Correspondence to: Kurt G. Beam, Department of Anatomy and Neurobiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523. Fax:(970) 491-7907 E-mail:kbeam{at}lamar.colostate.edu.
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Abstract |
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A defining property of L-type Ca2+ channels is their potentiation by both 1,4-dihydropyridine agonists and strong depolarization. In contrast, nonL-type channels are potentiated by neither agonist nor depolarization, suggesting that these two processes may by linked. In this study, we have tested whether the mechanisms of agonist- and depolarization-induced potentiation in the cardiac L-type channel (1C) are linked. We found that the mutant L-type channel GFP-
1C(TQ
YM), bearing the mutations T1066Y and Q1070M, was able to undergo depolarization-induced potentiation but not potentiation by agonist. Conversely, the chimeric channel GFP-CACC was potentiated by agonist but not by strong depolarization. These data indicate that the mechanisms of agonist- and depolarization-induced potentiation of
1C are distinct. Since neither GFP-CACC nor GFP-CCAA was potentiated significantly by depolarization, no single repeat of
1C appears to be responsible for depolarization-induced potentiation. Surprisingly, GFP-CACC displayed a low estimated open probability similar to that of the
1C, but could not support depolarization-induced potentiation, demonstrating that a relatively low open probability alone is not sufficient for depolarization-induced potentiation to occur. Thus, depolarization-induced potentiation may be a global channel property requiring participation from all four homologous repeats.
Key Words: ion channel modulation, facilitation, muscle
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INTRODUCTION |
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Voltage-gated Ca2+ channels respond to membrane depolarization by allowing Ca2+ into the cell, and, thus, mediate a variety of cellular responses in neurons and muscle, including transmitter release, neurite outgrowth, altered gene expression, exocytosis, and muscle contraction (
L-type Ca2+ channels show a shift in gating mode in response to either strong depolarization or 1,4-dihydropyridine (DHP)* agonist. After strong depolarization, the channel enters a state of higher open probability (Po) and long open times that can be detected by a number of different pulse paradigms. For example, after a strong, conditioning depolarization (e.g., +120 mV) followed by a 50150-ms return to the holding potential, a subsequent, moderate depolarization elicits a Ca2+ channel current that is about twofold larger than that measured without the conditioning depolarization (
The cardiac L-type channel, 1C, normally exhibits a low Po < 0.05 (
1A, exhibits a high Po of
0.6 (
1C that have an intrinsically low Po.
In an attempt to determine whether potentiation of 1C by DHP agonist and strong depolarization occurs via a common pathway, we have characterized wild-type and mutant
1C channels and chimeric channels composed of
1C and
1A sequence. The channels were fused at their amino termini to green fluorescent protein (GFP), expressed in dysgenic myotubes and examined using whole-cell patch clamp. For an
1C in which the agonist binding site was mutated (GFP-
1C(TQ
YM)), 10 µM Bay K 8644 had no significant effect on whole-cell currents, whereas depolarization-induced potentiation remained intact. A chimeric channel containing repeat II and the I-II linker of
1A sequence embedded in L-type background (GFP-CACC) could not support depolarization-induced potentiation but was potentiated by DHP agonist. Channels containing three (CACC), two (CCAA), or no (
1A) repeats of L-type sequence were not potentiated significantly by depolarization, suggesting that depolarization-induced potentiation cannot be localized to any single channel repeat. Interestingly, despite having a relatively low estimated Po comparable to that of
1C, GFP-CACC was not potentiated by depolarization, indicating that depolarization-induced potentiation must not be dependent solely on a low Po. Our results demonstrate that the mechanisms of DHP agonist- and depolarization-induced potentiation of
1C are distinct, and that depolarization-induced potentiation may be a global channel property requiring the participation of all four homology repeats.
