| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908
Submitted 12 May 2004; accepted in final form 10 July 2004
 |
ABSTRACT |
|---|
|
33). The goal of this study is to determine the functional consequences of these variations in the full-length channel. The cDNA encoding these regions were cloned using RT-PCR from human brain, and currents were recorded by whole cell patch clamp. Introduction of the 33 deletion slowed the rate of channel opening. Addition of exon 9 had little effect on kinetics, whereas its addition to 33 channels unexpectedly slowed both activation and inactivation kinetics. Modeling of neuronal firing showed that exon 9 or 33 alone reduced burst firing, whereas the combination enhanced firing. The major conclusions of this study are that the intracellular regions after repeats I and IV play a role in channel gating, that their effects are interdependent, suggesting a direct interaction, and that splice variation of Cav3.3 channels provides a mechanism for fine-tuning the latency and duration of low-threshold spikes. |
|
INTRODUCTION |
|---|
|
) and by alternative splicing of those genes (Bourinet et al. 1999). T-type Ca2+ channels open after small depolarizations of the plasma membrane and hence mediate low-threshold Ca2+ spikes. These spikes in turn mediate the opening of voltage-gated Na+ channels, and a second set of high-voltage-activated Ca2+ channels (reviewed in (Perez-Reyes 2003). Therefore neuronal firing can be fine-tuned by the kinetics and voltage sensitivity of T-type Ca2+ channels.
From the cloning and expression of their cDNAs, it is now known that there are three T-type channel genes. The channels encoded on these genes (Cav3.1, Cav3.2, and Cav3.3) have distinct kinetic features, suggesting that they play distinct roles in neuronal firing (Kozlov et al. 1999). Specifically, the slow activation and inactivation of Cav3.3 channels suggests they play a role in sustained firing, whereas the fast kinetics of Cav3.1 and Cav3.2 would lead to firing of short bursts (Chemin et al. 2002).
Alternative splicing of Ca2+ channel genes is known to play an important role in determining their channels' biophysical properties, their pharmacology, and their ability to be regulated by G proteins. Notable examples include the splicing of CACNA1C, which modulates its sensitivity to antihypertensive drugs (Welling et al. 1997), and splicing of CACNA1A, which modulates its sensitivity to spider toxins used to differentiate P- from Q-type channels (Bourinet et al. 1999). Completion of the human genome sequence facilitated study of alternative splicing by allowing identification of intron-exon boundaries and the design of PCR primers to span the splice junctions. Using this approach, Mittman and coworkers identified two sites of alternative splicing in the human CACNA1I gene (Mittman et al. 1999). One site is the variable inclusion of exon 9 (abbreviated +9 or 9), which adds 35 amino acids, whereas the second site is the use of an alternate acceptor in exon 33 (abbreviated +33 or 33), which leads to variable inclusion of 13 amino acids (Mittman et al. 1999). Although the effect of exon 33 splicing on human Cav3.3 channel activity was studied by Chemin and coworkers, these studies used a truncated channel that was missing exon 37 (see (Gomora et al. 2002). Exon 37 encodes half of the carboxy terminal sequence that follows the last transmembrane segment (IVS6) and has been shown to play a role in channel regulation (Gomora et al. 2002). The rat Cav3.3 gene is also alternatively spliced at the homologous region as exon 33, and the electrophysiological consequences of this splicing are modulated by the distal carboxy terminus (Murbartián et al. 2002). This splicing was found to occur in a brain-region-specific manner, suggesting that alternative splicing may provide a mechanism for fine tuning neuronal firing. Therefore the goals of this study were to investigate the electrophysiological consequences of exon 9 and 33 splicing in a full-length human Cav3.3 channel.
