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Journal of Physiology (2002), 540.1, pp. 3-14
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013269
1G, 1H and 1I) to neuronal excitability |
ABSTRACT |
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In several types of neurons, firing is an intrinsic property produced by specific classes of ion channels. Low-voltage-activated T-type calcium channels (T-channels), which activate with small membrane depolarizations, can generate burst firing and pacemaker activity. Here we have investigated the specific contribution to neuronal excitability of cloned human T-channel subunits. Using HEK-293 cells transiently transfected with the human
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INTRODUCTION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the nervous system, information is encoded primarily by the number and the frequency of action potentials. In several types of neurons, firing of action potentials is thought to be an intrinsic property produced by specific ion channels (Llinas, 1988; Connors & Gutnick, 1990). The low-voltage-activated or T-type Ca2+ channels (T-channels), a subclass of voltage-gated Ca2+ channels, are able to activate from small depolarizations near the resting membrane potential of cells and can generate neuronal spontaneous firing and pacemaker activities (for review see Huguenard, 1996). In rat thalamic relay neurons, T-channels mediate low threshold spikes that are involved in rebound burst firing (Llinas & Jahnsen, 1982). In thalamus, T-channels are involved in slow-wave sleep (Steriade et al. 1993; McCormick & Bal, 1997) and in the pathogenesis of epilepsy (Tsakiridou et al. 1995; Huguenard, 1999). At this stage, further understanding of the functions and diseased states involving T-channels requires molecular investigations of the channel properties, now made possible by their cloning. Three genes encoding the T-channel pore subunits were identified and designated 1G (CaV3.1), 1H (CaV3.2) and 1I (CaV3.3) (Cribbs et al. 1998; Perez-Reyes et al. 1998; Klugbauer et al. 1999; Lee et al. 1999; Williams et al. 1999; Monteil et al. 2000a, 2000b; McRory et al. 2001). T-currents generated by the 1I subunit display slow kinetics that differ markedly from the 1G and 1H currents which share the typical signature of native neuronal T-currents (Klöckner et al. 1999; Monteil et al. 2000b; McRory et al. 2001; for review see Lacinova et al. 2000). Northern blot analysis has shown that 1G and 1H mRNA is widely expressed in various tissues and especially in the the central nervous system (CNS) (Cribbs et al. 1998; Monteil et al. 2000a), while the 1I mRNA is restricted to the CNS as well as to thyroid and adrenal glands (Lee et al. 1999; Monteil et al. 2000b). In situ hybridization experiments have indicated that the three isotypes can coexist in neuronal tissues such as amygdala or hippocampus, while in the rat cerebellum the 1G subunit is predominant in the same way as 1H in sensory ganglia. In rat thalamus, the 1G and 1I subunits are both present but exhibit distinct expression patterns with respect to the various nuclei (Talley et al. 1999). Because Purkinje neurons of the cerebellum and thalamic neurons display intrinsic firing either in short burst or in tonic/sustained mode, we have examined the specific role of T-channel isotypes in these patterns of activity. Taking advantage of the ability to express pure populations of recombinant T-channels together with the use of voltage clamp protocols mimicking these neuronal activities, we describe here the behaviour of the three human T-channel isotypes in neuronal excitability. These channel properties were modelled to delineate the contribution of each cloned T-channel in promoting firing patterns. This study indicates that the 1I currents are preferentially recruited during the depolarizing after-potential (DAP) and can generate sustained electrical activity, while the 1G and 1H currents promote short burst firing.
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METHODS |
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Cell culture and transfection protocols
Human embryonic kidney cells (HEK-293 cell line; ATCC) were transfected as previously described (Chemin et al. 2001) with 0.3 µg of pBB14 plasmid encoding the reporter gene GFP (Brideau et al. 1998) and 2.7 µg of different pBK-CMV plasmid constructs that encode for 1G (1G-a ; Chemin et al. 2001), 1I (Monteil et al. 2000b) and 1H (HH7; Cribbs et al. 1998). Two to three days later, cells were harvested and plated at low confluence and electrophysiological recordings were performed between days 2 and 6 after transfection.