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MATERIALS AND METHODS |
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Construction of Chimeric and Mutant 1 cDNAs
A and C denote sequence derived from 1A (
1C (
1C and GFP-
1A were produced by fusing the
1 subunit of either the cardiac L-type channel (
1C) or neuronal P/Q-type channel (
1A) at the amino terminus to GFP as previously described (
1C and
1A subunits (
1C(TQ
YM) was created using overlapping PCR mutagenesis (
1C (Thr 1066 and Gln 1070), which were previously identified as essential components of the DHP binding site (
1A (Tyr 1393 and Met 1397). In brief, the SalI*-FspI fragment of
1C (nt -12C5054C) was subcloned into the SalI and SmaI sites of the pSP72 (Promega) polylinker. Overlapping PCR using GFP-
1C as the template yielded an 816-bp amplification product (nt 2638C3453C) carrying the point mutations, which was then cut with AflII (nt 2689) and AspI (nt 3385) and ligated into the AflII-AspI restriction sites of the pSP72 subclone. Finally, the SalI*-EcoRV fragment (nt -12C4348C) of the subclone was ligated into GFP-
1C at the corresponding restriction sites to yield the clone GFP-
1C(TQ
YM). The
1C/
1A chimera GFP-CACC consisted of repeat II and the I-II linker of
1A contained an
1C background (amino acids 1426C/352671A/7402171C). To produce GFP-CACC, the SalI*-EcoRV fragment of
1C (nt -12C4348C) was ligated into the corresponding sites of the plasmid pSP72. PCR mutagenesis was used to amplify a 980-bp product of
1A sequence, containing the introduced 5' BamHI* and 3' EcoRI* restriction sites at nt 1039 and 2015, respectively. The amplification product was first digested with BamHI, and then partially digested with EcoRI (to avoid cutting an internal EcoRI site), and the resulting fragment was ligated into the corresponding restriction sites of the pSP72/cardiac subclone. Finally, the SalI*-EcoRV fragment from this subclone was ligated into the SalI*-EcoRV restriction sites of GFP-
1C. The chimera GFP-CCAA was composed of repeats I and II (including the II-III linker) of
1C and repeats III and IV of
1A (amino acids 1-920C/1244-2424A). To create GFP-CCAA, the HindIII*-PvuII fragment (nt 3730A4216A) of chimera AL2 (
1A (nt 4216A5891A) into the corresponding restriction sites of the plasmid pSP72. Subsequently, the XhoI-HindIII* fragment (nt 1395A2758C) of clone AL5 (
1A to produce the intermediate clone GFP-ALC. The SalI*-AvrII fragment (nt -12C759C) of GFP-
1C was coligated with the AvrII-AocI fragment (nt 759C1752A) of AL5 into the SalI-AocI (5' polylinker-1752A) restriction sites of GFP-ALC, yielding the subclone GFP-C/3A. Finally, the ClaI-AflII fragment (nt 256C2689C) of
1C was ligated into the corresponding restriction sites of subclone GFP-C/3A to yield the final chimera GFP-CCAA. The integrity of all channel constructs was confirmed using automated sequence analysis (Macromolecular Resources).
Expression and Electrophysiological Analysis of Channels in Dysgenic Myotubes
1 wk after plating, primary cultures of mouse dysgenic myotubes (1S subunit (
1 subunits. 3652 h after injection, expressing myotubes were identified by green fluorescence and used for electrophysiology. Macroscopic Ca2+ currents were measured using the whole-cell patch-clamp method (
when filled with an internal solution containing 140 mM cesium aspartate, 10 mM Cs2EGTA, 5 mM MgCl2, and 10 mM HEPES, pH 7.4 with CsOH. The external bath solution contained 10 mM CaCl2, 145 mM TEA-Cl, and 10 mM HEPES, pH 7.4 with TEA-OH, plus 3 µM tetrodotoxin. Test currents were obtained by stepping from a holding potential of -80 to -30 mV for 1 s (to inactivate endogenous T-type Ca2+ current;
10 mV. Except for tail currents, data were sampled at 1 kHz. Tail currents, elicited by repolarizing to -50 mV for 125 ms, were recorded with fast sampling (10 kHz). Tail-current amplitude (Itail) was measured 0.5 ms after the onset of the repolarization from the test pulse to -50 mV. The rate of tail-current decay (
deact) was measured by fitting tail currents with a single exponential function. Maximal Ca2+ conductance (Gmax) and half-maximal activation potential (V1/2) were calculated by fitting peak inward current values with the equation:
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(1) |
where I is the peak inward Ca2+ current measured at the test potential (V), Vrev is the reversal potential, and kG is a slope factor. The values of Gmax and Vrev were used to calculate normalized conductance as a function of voltage (see Fig 1 A and 4 C) according to the equation: G (V) = I/[Gmax · (V - Vrev)].