Furthermore, the NEURON model was used to predict the functional impact that this splicing might have on neuronal firing (Hines and Carnevale 2001). This model has been used extensively to validate the role of T-type channels in neuronal excitability, in particular in neurons of the thalamocortical loop (Destexhe et al. 1994, 1996a, 1998a; Lytton et al. 1997). Notably, models have been developed for neurons of the reticular nucleus of the thalamus, where Cav3.3 mRNA is highly expressed. Burst firing of these neurons is mediated by a slowly inactivating T current (Huguenard and Prince 1992) and plays an important role in generating slow-wave (2–4 Hz) discharges observed in absence epilepsy patients and in other thalamocortical dysrhythmias (Jeanmonod et al. 1996).
|
|
METHODS |
|---|
|
cDNA was synthesized from 100 ng fetal brain poly(A)+ RNA (BD Biosciences Clontech, Palo Alto, CA) using 5 µM random nonamers, 0.5 mM of dNTP, 20 U SUPERase · In (Ambion, Austin, TX), and 100 U M-MLV reverse transcriptase (Ambion). Amplification reactions (25 µl volume) were performed in a Mastercycler gradient (Eppendorf, Westbury, NY) and contained 2.5 µl cDNA, 0.4 µM each primer, 0.2 mM dNTP, and 0.5 U Vent DNA polymerase (New England Biolabs, Beverly MA). After a 150-s denaturation step, reactions were cycled 35 times using 25 s for denaturation (94°C), annealing (62°C), and extension (72°C). PCR products were then treated with 1 U Taq DNA polymerase and 0.2 mM dATP and cloned into pCRII-TOPO kit (Invitrogen, Carlsbad, CA). Colonies were screened by PCR amplification with the respective PCR primers. Positive clones were further identified by their restriction enzyme map, and sequencing. Synthetic oligonucleotides used for DNA amplifications were obtained from Operon (Alameda, CA). Splicing of exon 9 was assessed using primers u5 and a22, which are located in exons 8 and 10 (Fig. 1A). Splicing of exon 33 was assessed using PCR primers n43 and r7, which are located in exons 32 and 34 (Fig. 1B).
|
). To facilitate addition of the exon 9 sequence, a silent mutation was introduced that would create a BspEI restriction enzyme site. This was accomplished in two PCR reactions: the first reaction used primers u5 and r22 and plasmid derived from the PCR product shown in Fig. 1C, and the second reaction used primers u10 and r21. The full-length cDNA was generated by ligating the following fragments: ClaI (polylinker)/BsaI (1220) from LT9, the BsaI/BspEI fragment for PCR1, the BspEI/AvrII fragment from PCR2, and the AvrII (2683)/EcoRI (polylinker) fragment from LT9. The 33 variation was introduced by single overlap extension (SOE) method (Horton et al. 1993) using 0.5 U Vent DNA polymerase. The full-length cDNA was generated by ligating the following fragments: EcoRI (polylinker)/HindIII (4755) from LT9, the HindIII/BamHI fragment of the SOE fragment, and the BamHI (5662)/XbaI (polylinker) fragment from LT9. Transfections
Two hundred ninety three cells (human embryonic kidney, No. CRL-1573, American Type Culture Collection, Manassas, VA) were transiently cotransfected with plasmid DNAs encoding each Cav3.3 variant and green fluorescent protein (GFP; pGreen Lantern, Invitrogen), at a molar ratio of 5:1, by the calcium phosphate method (CalPhos Maximizer Transfection Kit, Clontech). After 
24 h, GFP positive cells were selected for electrophysiological recordings. The results were obtained from 12 transfections, and each construct was tested in 
4 transfections.
Electrophysiology
Electrophysiological experiments were carried out using the whole cell configuration of the patch-clamp technique. Recordings were obtained using an Axopatch 200B amplifier, Digidata 1322A A/D converter, and pCLAMP 8.0 software (Axon Instruments, Union City, CA). Data were filtered at 2 kHz and digitized at 5 kHz. Tail currents were filtered at 10 kHz and digitized at 50 kHz. Whole cell Ca2+ currents were recorded using the following external solution (in mM): 5 CaCl2, 155 tetraethyl ammonium (TEA) chloride, and 10 HEPES, pH adjusted to 7.4 with TEA-OH. The internal pipette solution contained the following (in mM): 125 CsCl, 10 EGTA, 2 CaCl2, 1 MgCl2, 4 Mg-ATP, 0.3 Na3GTP, and 10 HEPES, pH adjusted to 7.2 with CsOH. Pipettes were made from TW-150-3 capillary tubing (World Precision Instruments, Sarasota, FL). Under these solution conditions, the pipette resistance was typically 2–3 M
. Access resistance in the whole cell configuration averaged 5.6 ± 0.2 M (n = 40) and was compensated between protocols to 70%. Cell capacitance averaged 10.9 ± 0.5 pF (n = 40). All experiments were performed at room temperature (22°C).
