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REPORT
1Departments of Neurology, 2Molecular Cell Biology-Group Neurophysiology, and 3Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands
Submitted 18 November 2005; accepted in final form 21 December 2005
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ABSTRACT |
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75% by the Cav2.1 channel blocker 
-agatoxin-IVA, which was less than the 95% reduction observed in wild-type. Release at Tg NMJs, but not at wild-type synapses, was reduced by 15% by SNX-482, a Cav2.3 channel blocker. No Cav2.2 channel involvement was found. Probably, there is a small reduction in functional presynaptic Cav2.1 channels at Tg NMJs, which is compensated for by Cav2.3 channels. The remaining Cav2.1 channels are likely to convey enlarged Ca2+ flux, because evoked ACh release at Tg NMJs, at low extracellular Ca2+ concentration, was approximately sixfold higher than at wild-type NMJs. This is the first report of compensatory expression of non-Cav2.1 channels at NMJs of mice with a single amino acid change in Cav2.1. |
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INTRODUCTION |
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; Fletcher et al. 1996), causing ataxia and epilepsy in homozygous animals. In humans, CACNA1A mutations cause familial hemiplegic migraine and other autosomal dominant neurological disorders (Imbrici et al. 2004; Jouvenceau et al. 2001; Ophoff et al. 1996).
High voltage-activated neuronal Ca2+ channels consist of Cav1 (L-type), Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) channels (Catterall 2000). P- and Q-type channels are splice variants (Bourinet et al. 1999) with different sensitivities to -agatoxin-IVA (Stea et al. 1994). Cav2.2 channels are blocked by -conotoxin-GVIA, Cav2.3 channels by SNX-482, and Cav1 channels by dihydropyridines (Catterall 2000).
Cav2.1 channels mediate neurotransmitter release at many central synapses and at the peripheral neuromuscular junction (NMJ), where they govern >90% of release (Uchitel et al. 1992). At mouse NMJs, synaptic effects of Cacna1a mutations can be studied with relative ease (Plomp et al. 2000; Van Den Maagdenberg et al. 2004). Previously, we showed abnormal spontaneous acetylcholine (ACh) release [approximately twofold increased miniature endplate potential (MEPP) frequency] at Tg NMJs, as well as reduced high-rate (40 Hz) evoked release [increased endplate potential (EPP) amplitude rundown]. However, low-rate (0.3 Hz) evoked release was unchanged (Plomp et al. 2000).
Cav2.1, -2, and -3 channels act in a mutually compensatory fashion. Thus transmitter release at Cav2.1 (null-)mutant NMJs and central synapses relies on Cav2.2 and -3 channels (Cao et al. 2004; Leenders et al. 2002; Urbano et al. 2002), whereas compensatory Cav2.1 expression occurs in Cav2.2 null-mutant neurons (Takahashi et al. 2004a,b). At Tg central synapses, compensatory Cav2.2 channels were shown (Leenders et al. 2002; Qian and Noebels 2000). We tested the hypothesis that compensatory, non-Cav2.1 channels contribute to ACh release at the Tg NMJ in electrophysiological experiments using Cav2 subtype-specific blocking toxins. We also measured low-rate (0.3 Hz) evoked ACh release at Tg NMJs at low extracellular Ca2+ to test whether effects of the Tg mutation that are not visible at normal Ca2+ concentration can be unmasked, similar to our recent finding in mice carrying the human familial hemiplegic migraine-associated mutations R192Q and S218L (Kaja et al. 2004, 2005; Van Den Maagdenberg et al. 2004).
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METHODS |
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All animal experiments were in accordance with national legislation, Leiden University guidelines, and the American Physiological Society's Guiding Principles in the Care and Use of Animals. Tg mice were raised from original breeder pairs obtained from Jackson Laboratories (Bar Harbor, ME). Animals were genotyped as described previously (Plomp et al. 2000). Homozygous Tg and wild-type mice were used at 6 wk of age, with the investigator blinded for genotype.