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Maximum immobilization-resistant charge movement (Qmax) was measured, after the addition of 0.5 mM Cd2+ and 0.1 mM La3+ to the bath, by integration of Qon for a 15-ms test pulse (exponentially rounded with a time constant of 100 µs) to +40 mV. Maximum channel Po was calculated from the average, measured values of and
according to the equation:
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(2) |
where =
- average dysgenic charge (2.5 nC/µF;
is the single-channel conductance in 10 mM Ca2+, assumed to be 4 pS for
1A (
1C (
Several different measures were used to quantify potentiation. For the DHP agonist (±)Bay K 8644, one measure was the ratio , where the numerator and denominator represent the peak currents elicited by depolarizing test pulses in the presence or absence of drug, respectively. Imaxcontrol was usually elicited by a Vtest of approximately +2030 mV, and ImaxBay K for a Vtest 2030 mV more hyperpolarized. Agonist-induced potentiation was also measured by means of the ratios
and
, where the tail current was produced by repolarizing to -50 mV from a Vtest of +40 mV. Depolarization-induced potentiation was quantified by the ratios
and
, where the numerator and denominator were determined from tail currents produced by repolarization to -50 mV after a Vtest of +90110 mV or +40 mV, respectively.
Statistical Analysis
Statistical significance was assessed using one-way analysis of variance (ANOVA) and SAS software (version 8). All data are presented as mean ± SEM.
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RESULTS |
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1C Is Potentiated by DHP Agonist and Strong Depolarization while
1A Is Potentiated by Neither
Fig 1 A illustrates representative whole-cell Ca2+ currents elicited by depolarizing dysgenic myotubes expressing either GFP-1C or GFP-
1A to the indicated potentials, followed by repolarization to -50 mV. Based upon steady-state activation calculated from peak currents during the test depolarizations (Fig 1 B), both channels were fully activated by test pulses to +40 mV and above. Consistent with this, the tail currents for GFP-
1A had a similar amplitude and time course after the depolarizations to either +40 or +60 mV (Fig 1 A). However, for GFP-
1C, the tail current after the +60-mV depolarization was larger and decayed more slowly than the tail after the +40-mV step. This behavior is an indication that strong depolarization caused
1C channels to enter a mode of gating having longer open times and increased Po.
Fig 2 compares currents produced by GFP-1C and GFP-
1A before and after exposure to 10 µM Bay K 8644. For GFP-
1A, application of Bay K 8644 had little effect on either the current elicited by a test depolarization to +20 mV or on the tail current after repolarization (Fig 2 A). Likewise, the average, peak current versus voltage relationship for GFP-
1A was not significantly (P > 0.1) affected by the agonist (Fig 2 B). By contrast, Bay K 8644 caused a hyperpolarizing shift in the test potential evoking maximum inward current for GFP-
1C, together with a substantial increase in the magnitude of this current. In addition to affecting the peak current, Bay K 8644 also caused an approximately threefold increase in tail-current amplitude (Itail) and in the time constant of tail-current deactivation (
deact) for GFP-
1C (Fig 2 A and Table 1). Overall, the effects of Bay K 8644 on GFP-
1C tail currents qualitatively resemble those of strong depolarization (compare Fig 1 A and 2 A).