Data analysis
PCR product formation was quantitated using densitometric analysis as described previously (Murbartián et al. 2002). Peak currents and exponential fits to currents were determined using Clampfit 8.0 software (Axon Instruments). Leak subtraction was performed off-line using the passive resistance algorithm in Clampfit. Fits to average data and statistical tests were performed using Prism software (Graphpad, San Diego, CA). Statistical tests included ANOVA, unpaired two-tailed Student's t-test for comparing two data sets, and the F test for comparing two models such as single- or double-exponential fits. Statistically significant differences are noted with a single asterisk if P < 0.05 and with a double asterisk if P < 0.01. Average data are presented as means ± SE.
Modeling
The NEURON model uses mathematical descriptors of ionic conductances to calculate whether a channel is open as a function of voltage, and does this by applying Hodgkin-Huxley-type equations (Destexhe et al. 1996b, 1998b). This requires a detailed characterization of the voltage dependence of activation and inactivation as well as the voltage dependence of their kinetics. The voltage dependence of activation (m
; mid-point V1/2; slope k) was determined by fitting the peak current-voltage (I-V) data from each cell with the following form of the Goldman-Hodgkin-Katz equation
 |
) was measured using 15-s prepulses to varying potentials and a 200-ms test pulse to –30 mV to measure channel availability. Data from each cell were fit with a Boltzmann equation then averaged. The voltage dependence of the time constants of activation (
m) and inactivation (h) were obtained using the following equation
 |
m and h were calculated using data from standard current-voltage (I-V) protocols, where the traces were fit with two exponentials. The beginning of the fit range was set to a time where the current was inward (typically 3 ms), thereby excluding an initial outward transient that may represent a gating current and any associated lag (Burgess et al. 2002; Kuo and Yang 2001). Values for m at more hyperpolarized potentials were obtained from tail current protocols. The tail current protocol included a short 2-ms step to +60 mV to open channels, followed by repolarization to varying voltages. As this evoked large currents (>5 nA), precautions were taken to minimize series resistance errors, such as use of low-resistance pipettes and series-resistance compensation. Under these conditions, the rise time to the peak of tail current was <30 µs. The fit region began at the peak of the tail current and ended 50 ms later. As noted previously (Frazier et al. 2001; Gomora et al. 2002), this deactivation kinetic was best fit with two exponentials, so a weighted (w) was calculated using the equation
 |
f) and slow components (s). The data used to determine the voltage dependence of h included the rate of recovery from inactivation at –100 and –90 mV, the development of inactivation at –70 and –60 mV, and the rate of inactivation at more depolarized potentials from I-V data. Development of inactivation was measured by holding the membrane for varying durations, and then channel availability was tested with a 55-ms pulse to –30 mV. These kinetics were also best fit with two exponentials, so a weighted h was calculated as in the preceding text. Channels were allowed to recover between sweeps by holding the membrane at –100 mV for 10 s. The data were corrected for a junction potential (–9.4 mV; Figs. 1–4 show uncorrected data) and surface charge screening (–4 mV). The data were collected at 24°C and the simulations were at 36°C. The model accounts for this difference using Q10 values determined previously (Coulter et al. 1989). The maximum permeability of T currents was left as in the original models. The experimentally determined values of the recombinant channels were then used to replace all other parameters for the T channel in the models of thalamic reticular neurons (Destexhe et al. 1996b). The current-clamp simulations used the three-compartment model with the current injected into the dendritic compartment and recorded from the virtual soma.
|
|
|
RESULTS |
|---|
|
9) and contain the full exon 33 sequence (abbreviated 9 + 33). The ratio of 9 to +9 transcripts was 10:1. Transcripts containing exon 9 were readily detected using a reverse primer based on the exon 9 DNA sequence. PCR products containing the complete exon 33 sequence were more abundant than those resulting from the internal acceptor site (33). The ratio of +33 to 33 was 5:1. The PCR products were subcloned, and the plasmid DNA from 48 colonies was analyzed by restriction mapping and sequencing of representative clones. Of these, 41 were +33 and 7 were 33. These results confirm the identity of the PCR products and their relative abundance.