Ex vivo NMJ electrophysiology
Mice were killed by carbon dioxide inhalation. Hemi-diaphragms with phrenic nerve were dissected and kept in Ringer medium (in mM: 116 NaCl, 4.5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 23 NaHCO3, 11 glucose, pH 7.4) at room temperature. Intracellular recordings of MEPPs and EPPs were made at NMJs at 28°C using standard microelectrode equipment. At least 30 MEPPs and EPPs were recorded at each NMJ, and 5–10 NMJs were sampled per experimental condition per muscle. Muscle action potentials were blocked by 3 µM µ-conotoxin GIIIB (Scientific Marketing Associates, Barnet, UK). For EPP recording, the nerve was stimulated at 0.3 or 40 Hz. Procedures for analysis of MEPPs and EPPs and calculation of quantal contents, i.e., the number of ACh quanta released per nerve impulse, have been described before (Van Den Maagdenberg et al. 2004). EPPs and MEPPs were also measured in presence of the specific Ca2+ channel blockers -agatoxin-IVA (Cav2.1, 200 or 400 nM, as indicated), -conotoxin-GVIA (Cav2.2, 2.5 µM), and SNX-482 (Cav2.3, 1 or 2 µM, as indicated), after a 20-min preincubation period. Toxins were from Scientific Marketing Associates. We also probed for the presence of Cav2.2 channels more distant from the Ca2+ sensor at release sites by testing the effect of -conotoxin-GVIA on the quantal content in the presence of 50 µM of the K+ channel blocker 4-aminopyridine (4-AP, Sigma-Aldrich, Zwijndrecht, The Netherlands) in a 0.5 mM Ca2+/5.5 mM Mg2+-Ringer medium, according to the methods described by Urbano et al. (2003).
Statistical analysis
Paired or unpaired Student's t-tests were used where appropriate, on grand mean values, with n as the number of mice tested, and 5–10 NMJs tested per muscle per condition. P < 0.05 was considered to be statistically significant. Data are presented as means ± SE.
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RESULTS |
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-agatoxin-IVA sensitivity of evoked ACh release at Tg NMJs
Total 0.3-Hz stimulation-evoked ACh release at normal (2 mM) Ca2+ concentration was the same at Tg and wild-type NMJs, in line with earlier observations (Plomp et al. 2000). The quantal content was 32 (Fig. 1). -Agatoxin-IVA (200 nM) reduced quantal content at NMJs of wild-type muscles by 94%, from 32.5 ± 1.6 to 1.8 ± 0.8 (n = 4 muscles, 7–10 NMJs per muscle, P < 0.001; Fig. 1A). However, at Tg NMJs, it decreased only by 73% (from 33.9 ± 1.9 to 9.0 ± 2.0, n = 4 muscles, 7–10 NMJs per muscle, P < 0.001; Fig. 1A). -Agatoxin-IVA decreased EPP amplitude by 93% (from 22.8 ± 1.1 to 1.6 ± 0.7 mV) in wild-type, but only by 69% (from 27.2 ± 0.8 to 8.3 ± 1.0 mV) at Tg NMJs (n = 4 muscles, 7–10 NMJs per muscle, P < 0.01; Fig. 1B). In the presence of -agatoxin-IVA, EPP failure on a stimulus was frequently observed at wild-type but almost never at Tg NMJs (29.8 ± 11.8 and 1.5 ± 1.5% of the stimuli, respectively, n = 4 muscles, 7–10 NMJs per muscle, P < 0.05; Fig. 1, B and C). MEPP amplitudes and kinetics were unaffected by -agatoxin-IVA (data not shown). MEPP frequency was reduced by 50% at both Tg and wild-type NMJs (Table 1), as shown previously (Plomp et al. 2000).