Electrophysiology
Macroscopic currents were recorded by the whole-cell patch clamp technique using an Axopatch 200B amplifier (Axon Instruments, CA, USA) at room temperature (~25 °C) as previously described (Chemin et al. 2001). Extracellular solution contained (mM): 2 CaCl2, 160 TEACl and 10 Hepes (pH adjusted to 7.4 with TEAOH). Borosilicate glass pipettes have a typical resistance of 1-2 M
when filled with an internal solution containing (mM): 110 CsCl, 10 EGTA, 10 Hepes, 3 Mg-ATP and 0.6 GTP (pH adjusted to 7.2 with CsOH). For action potential clamp studies we have used: (i) a generic action potential (J. Pancrasio, Axon Instruments website); (ii) a regular train of spikes and a fast burst activity recorded in Purkinje neurons of the cerebellum (Raman & Bean, 1997), generously provided by Dr B. P. Bean (Harvard Medical School, Boston, MA, USA); and (iii) a firing activity typical of those measured on the thalamocortical relay (rTC) neurons generated by the NEURON model (Hines & Carnevale, 1997), described below. Records were filtered at 5 kHz. Cell capacitance was 12.9 ± 2.7 pF (n = 45). Series resistance (Rs) was 14.2 ± 0.6 M (n = 45) and the voltage error factor before compensation was 0.026 ± 0.0016 (n = 45) according to the equation Vm = Vc(1 - Rs/(Rs + Rm)), where Vm is the membrane potential, Vc the voltage command and (Rs/(Rs + Rm)) the voltage error factor. Capacitance and Rs were compensated by 90-100 % using the whole-cell parameters of the Axopatch 200B amplifier. Leak and residual capacitive currents were subtracted using a P/-5 procedure for tail current recordings and action potential clamp experiments. Data were analysed as previously described (Chemin et al. 2001) using pCLAMP6 (Axon Instruments), Excel (Microsoft) and GraphPad Prism (GraphPad Inc.) software. One-way ANOVA combined with a Student-Newman-Keuls post hoc test was used to compare the different values, and differences were considered significant at P < 0.05. Results are presented as the means ± S.E.M., and n is the number of cells used.
Modelling
The impact of the expression of 1G, 1H and 1I channels on the firing of thalamocortical relay neurons and thalamic reticular neurons was estimated using the NEURON model (described in detail by Hines & Carnevale, 1997). This model was modified by Destexhe et al. (1998) for thalamocortical relay cells and by Destexhe et al. (1996) for the thalamic reticular neurons. Both models were downloaded from the model database at Yale University (http://senselab.med.yale.edu/senselab/neurondb/). The parameters used in our experiments were the 'three-compartment model configuration of burst behaviour' as described in detail by Destexhe et al. (1996). The electrophysiological properties of the 1G, 1H and 1I channels were modelled using Hodgkin-Huxley equations as described by Huguenard & McCormick (1992) and the values obtained for the various 1 isoforms were substituted for the corresponding values of native T-channels of thalamocortical relay cells (Huguenard & McCormick, 1992) or thalamic reticular neurons (Huguenard & Prince, 1992). All these values are presented in Table 1 except voltage-independent 
of activation, which is 0.8 ± 0.1 (n = 18), 1.34 ± 0.1 (n = 8) and 7.2 ± 0.8 ms (n = 18) for 1G, 1H and 1I channels, respectively. To match the voltage clamp data, the modelling experiments were performed at 28 °C. Firing was triggered by injecting the virtual soma with a 0.18 nA depolarizing current over 100 ms; the resulting activity and the Ca2+ entry via T-channels recorded in the soma are presented in Fig. 7 and Fig. 8. For voltage clamp experiments, a rTC neuronal firing pattern (using native T-channel parameters) was produced by a 0.3 nA current injection into the virtual soma over 700 ms, then converted into a pCLAMP stimulation file and further applied to the transfected HEK-293 cells.