To assess the electrophysiological consequences of alternative splicing on Cav3.3 channel activity, full-length cDNAs were constructed, transfected into HEK-293 cells, then studied using patch-clamp electrophysiology. The variants were introduced into the full-length Cav3.3 clone LT9, which lacks exon 9, contains the full exon 33 (9 + 33), and includes exon 37 (Gomora et al. 2002). All four splice constructs led to the induction of robust low-voltage-activated currents with peak current densities >100 pA/pF. The current-voltage (I-V) relationships were identical for all variants (Table 1). Visual inspection of normalized current traces (Fig. 2A) revealed that truncation of exon 33 (33) led to channels that activated slower. To quantitate this effect, the inward currents were fit with two exponentials, where one describes activation and the other inactivation. On average, 933 channels activated 40% slower than those with the full exon 33 (9 + 33) during pulses between –30 and 0 mV (Fig. 2B). Deactivation kinetics of the tail current were not significantly different. Activation and deactivation kinetics were then fit simultaneously to determine the voltage dependence of m (equation described in METHODS; results in Table 1). Exon 33 variants showed nearly identical rates of inactivation at subthreshold voltages and at the more depolarized potentials tested during the I-V protocol as well as similar rates of recovery from inactivation (Fig. 2C).
|
|
9 + 33) revealed that at depolarized potentials they activate and inactivate with virtually the same kinetics and voltage dependence. However, there were differences at potentials closer to the resting membrane potential of most neurons with +9 channels showing a steeper voltage dependence of steady-state inactivation (Table 1) and faster recovery from inactivation (Fig. 3, A and B; Table 1). Inactivation was induced by holding the membrane potential at –60 mV for 10 s, and recovery was measured by varying the duration at –90 mV before testing current availability at –30 mV. Representative traces are shown (Fig. 3A) where the currents were normalized to the maximum current recovered. Average results show that +9 channels recovered significantly faster.
|
33 variation. In contrast to the previous results, addition of exon 9 now had a profound affect on activation and inactivation kinetics (Fig. 4, B and C). The greatest effect on activation kinetics occurred at depolarized potentials (–30 to +10 mV), where channels opened 60% faster. The effect on inactivation rates was also greater at depolarized potentials (–30 to +20 mV), where +933 channels inactivated 26% faster than 933. Other measures of h and m were unaffected.
The functional impact of Cav3.3 splicing was then tested using the NEURON model (Hines and Carnevale 2001). Because the model calculates the probability of channel opening as a function of voltage, one must first calculate the voltage dependence of activation, inactivation, and the voltage dependence of these kinetics (equations detailed in METHODS and fits shown in Fig. 2– 4). We used the model developed by Destexhe and coworkers (1996b) for thalamic reticular (nRT) neurons because Cav3.3 mRNA is abundantly expressed in these neurons and because recombinant Cav3.3 channels are quite similar to native currents recorded from these neurons (Huguenard and Prince 1992). The model has been previously validated by showing that it could replicate the rebound burst firing observed in native nRT neurons (Destexhe et al. 1996b). Introduction of the biophysical parameters of the recombinant Cav3.3 channels also led to rebound burst firing (Fig. 5, A–D, a–c). Addition of either exon 9, or truncation of exon 33 from the most predominant isoform, –9 + 33, reduced the number of spikes per burst after mild hyperpolarizations. In contrast, +933 channels are predicted to increase neuronal excitability by producing bursts after smaller hyperpolarizations. The splice variants also differed in how rapidly they produced the depolarizing LTS, and this was quantitated by measuring the latency to the first Na-dependent action potential (Fig. 5Eb). The rank order between the variants in their latency to fire correlated well with their activation kinetics at these potentials (Table 1). Robust burst firing could be observed in all splice variants after strong hyperpolarizations (>0.4 nA). Because significant differences were also found for the rates at which the variants recover from inactivation (Fig. 3, Table 1), the effect of varying the duration of the hyperpolarizing pulse was modeled. A pattern similar to that of the rebound bursts was observed in terms of both the number and latency of the Na spikes. To model firing after excitatory postsynaptic potentials, the effect of depolarizing current injections were studied (representative traces shown in A–D, d). Under these conditions, the –9 + 33, +9 + 33, and +933 channels behaved similarly, whereas the +9 + 33 channels showed a reduced tendency to fire, requiring stronger depolarization and producing a slowly developing LTS (Fig. 5G, a and b). Simulations using properties recorded from rat nRT neurons were similar but distinct from the human variants (Destexhe et al. 1996b; Huguenard and Prince 1992). These studies predict that modest changes in channel biophysics can lead to pronounced changes in neuronal activity.