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-agatoxin-IVA, although such an effect is not very likely in view of the distant localizations of the Tg mutation (amino acid 601, P-loop of repeat II) and the -agatoxin-IVA binding site (amino acid 1658, C-terminal end of S3, repeat IV, Bourinet et al. 1999; Winterfield and Swartz 2000). However, such an effect could explain the lesser reduction of quantal content by -agatoxin-IVA compared with wild-type NMJs. We therefore experimentally tested this possibility by measuring ACh release at wild-type and Tg NMJs also in the presence of a higher -agatoxin-IVA concentration (400 nM), which is more than five times the IC50 for wild-type quantal content, as determined in our laboratory (75 nM, unpublished data). No extra reduction was observed compared with that in the presence of 200 nM toxin (n = 3–4 muscles, 10 NMJs per muscle, P = 0.85 in wild type and P = 0.34 in Tg; Fig. 1A), excluding the possibility of a reduced toxin sensitivity of Tg-mutated Cav2.1 channels. Cav2.3 channels contribute to evoked ACh release at Tg NMJs
Hence, in view of the reduced -agatoxin-IVA sensitivity of evoked ACh release at Tg NMJs, compensatory involvement of non-Cav2.1 channels is likely. Both Cav2.2 and Cav2.3 channels are known to mediate neurotransmitter release (Reid et al. 2003) and both partially compensate for the loss of Cav2.1 channels at NMJs of Cacna1a null-mutant mice (Urbano et al. 2003; S. Kaja and J. J. Plomp, unpublished observations). To study compensatory involvement of Cav2.2 channels, we recorded MEPPs and EPPs at NMJs of Tg and wild-type muscles before and after application of the selective blocker -conotoxin-GVIA (2.5 µM). In either genotype, the toxin affected neither evoked nor spontaneous ACh release (Fig. 1D; Table 1). Quantal contents were similar (31.8 ± 1.2 and 32.9 ± 2.5 at wild-type NMJs, n = 4 muscles, 7–10 NMJs per muscle, P = 0.73, and 31.4 ± 1.2 and 30.0 ± 3.2 at Tg NMJs, n = 4 muscles, 7–10 NMJs per muscle, P = 0.71, before and in presence of the toxin, respectively). The possibility exists that compensatory expressed Cav2.2 channels are localized more distantly from the Ca2+ sensor at release sites, as proposed for Cacna1a null-mutant NMJs (Urbano et al. 2003). We tested this hypothesis using the protocol described in Urbano et al. (2003). The quantal content was first measured in Ringer medium containing 0.5 mM Ca2+/5.5 mM Mg2+ and was similar at wild-type and Tg NMJs (0.58 ± 0.09, and 0.51 ± 0.09, respectively, n = 5–6 muscles, 10 NMJs per muscle, P = 0.64). Addition of 50 µM 4-AP increased quantal content equally (37-fold) in both genotypes (Fig. 1E). Further addition of 2.5 µM -conotoxin-GVIA did not affect quantal content (19.1 ± 2.3 and 20.8 ± 2.4 before and during toxin in wild-type, n = 5 muscles, 10 NMJs per muscle, P = 0.18; 19.5 ± 2.3 and 17.3 ± 3.0 before and during toxin in Tg, n = 7 muscles, 10 NMJs per muscle, P = 0.24; Fig. 1E). These results further indicated absence of compensatory expression of Cav2.2 channels at Tg NMJs, even at distant sites.
Next, we tested the Cav2.3 channel blocker SNX-482 (1 µM). Quantal content of wild-type NMJs was unchanged (29.4 ± 0.9 before and 30.0 ± 1.4 in presence of SNX-482, n = 4 muscles, 7–10 NMJs per muscle, P = 0.51; Fig. 1F). However, at Tg NMJs, SNX-482 reduced quantal content by 15% (from 30.5 ± 1.6 to 25.8 ± 1.7, n = 4 muscles, 7–10 NMJs per muscle, P < 0.05; Fig. 1F). Together, Cav2.1 and Cav2.3 channels mediate 90% (75% and 15%, respectively) of the 0.3-Hz evoked ACh release. Thus the apparent reduction of Ca2+ influx through -agatoxin-IVA-sensitive channels at the Tg NMJ is almost fully compensated for by Ca2+ influx through SNX-482-sensitive channels, i.e., Cav2.3, because Cav2.1 channels mediate 90–95% of 0.3-Hz evoked release at wild-type channels. SNX-482 did not statistically significantly affect spontaneous release in either genotype, although there was a tendency for reduction at Tg NMJs (Table 1).
