 4). Although Kv2.1DN expression does not affect the distribution of firing patterns in SCG neurons, action potentials in tonic cells expressing Kv2.1DN are prolonged (B). Action potential durations in phasic and adapting cells (B), in contrast, are unaffected by Kv2.1DN expression (Table 4). wt, Wild type.
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Table 4. Effects of Kv2.1DN and Kv2.2DN expression on SCG neuron firing propertiesa Kv2.2-encoded IK channels regulate action potential threshold and repolarization
To explore the functional role of Kv2.2-encoded IK channels, current-clamp recordings were obtained from SCG cells expressing Kv2.2DN (and EGFP). Similar to the findings with Kv2.1DN, all three firing patterns are observed in Kv2.2DN-expressing cells (Fig. 8). Also, similar to the findings with Kv2.1DN, action potential durations at 90% repolarization in tonic cells expressing Kv2.2 are significantly (p < 0.02) longer than in tonic wild-type cells (Table 4). However, in contrast to the findings with Kv2.1DN, expression of Kv2.2DN results in an increase in the percentage of adapting cells and a decrease in the percentage of phasic cells (Fig. 8, Table 4). Elimination of Kv2.2-encoded IK channels also has marked effects on the excitability of SCG neurons. In adapting and tonic cells expressing Kv2.2DN, for example, resting membrane potentials are depolarized significantly (p < 0.001) compared with resting membrane potentials in wild-type adapting and tonic SCG cells (Table 4). In addition, the current thresholds for action potential generation are reduced significantly in phasic (p < 0.002) and in tonic (p < 0.02) firing SCG cells expressing Kv2.2DN. However, input resistances and action potential amplitude are unaffected by Kv2.2DN expression (Table 4). Together, these results suggest that, unlike Kv2.1-encoded IK channels, Kv2.2-encoded IK channels play a role in setting resting potentials and in regulating action potential generation.
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Figure 8. Expression of Kv2.2DN in SCG neurons increases the number of adapting cells. In SCG neurons transfected with Kv2.2DN (and EGFP), action potentials and repetitive firing patterns were recorded, and cells were classified as phasic (top), adapting (middle), or tonic (bottom), as described in the legend to Figure 7. Similar to the results obtained with Kv2.1DN, expression of Kv2.2DN prolongs action potential durations in tonic cells (B). In addition, Kv2.2DN expression alters the distribution of firing patterns: the percentage of phasic cells is reduced (by ~40%), and the percentage of adapting cells is increased (Table 4). wt, Wild type.
Elimination of IK prolongs action potentials and reduces tonic firing
The voltage-clamp experiments detailed above revealed that expression of either Kv2.1DN or Kv2.2DN reduces IK density in type I SCG cells by ~50%, whereas only Kv2.1DN expression attenuates IK in type II cells. In addition, coexpression of Kv2.1DN and Kv2.2DN eliminates IK in all type I cells and reduces IK density in type II cells. Together with the biochemical data presented (Fig. 5), these results suggest that there are two distinct populations of Kv2.x-encoded IK channels, and that these channels are differentially expressed: Kv2.1-encoded IK channels in both type I and type II cells and Kv2.2-encoded IK channels in type I cells. To determine the effects of removing both populations of Kv2
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Figure 9. Coexpression of Kv2.1DN and Kv2.2DN markedly reduces tonic firing. Isolated SCG neurons were transfected with Kv2.1DN, Kv2.2DN, and EGFP. Action potentials and repetitive firing patterns were recorded, and cells were classified as phasic (top), adapting (middle), or tonic (bottom), as described in the legend to Figure 7. In contrast to the results obtained with expression of Kv2.1DN or Kv2.1DN alone (Figs. 7, 8), expression of both dominant negative constructs markedly decreases the percentage of tonic firing cells (Table 4).
Expression of wild-type Kv2.1
The results of the experiments described here suggest that Kv2-encoded IK channels play a role in determining tonic firing. To explore this hypothesis further, action potentials and repetitive firing patterns were recorded from SCG cells transfected with wild-type Kv2.1 and EGFP (Fig. 10). These experiments revealed that the membrane properties of Kv2.1-transfected and wild-type phasic, adapting, and tonic SCG cells are indistinguishable (see on-line Table, available at www.jneurosci.org). However, expression of Kv2.1 markedly reduces the number of phasic (~25%) and adapting (~25%) cells and increases the number of tonic cells (50%). In addition, mean ± SEM action potential durations at 50 and 90% repolarization in Kv2.1-expressing tonic cells are significantly (p < 0.02) briefer than in phasic and adapting cells.
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Figure 10. Expression of Kv2.1 in SCG neurons increases tonic firing. Action potentials and repetitive firing patterns, recorded as described in the legend to Figure 7, were obtained from isolated SCG neurons 24 hr after transfection with wild-type Kv2.1 and EGFP. As in wild-type cells, the phasic (top), adapting (middle), and tonic (bottom) firing patterns were seen in recordings from cells transfected with Kv2.1. However, the percentage of tonic cells is higher and the percentages of adapting and phasic cells are lower in Kv2.1-expressing cells than in either wild-type, Kv2.1DN-expressing (Fig. 7), or Kv2.2DN-expressing (Fig. 8) SCG cells (Table 4).