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Fig 3 illustrates a more detailed characterization of tail currents in cells expressing GFP-1C or GFP-
1A. Fig 3 A shows the standard protocol for quantifying depolarization-induced potentiation, which was determined as the ratio of either Itail or
deact for a tail current after a Vtest of +90 mV, to the corresponding values for a tail current after a Vtest of +40 mV. By both measures, GFP-
1C showed substantial depolarization-induced potentiation, whereas GFP-
1A did not (Fig 3 A and Table 1). Fig 3 B illustrates the dependence of Itail on prior test potentials ranging from -40 to +80 mV. For GFP-
1A, Itail reached a maximum after a Vtest of +30 mV, in good agreement with the conductance versus voltage curve calculated from peak currents (Fig 1 B). For still stronger depolarizations, Itail became smaller for GFP-
1A, as expected for a channel undergoing voltage-dependent inactivation that becomes faster with stronger depolarization. In contrast to GFP-
1A, Itail for GFP-
1C increased monotonically over the entire range of test potentials. This monotonic increase differs from the saturating conductance versus voltage relationship (Fig 1 B) and is consistent with entry into a potentiated state having high Po. This monotonic voltage dependence also suggests that depolarization-induced potentiation is not dependent upon Ca2+ entry during the prepulse. The application of 10 µM Bay K 8644 caused a still further increase in Po, which is indicated by a substantial increase in Itail for GFP-
1C at any given test potential (Fig 3 B). In the presence of agonist, Itail was still increased by stronger test depolarizations, up to at least +70 mV. Table 1 summarizes the effects of DHP agonist and strong depolarization on tail currents for GFP-
1C and GFP-
1A.
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Mutation of T1066 and Q1070 of 1C Eliminates Agonist- but Not Depolarization-induced Potentiation
The observation that GFP-1C is potentiated by both agonist and strong depolarization, whereas GFP-
1A is potentiated by neither, raises the possibility that agonist- and depolarization-induced potentiation are linked. As one test of this hypothesis, we created GFP-
1C(TQ
YM), in which two residues of IIIS5 that are critical for the DHP sensitivity of
1C (
1A. Currents produced by GFP-
1C(TQ
YM) were not affected by the addition of 10 µM Bay K 8644 (Fig 4 A), whereas depolarization-induced potentiation was intact (Fig 4 B). In particular, tail currents were larger and decayed more slowly after a Vtest of +90 mV compared with a Vtest of +40 mV (Fig 4 B, top) and the tail-current amplitude increased monotonically as a function of test potential (Fig 4 B, bottom). On average, GFP-
1C(TQ
YM) was quantitatively similar to GFP-
1C with respect to depolarization-induced potentiation, but was indistinguishable from GFP-
1A in the lack of agonist-induced potentiation (Table 1). Interestingly, mutation of T1066 and Q1070 in the IIIS5 transmembrane segment of
1C resulted in a decreased steepness, and positive shift, of the steady-state activation curve in comparison to GFP-
1C (Fig 4 C), indicating that these residues can affect activation gating. Taken together, the data of Fig 4 demonstrate that depolarization-induced potentiation still occurs in a mutant
1C lacking a response to DHP agonist.
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Agonist-induced Potentiation Persists in the Absence of Depolarization-induced Potentiation
In an attempt to determine whether a single repeat of 1C is sufficient to allow depolarization-induced potentiation, we constructed the chimeras GFP-CACC and GFP-CCAA and tested them for agonist- and depolarization-induced potentiation (Fig 5). GFP-CACC consists of repeat II and the I-II linker of
1A in an otherwise
1C background (Fig 5A and Fig C), and thus contains an intact DHP agonist binding site (
1C (Table 1). However, this chimera failed to show the large depolarization-induced potentiation characteristic of either GFP-
1C or GFP-
1C(TQ
YM) (Table 1; also, compare Fig 5 C with Fig 3 B and 4 B). Thus agonist-induced potentiation can be present in a channel construct that lacks significant depolarization-induced potentiation. The chimera GFP-CCAA (Fig 5B and Fig D) consists of the first two repeats and the II-III linker of
1C fused to repeats III and IV of
1A. GFP-CCAA lacked both agonist- and depolarization-induced potentiation (Fig 5B and Fig D, and Table 1). Fig 6 summarizes the effects of strong depolarization and DHP agonist on the constructs GFP-
1C, GFP-
1C(TQ
YM), GFP-CACC, and GFP-
1A. The asterisks indicate a significant (P < 0.05) difference from onefold (where onefold indicates a lack of potentiation). Fig 6 demonstrates that depolarization-induced potentiation can persist in the absence of potentiation by agonist (i.e., GFP-
1C(TQ
YM)), and potentiation by agonist can occur in the absence of depolarization-induced potentiation (i.e., GFP-CACC); therefore, the two processes likely occur via distinct mechanisms.