|
|
|
DISCUSSION |
|---|
|
1 subunits of T-type channels and their splice variants (Perez-Reyes 2003). The CACNA1I gene encodes Cav3.3 channels, and the gene is alternatively spliced at exons 9 and 33 (Mittman et al. 1999). The present study confirms the existence of these splice variants and provides the first description of the functional impact this splicing has on the full-length channel.
Transcripts encoding the exon 9 and 33 variants were readily detected using PCR. The most abundant mRNA transcripts were those that skipped exon 9 and contained the full exon 33 sequence (9 + 33). This result indicates that the original human Cav3.3a variant cloned is the predominant isoform (Gomora et al. 2002; Monteil et al. 2000). Both alternative splicing events would lead to changes in the sequence of cytoplasmic loops immediately after the last transmembrane segment of repeats I and IV. The analogous regions in HVA channels are very important for channel regulation by Ca2+ channel 
subunits, G protein 
dimers, and calmodulin. Therefore we hypothesized that they might be important in Cav3.3 channel function as well. This hypothesis was supported by previous studies of exon 33 splicing using rat and human Cav3.3 channels (Chemin et al. 2001; Murbartián et al. 2002). When expressed in 293 cells, the human Cav3.3 exon 33 variants were found to deactivate at different rates but activate and inactivate at essentially the same rate (Chemin et al. 2001). In contrast, the present study found that these variants differ in activation kinetics with no significant difference in deactivation and inactivation kinetics. These disparate results might be explained by the previous study using a channel that was missing the last 214 amino acids of the carboxy terminus encoded by exon 37 (Gomora et al. 2002). Support for this notion comes from studies on rat Cav3.3 splice variants, where the effects of splicing in the analogous region as exon 33 were dependent on whether the channel had a short or long carboxy terminus (Murbartián et al. 2002). Alternative splicing might explain why two distinct sized isoforms of Cav3.3 channels were detected during Western blot studies of mouse brain (Yunker et al. 2003). Notably, these variants were found to vary in a tissue-specific and developmental pattern.
Chimeric studies between Cav3.1 and Cav1.2 identified a negatively charged region in the proximal carboxy terminus (23 amino acids after IVS6) as having an important role in channel inactivation (Staes et al. 2001). This region is conserved in all three Cav3 channels, suggesting that its role is conserved. Exon 33 splicing occurs 67 amino acid residues after IVS6, and the present results indicate this region has a greater role in Cav3.3 channel activation kinetics. Taken together, these results indicate that the carboxy termini of Cav3 channels modulate channel activity and may provide a site for binding of auxiliary subunits or posttranslation processing.
An unexpected finding of this study was that the effect of exon 9 splicing was highly dependent on the sequence at exon 33. Addition of exon 9 to channels containing the full exon 33 sequence had modest effects on channel gating, slowing recovery from inactivation and the voltage dependence of inactivation (comparison of –9 + 33 vs. +9 + 33). These results indicate that exon 9 has little effect on the final transitions between open and inactivated states, acting instead on transitions between inactivated and closed states. In contrast, addition of exon 9 to channels containing the partial exon 33 sequence led to pronounced changes in channel activation and inactivation rates (comparison of –933 vs. +933). Now exon 9 affected transitions between closed states and the open state and transitions from the open to inactivated state. Such interactions could result from two distinct mechanisms. One, gating transitions of domains I and IV may be transmitted allosterically, as they likely face each other in the three-dimensional structure. Or two, the intracellular loops interact directly, thereby modulating the effects the other has on gating. We favor the second hypothesis based on the degree of structure-function conservation between LVA and HVA channels, and the physical interactions these loops have in Cav2.1 channels (Geib et al. 2002).