It might be speculated that the Tg mutation brings (some) SNX-482 sensitivity onto Cav2.1 channels by creating a (low-affinity) receptor site in the Cav2.1 protein instead of indirectly inducing the expression of compensatory Cav2.3 channels. In that case, the 15% reduction of quantal content by 1 µM SNX-482 at Tg NMJs could be regarded as a suboptimal inhibition of SNX-482-sensitive Tg-mutated Cav2.1 channels. Although this possibility is not very likely in view of the distant localizations of the Tg mutation in Cav2.1 (in P-loop of repeat II) and the SNX-482 binding site in Cav2.3 (on repeats III and IV, presumably in S3-4 regions; Bourinet et al. 2001), we nevertheless tested it by exposing Tg NMJs to a doubled SNX-482 concentration (2 µM). However, no extra reduction of quantal content at Tg NMJs occurred compared with that observed on incubation with 1 µM SNX-482 (n = 3–4 muscles, 10 NMJs per muscle, P = 0.85; Fig. 1F), indicating that it is very unlikely that the Tg mutation rendered the Cav2.1 channel sensitive to SNX-482. We also added 400 nM -agatoxin-IVA to the Tg preparations that were incubated in 2 µM SNX-482 and observed an almost complete block of quantal content (to only 2.3% of the quantal content before the toxins, n = 3 muscles, 10 NMJs per muscle, P < 0.01; Fig. 1F), again showing that ACh release at Tg NMJs is governed exclusively by Cav2.1 and Cav2.3 channels.
Increased 0.3-Hz evoked ACh release at Tg NMJs in low Ca2+
We studied 0.3-Hz evoked ACh release at Tg NMJs in low (0.2 mM) extracellular Ca2+. At wild-type NMJs, quantal content was 1.7 ± 0.4 (n = 4 muscles, 7–10 NMJs per muscle). However, at Tg NMJs, it was approximately sixfold higher (10.7 ± 0.9, n = 4 muscles, 7–10 NMJs per muscle, P < 0.01; Fig. 2A). EPP amplitudes were 1.8 ± 0.6 and 9.1 ± 0.8 mV at wild-type and Tg NMJs, respectively (n = 4 muscles, 7–10 NMJs per muscle, P < 0.01; Fig. 2, B and D). In 0.2 mM Ca2+, EPP failure upon a nerve stimulus was regularly observed at wild-type but not Tg NMJs (48.7 ± 5.7 and 1.3 ± 1.3% of the stimuli, respectively, n = 4 muscles, 7–10 NMJs per muscle, P < 0.001; Fig. 2, B and C). MEPP amplitudes did not differ between genotypes (1.00 ± 0.10 and 0.96 ± 0.05 mV at wild-type and Tg NMJs, respectively, n = 4 muscles, 7–10 NMJs per muscle, P = 0.73).
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-agatoxin-IVA (Fig. 2E). Cav2.3 channels do not contribute disproportionally to rundown of EPP amplitude during 40-Hz nerve stimulation
Previously, at normal extracellular Ca2+ concentration, we have shown that rundown of EPP amplitude during tetanic (40 Hz) nerve stimulation is somewhat more pronounced at Tg NMJs (the rundown plateau level, expressed as percentage of the 1st EPP, was 8% lower; Plomp et al. 2000). Specific Cav2.3 channel behavior, for instance a relatively large use-dependent inhibition, might underlie such increased rundown. To test this hypothesis, we recorded and quantified 40-Hz EPP rundown at Tg NMJs in the presence of either no channel blockers, -agatoxin-IVA (200 or 400 nM), SNX-482 (1 or 2 µM), or both toxins in combination at normal Ca2+ level (Fig. 3, A and B). Normalized EPP rundown in the presence of -agatoxin-IVA and in the presence of SNX-482 was similar (to 77% of the 1st EPP in the train, n = 3–6 muscles, 7–10 NMJs per muscle, P = 0.65; Fig. 3C), which does not differ from the control condition without toxins. This indicates that Cav2.3 channels do not contribute disproportionally to EPP rundown at Tg NMJs.
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DISCUSSION |
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-agatoxin-IVA reduces quantal content by >90%, indicating that ACh release is almost exclusively mediated by Cav2.1 channels (Giovannini et al. 2002; Kaja et al. 2005; Uchitel et al. 1992). The lesser inhibition found at Tg NMJs (only 75%) suggests compensatory involvement of non-Cav2.1 channels. The experiments with SNX-482, a selective blocker of Cav2.3 channels, show that 15% of the total release at Tg NMJs is mediated by Cav2.3 channels. ACh release at NMJs of Cacna1a null-mutant mice, which lack Cav2.1 channels and die at 3 wk of age, becomes dependent on Cav2.3 as well as Cav2.2 channels (Urbano et al. 2003). However, we could exclude compensatory involvement of Cav2.2 channels at Tg NMJs by showing insensitivity of quantal content to -conotoxin-GVIA, even in a protocol testing for Cav2.2 channels localized more distantly from the Ca2+ sensor at release sites. Thus we showed that Cav2.3 channels are recruited first as compensatory channels at Tg NMJs. This is the first report of compensatory expression of non-Cav2.1 channels at NMJs of mice with a single amino acid change in Cav2.1. On the basis of the results of this extensive study, we have to revise our earlier, preliminary view of similar -agatoxin-IVA sensitivity of evoked ACh release at wild-type and Tg NMJs, which was based on only a single experiment per genotype (Plomp et al. 2000).