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No Single Repeat of 1C Is Sufficient for Depolarization-induced Potentiation
Fig 7 shows that in terms of both amplitude of tail currents (A) and deact (B), the chimeras GFP-CACC and GFP-CCAA, like
1A, lacked depolarization-induced potentiation. Because GFP-CACC lacked depolarization-induced potentiation, none of the three cardiac repeats contained in this construct (i.e., I, III, and IV) appears to be sufficient, individually or in concert, to mediate this process. In addition, because GFP-CCAA contains a cardiac repeat II and lacked depolarization-induced potentiation, a cardiac repeat II does not appear to be sufficient, either alone or in combination with repeat I. In conclusion, no single repeat of
1C seems to be sufficient for depolarization-induced potentiation, which may instead represent a more global property. Several combinations of multiple repeats are tested by the chimeras examined in this paper, and others cannot be tested because not all possible combinations of repeats of L-type and nonL-type sequence produce functional channels (
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Because potentiation is defined by a shift into a gating mode of substantially increased Po, it seems likely that potentiation could not occur in a channel already having a relatively high Po. Therefore, it is of interest to know whether the lack of depolarization-induced potentiation in GFP-CACC and GFP-CCAA is a consequence of an already high Po. We used Equation 2 (MATERIALS AND METHODS) to estimate Po for GFP-1C, GFP-CACC, GFP-CCAA, and GFP-
1A from measured values of Gmax and Qmax. The values estimated by this approach for both GFP-
1C and GFP-
1A (Table 2) are in reasonable agreement with values determined from single-channel measurements for
1C (<0.05;
1A (0.6;
1A was about 30-fold higher than for GFP-
1C. The estimated Po for GFP-CCAA was similar to that of GFP-
1A, but estimated Po for GFP-CACC was much closer to that of GFP-
1C (Fig 7 C and Table 2). The absence of depolarization-induced potentiation for GFP-CACC indicates that a low Po alone is not a sufficient condition for this process to occur. The lack of depolarization-induced potentiation for GFP-CACC is even more striking given that this construct can be strongly potentiated by agonist.
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DISCUSSION |
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In the present study, we have examined DHP- and depolarization-induced potentiation of L-type Ca2+ channels by expressing GFP-tagged cardiac (1C) and neuronal (
1A)
1 subunits in dysgenic myotubes. For GFP-
1C, both strong depolarization and agonist (10 µM Bay K 8644) caused tail currents to become larger and to decay more slowly, whereas tail currents for GFP-
1A were not affected by either manipulation. Introduction of two point mutations (T1066Y and Q1070M) into GFP-
1C abolished potentiation by agonist without any evident effect on potentiation by depolarization. Conversely, agonist but not depolarization caused potentiation of a chimera of
1C and
1A (GFP-CACC). Because depolarization-induced potentiation was absent for both GFP-CACC and the chimera GFP-CCAA, it appears that no single repeat of
1C can be responsible for this process. GFP-CACC displayed a relatively low estimated Po, quite similar to that of GFP-
1C, whereas the estimated Po for both GFP-CCAA and GFP-
1A was much higher. Therefore, a channel that displays a low Po (and is potentiated by agonist) can fail to be potentiated by depolarization.