Recordings from neurons have revealed a large heterogeneity in the kinetics of T-type currents (Perez-Reyes 2003). Slow T-type currents have been described in neurons from thalamic reticular nucleus and lateral habenula, where they are thought to play a unique role in generating long bursts of firing (Huguenard et al. 1992, 1993). Because these regions also express abundant Cav3.3 mRNA and recombinant Cav3.3 channels display similar slow inactivation kinetics, it is likely that alternative splicing of CACNA1I plays an important role in these neurons (Talley et al. 1999). Subtype-specific antibodies might validate this hypothesis.
Modeling has provided a useful tool to infer the functional roles of ion channels in determining the firing patterns of neurons. Models of thalamic reticular and relay neurons have provided insight into the role of T-type Ca2+ channels in generating low-threshold spikes and how their kinetics, levels of expression, and distribution within the neuron can affect firing (Destexhe et al. 1996b; Huguenard and Prince 1994; McCormick and Huguenard 1992). Introduction of recombinant T-type channel parameters into these models confirms that fast inactivating channels such as Cav3.1 and Cav3.2 produce short spikes in response to depolarizing stimuli, whereas Cav3.3 channels produce longer-lasting spikes that lead to sustained burst firing (Chemin et al. 2002). The present study shows that recombinant Cav3.3 channel behavior can also be used to model rebound burst firing. Introduction of parameters for alternatively spliced variants indicated that these channels will produce distinct firing patterns. In general, the effect of alternative splicing is to decrease the ability of these channels to produce rebound spikes when compared with the predominant isoform (9 + 33). One of the most critical determinants of rebound burst firing is the rate of activation. Channels that activate fast (e.g., 9 + 33) are capable of producing a LTS that triggers bursts that occur sooner and with more Na spikes in comparison to slowly activating channels (e.g., 933). Although a significant difference was noted in the rate of recovery from inactivation for the +9 + 33 channel, this difference did not appear to have much effect because varying the duration of the hyperpolarizing pulse did not alter the rank order of the channels to trigger firing. Channel availability is another important determinant of burst firing. A significant difference in the steady-state inactivation curve was noted for +9 + 33 channels, and this provides a likely explanation for why this variant triggered less firing after depolarizing pulses. Consistent with this hypothesis, all four variants behaved similarly to depolarizing pulses if the resting membrane potential was –85 mV. These results indicate alternative splicing modifies important channel transitions that determine neuronal excitability and that studies on T-type channel gating should focus more on the transitions that occur near the resting membrane potential of most neurons.
|
|
GRANTS |
|---|
|
|
|
ACKNOWLEDGMENTS |
|---|
|
|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: E. Perez-Reyes, Dept. of Pharmacology, P.O. Box 800735, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0735 (E-mail: eperez{at}virginia.edu).
|
|
REFERENCES |
|---|
|
1A subunit gene generates phenotypic variants of P- and Q-type calcium channels. Nat Neurosci 2: 407–415, 1999.[CrossRef][Web of Science][Medline]Burgess DE, Crawford O, Delisle BP, and Satin J. Mechanism of inactivation gating of human T-type (low-voltage activated) calcium channels. Biophys J 82: 1894–1906, 2002.[Web of Science][Medline]
Chemin J, Monteil A, Dubel S, Nargeot J, and Lory P. The 1I T-type calcium channel exhibits faster gating properties when overexpressed in neuroblastoma/glioma NG 108–15 cells. Eur J Neurosci 14: 1678–1686, 2001.[CrossRef][Web of Science][Medline]
Chemin J, Monteil A, Perez-Reyes E, Bourinet E, Nargeot J, and Lory P. Specific contribution of human T-type calcium channel isotypes (1G, 1H and 1I) to neuronal excitability. J Physiol 540: 3–14, 2002.