The studies on Cacna1a null-mutant NMJs have led to the hypothesis that ACh release sites have "slots" that are preferentially filled with Cav2.1 channels, but in their absence, become occupied by Cav2.3 channels (Urbano et al. 2003). This would suggest a small reduction in the presynaptic membrane expression of Cav2.1 channels at Tg NMJs, leaving slots available for Cav2.3 channels. However, although reduced Tg-mutated Cav2.1 channel expression at nerve terminals has indeed been proposed (Leenders et al. 2002), such a reduction was not supported by our previous finding of a twofold increased spontaneous ACh release at Tg NMJs that remains sensitive to -agatoxin-IVA (Plomp et al. 2000). The explanation may be that the effect of a small reduction in Tg Cav2.1 channel expression is masked by increased Ca2+ flux through the remaining channels, because of a mutation-induced shift of their activation voltage toward more negative values, as proposed by us earlier (Plomp et al. 2000). Extra Ca2+ influx through Tg-mutated Cav2.1 channels was further substantiated here by the finding that ACh release in low extracellular Ca2+ was approximately sixfold increased at Tg NMJs. This increase was solely caused by the Tg-mutated Cav2.1 channels and not due to compensatory Cav2.3 channels, because it was unaffected by SNX-482 and almost completely inhibited by -agatoxin-IVA. There is an interesting parallel between the electrophysiology of Tg NMJs and those of R192Q and S218L Cacna1a-mutated mice (knock-in models for human familial hemiplegic migraine), in that we have recently observed similar increases in MEPP frequency and low-Ca2+ quantal content at those NMJs (Kaja et al. 2004, 2005; Van Den Maagdenberg et al. 2004). Voltage-clamp measurements in (transfected) primary cultured neurons and heterologous expression systems showed a clear negative shift of activation voltage for both R192Q- and S218L-mutated channels (Tottene et al. 2005; Van Den Maagdenberg et al. 2004). These parallels with Tg synapses further suggest that Tg-mutated Cav2.1 channels at NMJs may have a negatively shifted activation voltage. However, enigmatically, Wakamori et al. (1998) showed normal activation voltage for Tg channels in Purkinje cell bodies and transfected baby hamster kidney cells. It may, however, be that extrapolation of data obtained at cell body channels to behavior of presynaptic channels is not justifiable because of the specific interactions of presynaptic channels with their native environment at transmitter release sites.
Besides a shift in activation voltage, Tg channels may have altered modulatory properties after a mutation-induced change of interaction with factors such as calmodulin or G proteins (Catterall 2000; Lee et al. 1999), leading to increased Ca2+ flux. However, such an explanation is not very likely in view of the very different localizations of the Tg mutation and known binding and effector sites of modulatory factors (Zhong et al. 2001).
As yet, it is unclear how compensatory presynaptic Cav2.3 channels are recruited. At wild-type NMJs, they do not contribute to ACh release and are undetectable with immunohistochemistry (Westenbroek et al. 1998). It may be that protein expression must first be triggered, e.g., by the changed Ca2+ influx caused by Tg Cav2.1 mutation. Alternatively, membrane insertion may normally fail because of absence of available slots at ACh release sites, but become successful when Tg Cav2.1 mutation results in some free slots.
The lack of compensatory involvement of Cav2.2 channels at Tg NMJs, as opposed to Cacna1a null-mutant NMJs, may be explained by the presence of remaining Cav2.1 channels. For instance, expression of syntaxin-1A is dependent on selective Cav2.1-mediated Ca2+ influx (Sutton et al. 1999). This presynaptic protein can inhibit the function of Cav2.1 and Cav2.2 channels (Bezprozvanny et al. 1995) but not that of Cav2.3 channels. Thus if altogether present at the NMJ, Cav2.2 channels in the Tg presynaptic membrane may be silenced. The situation at central synapses seems different. Compensatory Cav2.2 channels have been shown at Tg hippocampal synapses (Qian and Noebels 2000) and forebrain synaptosomes (Leenders et al. 2002).