Independent Pathways for Potentiation by DHP Agonist and Depolarization
Unitary records of L-type Ca2+ channels have been described as having three modes of gating upon depolarization: mode 0 (null sweeps); mode 1 characterized by brief openings (<1 ms) in bursts; and mode 2 defined by longer openings and high Po (
Bay K 8644 is well-known to shift activation in the hyperpolarizing direction (Fig 2; 1C not only ablates the response to agonist, but also shifts the voltage dependence of activation oppositely, in the depolarizing direction. On this basis, one could hypothesize that Bay K 8644 promotes a conformation of these two residues that stabilizes open states of the channel, and mutation of these residues destabilizes this conformation.
Role of Accessory Subunits and of Phosphorylation in Depolarization-induced Potentiation
The accessory ß subunit has been shown to influence modal gating of 1C. In particular, comparison of
1C expressed with or without the ß2a subunit showed that ß2a increased both open times and the proportion of long openings (
1C was coexpressed with the ß1, ß3, or ß4 subunits (
1C, and perhaps in potentiation as well. However, others have found that depolarization-induced potentiation of the smooth muscle
1C occurs in the absence of any ß subunit (
1 subunit itself, because all of the
1 constructs examined in this study have a conserved "alpha interaction domain" (site of ß subunit binding;
Evidence has been presented that PKA-dependent phosphorylation occurring during depolarizing prepulses is necessary for depolarization-induced facilitation of 1S (
1C (
1C (
1C, although basal phosphorylation may be required (
1C directly because truncation of the consensus PKA sites (
1C carboxyl tail does not eliminate depolarization-induced facilitation (
1C.
Structural Determinants of Depolarization-induced Potentiation and Low Po
As discussed above, brief openings predominate during activation of 1C by modest depolarizations applied from a negative holding potential (mode 1 gating). The conformational changes responsible for activation of these brief openings occur rapidly (macroscopic activation occurs with a time constant of several ms at +30 mV;
1C appears to require large movements of charge distributed throughout the protein.
L-type channels like 1S and
1C differ from
1A channels in that the L-type channels display agonist- and depolarization-induced potentiation, and also have a much lower Po, raising the possibility that the structural determinants of potentiation and low Po reside in similar structures. However, the chimera GFP-CACC had a relatively low Po, yet did not display significant depolarization-induced potentiation. Because the chimera GFP-CCAA displayed a high Po, the amino-terminal half of
1C (repeats I and II) does not appear to be an important determinant of low Po; instead, structural requirements for low Po may reside in the carboxyl half of the protein. Certainly, it is attractive to hypothesize that repeats III and IV are important for the intrinsic, low Po of L-type channels since these same two repeats play an essential role in agonist binding, which increases Po. A role for the carboxyl tail in determining Po is suggested by previous work showing that Po of
1C is markedly increased by partial truncation of the carboxyl tail (
As stated earlier, the Po of the L-type channels containing 1C (<0.05;
1A (0.6;
1B (0.5;
1C] Kokobun and Reuter, 1984; [
1A]
1B]
1:1 correspondence between the plasmalemmal L-type channels and the intracellular ryanodine receptors. Perhaps this correspondence is best served by a relatively high density of low Po channels. Conversely, a high Po and relatively low channel density would be advantageous when it is critical that a cellular response be triggered by the activation of only a few channels. Important goals for future work will be to better define the structures determining the differences in Po between
1C and neuronal channels like
1A and
1B, and to identify the conformational rearrangements that occur during potentiation of L-type channels.
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Footnotes |
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* Abbreviations used in this paper: DHP, 1,4-dihydropyridine; GFP, green fluorescent protein; nt, nucleotide; Po, open probability.
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Acknowledgements |
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We thank Kathy Parsons and Lindsay Grimes for performing the tissue culture.
This work was supported by the National Institutes of Health grant NS24444 to K.G. Beam, an NIH predoctoral fellowship MH12512 to C.M. Wilkens, and by the Fonds zur Förderung der Wissenschaftlichen Forschung, Austria (J01242-GEN) to M. Grabner.
Submitted: 26 June 2001
Revised: 24 September 2001
Accepted: 26 September 2001
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