Coulter DA, Huguenard JR, and Prince DA. Calcium currents in rat thalamocortical relay neurones: kinetic properties of the transient, low-threshold current. J Physiol 414: 587–604, 1989.
Destexhe A, Bal T, McCormick DA, and Sejnowski TJ. Ionic mechanisms underlying synchronized oscillations and propagating waves in a model of ferret thalamic slices. J Neurophysiol 76: 2049–2070, 1996a.
Destexhe A, Contreras D, Sejnowski TJ, and Steriade M. A model of spindle rhythmicity in the isolated thalamic reticular nucleus. J Neurophysiol 72: 803–818, 1994.
Destexhe A, Contreras D, and Steriade M. Mechanisms underlying the synchronizing action of corticothalamic feedback through inhibition of thalamic relay cells. J Neurophysiol 79: 999–1016, 1998a.
Destexhe A, Contreras D, Steriade M, Sejnowski TJ, and Huguenard JR. In vivo, in vitro, and computational analysis of dendritic calcium currents in thalamic reticular neurons. J Neurosci 16: 169–185, 1996b.
Destexhe A, Neubig M, Ulrich D, and Huguenard J. Dendritic low-threshold calcium currents in thalamic relay cells. J Neurosci 18: 3574–3588, 1998b.
Frazier CJ, Serrano JR, George EG, Yu X, Viswanathan A, Perez-Reyes E, and Jones SW. Gating kinetics of the 1I T-type calcium channel. J Gen Physiol 118: 457–470, 2001.
Geib S, Sandoz G, Cornet V, Mabrouk K, Fund-Saunier O, Bichet D, Villaz M, Hoshi T, Sabatier J-M, and De Waard M. The interaction between the I-II loop and the III-IV loop of Cav2.1 contributes to voltage-dependent inactivation in a -dependent manner. J Biol Chem 277: 10003–10013, 2002.
Gomora JC, Murbartián J, Arias JM, Lee J-H, and Perez-Reyes E. Cloning and expression of the human T-type channel Cav3.3: insights into prepulse facilitation. Biophys J 83: 229–241, 2002.[Web of Science][Medline]
Hines ML and Carnevale NT. NEURON: a tool for neuroscientists. Neuroscientist 7: 123–135, 2001.
Horton RM, Ho SN, Pullen JK, Hunt HD, Cai Z, and Pease LR. Gene splicing by overlap extension. Methods Enzymol 217: 270–279, 1993.[Web of Science][Medline]
Huguenard JR, Gutnick MJ, and Prince DA. Transient Ca2+ currents in neurons isolated from rat lateral habenula. J Neurophysiol 70: 158–166, 1993.
Huguenard JR and Prince DA. Intrathalamic rhythmicity studied in vitro: nominal T-current modulation causes robust antioscillatory effects. J Neurosci 14: 5485–5502, 1994.[Abstract]
Huguenard JR and Prince DA. A novel T-type current underlies prolonged Ca2+-dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus. J Neurosci 12: 3804–3817, 1992.[Abstract]
Jeanmonod D, Magnin M, and Morel A. Low-threshold calcium spike bursts in the human thalamus. Common physiopathology for sensory, motor and limbic positive symptoms. Brain 119: 363–375, 1996.
Kozlov AS, McKenna F, Lee JH, Cribbs LL, Perez-Reyes E, Feltz A, and Lambert RC. Distinct kinetics of cloned T-type Ca2+ channels lead to differential Ca2+ entry and frequency dependence during mock action potentials. Eur J Neurosci 11: 4149–4158, 1999.[CrossRef][Web of Science][Medline]
Kuo CC and Yang S. Recovery from inactivation of T-type Ca2+ channels in rat thalamic neurons. J Neurosci 21: 1884–1892, 2001.
Llinás RR. The intrinsic electrophysiological properties of mammalian neurons: insights into central nervous system function. Science 242: 1654–1664, 1988.