DISCUSSION
Two distinct components of IK encoded by Kv2.1 and Kv2.2 in SCG neurons
The studies described here were designed to probe the role of Kv2
The experiments here also revealed that the effects of Kv2.1DN and Kv2.2DN are additive. Coexpression of Kv2.1DN and Kv2.2DN, for example, eliminates IK in type I SCG cells (Fig. 4, Table 2). Importantly, in these experiments, the total amount of cDNA used in the transfections was the same as with Kv2.1DN or Kv2.2DN alone. The additivity of the effects of Kv2.1DN and Kv2.2DN, therefore, does not simply reflect a dose-dependent attenuation of the current. Rather, the experimental observations suggest that Kv2.1DN and Kv2.2DN affect different populations of IK channels, consistent with the hypothesis that Kv2.1 and Kv2.2 do not coassemble in type I SCG neurons. The results presented here also reveal that Kv2-encoded K+ channels underlie IK in all type I SCG neurons. In type II cells, in contrast, IK is not eliminated with Kv2.1DN and Kv2.2DN coexpression. This might reflect the slow turnover rate of some Kv2.x-encoded K+ channels in type II SCG neurons. Alternatively, it is also possible that the expression levels of the dominant negative constructs were too low in these experiments to effectively eliminate IK in type II cells. Both of these hypotheses seem unlikely, however, in light of the results obtained in type I cells and the similarities in the reductions in IK in Kv2.1DN- and Kv2.1DN plus Kv2.2DN-expressing type II cells. It seems more likely that only a subset of IK channels in type II cells are encoded by Kv2 (Kv2.1)
Kv2.1- and Kv2.2-encoded IK channels are differentially expressed in type I and type II SCG cells: Kv2.1-encoded IK is expressed in both type I and type II cells, whereas Kv2.2-encoded IK channels are only evident in type I cells (Table 2). Although the numbers and the percentages of type III cells are small, the experiments completed to date suggest that IK density is reduced in type III cells expressing Kv2.2DN but not in cells expressing Kv2.1DN (Table 2). No type III cells were seen in recordings from cells coexpressing Kv2.1DN and Kv2.2DN, an observation that likely reflects the small number (n = 16) of cells studied in these experiments.
IK density regulates action potential repolarization and tonic firing
Expression of either Kv2.1DN or Kv2.2DN selectively increases action potential durations in tonic cells, suggesting that IK channels contribute to action potential repolarization in these cells (Table 4). Although expression studies in Xenopus oocytes suggest relatively depolarized (~0 mV) thresholds for activation (Fink et al., 1996
Although IK shapes individual action potential waveforms in tonic cells, reductions in IK density do not affect the percentage of cells that fire tonically (Table 4), revealing that brief action potentials are not the sole determinant of tonic firing. However, coexpression of Kv2.1DN and Kv2.2DN and the resulting reductions (in type II cells) or elimination (in type I cells) of IK markedly reduce the percentage of tonic firing cells (Fig. 9). Although action potential repolarization in phasic and adapting cells is not affected by removal of either Kv2.1- or Kv2.2-encoded IK, elimination of both Kv2.1- and Kv2.2-encoded IK significantly (p < 0.001) increases action potential durations in these cells (Table 4). Together, therefore, these observations suggest that IK channels contribute to action potential repolarization in phasic, adapting, and tonic SCG cells, and that total IK density is a critical determinant of the tonic firing pattern. Consistent with this hypothesis, expression of Kv2.1 increases IK density and the percentage of tonic cells (see Fig. 10 and on-line Table, available at www.jneurosci.org).
Kv2.2-encoded IK regulates neuronal excitability
Although both Kv2.1- and Kv2.2-encoded IK channels play roles in action potential repolarization in SCG neurons, only Kv2.2-encoded IK channels appear to contribute to the regulation of (resting) membrane excitability and to affect action potential firing. Expression of Kv2.2DN, for example, depolarizes all SCG cells and reduces the current thresholds for action potential generation in phasic and tonic cells (Table 4). Thus, unlike Kv2.1-encoded channels, Kv2.2-encoded IK channels contribute to setting the resting membrane potentials of SCG neurons and function to regulate membrane excitability. Interestingly, the properties of Kv2.1- and Kv2.2-encoded K+ channels are very similar in heterologous expression systems (Figs. 1, 2), suggesting that there are cell-type-specific regulatory pathways that differentially modulate Kv2.1- and Kv2.2-encoded K+ channels. The functional distinctions between the Kv2.1- and Kv2.2-encoded IK channels revealed in the experiments reported here suggest that differential regulation of these two IK components, either by post-translational modification (Summers and Gelband, 1998
Previous studies have shown that the current thresholds for action potential generation are lower in adapting than in phasic or tonic cells, which appears to reflect, at least in part, the reduced density of the fast transient current IAf in adapting cells (Malin and Nerbonne, 2000
FOOTNOTES Received July 24, 2002; revised Sept. 10, 2002; accepted Sept. 10, 2002.
Financial support was provided by the National Science Foundation (predoctoral fellowship to S.A.M.) and the National Institutes of Health (NS-30676).
Correspondence should be addressed to Jeanne M. Nerbonne, Washington University Medical School, 660 South Euclid, Box 8103, St. Louis, MO 63110. E-mail: jnerbonn{at}pcg.wustl.edu.
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