The changes at Tg NMJs partly resemble those found at NMJs of the R192Q knock-in mouse model for familial hemiplegic migraine (Kaja et al. 2005; Van Den Maagdenberg et al. 2004): increased spontaneous ACh release and evoked release that is normal at physiological extracellular Ca2+ but strongly increased, compared with wild-type, at low Ca2+. However, clear differences exist. There is no compensatory involvement of Cav2.3 channels at R192Q NMJs, because -agatoxin-IVA reduces quantal content by the same extent (>90%) as in wild-types. Furthermore, the reduction in high-rate evoked ACh release at Tg NMJs (Plomp et al. 2000) is more pronounced than that at R192Q NMJs (Kaja et al. 2005).
The Tg NMJ displays some extra rundown of high-rate evoked ACh release, compared with wild-type (Plomp et al. 2000). It may be that Cav2.3 channel behavior contributes to this phenomenon. Normal rundown at wild-type NMJs is likely caused by a combination of Cav2.1 channel inactivation, its recovery, and the replenishment of releasable ACh vesicles. A relatively large degree of use-dependent inhibition of Cav2.3, compared with that of (Tg-)Cav2.1 channels, e.g., induced by faster inactivation (Williams et al. 1994), might add disproportionally to the EPP rundown at Tg NMJs. However, normalized EPP rundown at Tg NMJs in the presence of either SNX-482 or -agatoxin-IVA did not differ. This indicates that the contribution of use-dependent inhibition of Cav2.3 channels to EPP rundown is either similar to that of (Tg-)Cav2.1 channels or that use-dependent inhibition of Cav2 channels is not at all a factor contributing to rundown at normal Ca2+ level. It may be that the replenishment rate of ACh vesicles at release sites is the major determinant of EPP rundown under these conditions.
The mechanism of increased ACh release becoming unmasked at low extracellular Ca2+ concentration at Tg as well as R192Q NMJs is unclear. The Ca2+ influx through mutant channels at physiological extracellular Ca2+ may be of such magnitude that presynaptic sensors saturate. Alternatively, Ca2+/calmodulin-dependent Cav2.1 inactivation (Lee et al. 1999) may be increased at mutant synapses, because of increased Ca2+ influx or, although not very likely (as discussed above), a direct change in Cav2.1 modulatory characteristics. Alternatively, the localization of the different types of Cav2 channels relative to the Ca2+ sensor of the neuroexocytotic mechanism may play a role (Urbano et al. 2003; Wu et al. 1999). Tg-mutated Cav2.1 channels might be more closely localized than wild-type channels and therefore contribute more efficiently to ACh release. This may also (partly) explain the higher MEPP frequency at Tg NMJs, compared with wild-type, at normal extracellular Ca2+ concentration. Similarly, a closer localization of Tg-Cav2.1 channels to release sites than Cav2.3 channels may explain the lack of contribution of Cav2.3 channels to evoked ACh release under the condition of low extracellular Ca2+.
It is unclear whether compensatory Cav2.3 channel-mediated transmitter release at Tg CNS synapses, as present at the NMJ, influences the symptoms of ataxia and epilepsy. Central neurons can upregulate Cav2.3 channels after partial downregulation of Cav2.1 channels, as shown in cerebellar Purkinje cells (Pinto et al. 1998). However, total genetic Cav2.1 ablation results in unaltered or reduced Cav2.3 current density in cerebellar neurons (Fletcher et al. 2001; Jun et al. 1999). The unidentified residual Ca2+ current shown at Tg hippocampal presynapses after blocking Cav2.1 and -2 channels (Qian and Noebels 2000) may be due to Cav2.3 channel expression. It would be interesting to cross-breed Tg with Cacna1e mice (Wilson et al. 2000) to test Cav2.3 channel involvement.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. J. Plomp, Dept. of Molecular Cell Biology-Group Neurophysiology, Research Building, Leiden University Medical Centre, PO Box 9600, NL-2300 RC Leiden, The Netherlands (E-mail: j.j.plomp{at}lumc.nl)
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