Lytton WW, Contreras D, Destexhe A, and Steriade M. Dynamic interactions determine partial thalamic quiescence in a computer network model of spike-and-wave seizures. J Neurophysiol 77: 1679–1696, 1997.
McCormick DA and Huguenard JR. A model of the electrophysiological properties of thalamocortical relay neurons. J Neurophysiol 68: 1384–1400, 1992.
Mittman S, Guo J, Emerick MC, and Agnew WS. Structure and alternative splicing of the gene encoding 1I, a human brain T calcium channel 1 subunit. Neurosci Lett 269: 121–124, 1999.[CrossRef][Web of Science][Medline]
Monteil A, Chemin J, Leuranguer V, Altier C, Mennessier G, Bourinet E, Lory P, and Nargeot J. Specific properties of T-type calcium channels generated by the human 1I subunit. J Biol Chem 275: 16530–16535, 2000.
Murbartián J, Arias J, Lee J, Gomora J, and Perez-Reyes E. Alternative splicing of the rat Cav3.3 T-type calcium channel gene produces variants with distinct functional properties. FEBS Lett 528: 272, 2002.[CrossRef][Web of Science][Medline]
Perez-Reyes E. Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev 83: 117–161, 2003.
Staes M, Talavera K, Klugbauer N, Prenen J, Lacinová L, Droogmans G, Hofmann F, and Nilius B. The amino side of the C-terminus determines fast inactivation of the T-type calcium channel 1G. J Physiol 530: 35–45, 2001.
Talley EM, Cribbs LL, Lee JH, Daud A, Perez-Reyes E, and Bayliss DA. Differential distribution of three members of a gene family encoding low-voltage-activated (T-type) calcium channels. J Neurosci 19: 1895–1911, 1999.
Welling A, Ludwig A, Zimmer S, Klugbauer N, Flockerzi V, and Hofmann F. Alternatively spliced IS6 segments of the 1C gene determine the tissue-specific dihydropyridine sensitivity of cardiac and vascular smooth muscle L-type Ca2+ channels. Circ Res 81: 526–532, 1997.
Yunker AM, Sharp AH, Sundarraj S, Ranganathan V, Copeland TD, and McEnery MW. Immunological characterization of T-type voltage-dependent calcium channel Cav3.1 (alpha1G) and Cav3.3 (alpha1I) isoforms reveal differences in their localization, expression, and neural development. Neuroscience 117: 321–335, 2003.[CrossRef][Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  K. L. Powell, S. M. Cain, C. Ng, S. Sirdesai, L. S. David, M. Kyi, E. Garcia, J. R. Tyson, C. A. Reid, M. Bahlo, et al. A Cav3.2 T-Type Calcium Channel Point Mutation Has Splice-Variant-Specific Effects on Function and Segregates with Seizure Expression in a Polygenic Rat Model of Absence Epilepsy J. Neurosci., January 14, 2009; 29(2): 371 - 380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Nie, J. Zhu, M. A. Gratton, A. Liao, K. J. Mu, W. Nonner, G. P. Richardson, and E. N. Yamoah Molecular Identity and Functional Properties of a Novel T-Type Ca2+ Channel Cloned From the Sensory Epithelia of the Mouse Inner Ear J Neurophysiol, October 1, 2008; 100(4): 2287 - 2299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Cataldi, V. Lariccia, V. Marzaioli, A. Cavaccini, G. Curia, D. Viggiano, L.M.T. Canzoniero, G. di Renzo, M. Avoli, and L. Annunziato Zn2+ Slows Down CaV3.3 Gating Kinetics: Implications for Thalamocortical Activity J Neurophysiol, October 1, 2007; 98(4): 2274 - 2284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Hildebrand, L. S. David, J. Hamid, K. Mulatz, E. Garcia, G. W. Zamponi, and T. P. Snutch Selective Inhibition of Cav3.3 T-type Calcium Channels by G{alpha}q/11-coupled Muscarinic Acetylcholine Receptors J. Biol. Chem., July 20, 2007; 282(29): 21043 - 21055. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
TOP
ABSTRACT
INTRODUCTION
and 
, the value of 
and 
, the values from I-V protocols (n = 7–11). C: average 
, 
) or the partial deletion (