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RESULTS |
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Biophysical properties of the three human cloned T-channels
The T-type Ca2+ currents generated by the cloned human 1G, 1H and 1I subunits were studied comparatively in HEK-293 cells. The three subunits produced robust Ca2+ currents, as illustrated in Fig. 1A. The current-voltage relationships (I-V curves) were normalized (Fig. 1B), revealing a 7 mV difference in the peak of the I-V curve of the 1I current. Steady-state activation curves were deduced from the I-V curves (Fig. 1C), which were fitted using a combined Boltzmann and linear Ohmic relationships as described previously (Chemin et al. 2001). Both steady-state activation and inactivation curves of the 1I current were moved towards positive voltages (7 and 6 mV, respectively), compared to the 1G and 1H currents (Table 1). As a consequence, a ~5 mV positive shift of the window current component of 1I was observed. The three subunits also produced currents that were distinct in their kinetics. Besides the large kinetic differences between the 1G and 1I currents, it is important to note that the 1G subunits generated faster Ca2+ currents in both activation and inactivation kinetics compared to the 1H subunit (Fig. 2A and B, see also Table 1). Furthermore, the major difference between 1G and 1H channels was the recovery from short inactivation, which was significantly slower (3 times) for the 1H subunit, compared to the 1G subunit (Fig. 2C and Table1). A specific electrophysiological feature of T-channels, compared to high-voltage-activated (HVA) Ca2+ channels, is their slow deactivation kinetics (Armstrong & Matteson, 1985). Again, significant differences were found among the three human isotypes (Fig. 3B), with 1I channels generating the fastest deactivation kinetics as illustrated in Fig. 3A while the 1H currents presented the slowest deactivation kinetics (Fig. 3B and Table 1). In each case, deactivating tail currents were fitted using a monoexponential function and the rate of deactivation was independent of the current amplitude (not shown), indicating further that accurate voltage control was obtained.
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Figure 1 A, typical currents generated by the cloned human | ||
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Figure 2 A, activation kinetics were presented as the time for the current to rise from 10 to 90 % (Rise 10-90 %) as function of the test potential. In order to better visualize the difference in activation kinetics between | ||
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Figure 3 A, and B, deactivation kinetics. A, examples of normalized deactivating currents at -60 mV elicited after a -30 mV TP from an HP of -110 mV. For each subunit, the TP duration was adapted in order to trigger maximal deactivating currents (4 ms for | ||
Single action potential clamp studies
Action potential (AP) clamp studies were then performed in order to investigate the specific behaviour of the three T-channel isotypes during neuronal activities. First, we evaluated the contributions of each cloned T-channel during a single AP (Fig. 3C). For the three channel isotypes, the onset of Ca2+ entry occurred during the repolarization phase of the AP. However, their behaviour during an AP stimulation was markedly distinct. The 1I subunit generated a rapidly inactivating current of small amplitude, revealing that this channel modestly contributed to Ca2+ entry during a single AP. In contrast, the 1G and 1H subunits generated larger and sustained currents (Fig. 3C). Channel maximum open probability during the AP was evaluated by calculating the ratio (r) of the maximal current amplitude induced by the AP to the maximal slope conductance from the I-V curve (see Fig. 1) recorded in the same cell. Maximum open probability (Fig. 3D) was larger for the 1G channels (r = 85.1 ± 7.4; n = 13) compared to the 1H channels (r = 60.4 ± 10.8; n = 12) and to the 1I channels (r = 16.1 ± 0.9; n = 11). In order to evaluate the Ca2+ entry duration triggered by the AP, we have calculated the percentage of current remaining 10 ms after the beginning of the AP (Fig. 3E). The Ca2+ entry duration, which reflects deactivation kinetics (Fig. 3B), was larger for 1G and 1H channels (percentage of current remaining: 19 ± 1 %, n = 13; and 23 ± 1 %, n = 12, respectively) compared to 1I channels (6 ± 2 %, n = 11). Overall, these data indicated that 1G and 1H channels produce larger and sustained Ca2+ entry during a single AP when compared to 1I channels.
Purkinje neuron and thalamocortical relay neuron action potential clamp studies
To further understand the role of the three T-channel isotypes in neuronal excitability, we performed AP clamp studies using several patterns of neuronal activity that occur in Purkinje neurons of the cerebellum (Fig. 4 and Fig. 5) and in relay neurons of the thalamus (Fig. 6). Although these neuronal activities were recorded in rats, we can predict that our approach is valid since the major T-current properties are conserved among rat and human T-channels (see Klöckner et al. 1999). We first applied a typical regular train of spikes (50 Hz) recorded in Purkinje cells (Raman & Bean, 1999a) that exhibited sustained spontaneous firing (Fig. 4A). In these neurons, APs are generated from a short DAP (18 ms). Several important differences could be observed among the Ca2+ currents produced by the three cloned T-channels. The 1G channels rapidly inactivated and only a very small current remained after the tenth spike (Fig. 4A and C). In contrast, the 1I channels generated a small and transient current during the first interspike interval (~4 times less than the 1G and 1H channels), then increased during the next three spikes (Fig. 4A and C), suggesting an apparent facilitation of channel activity. After four to five spikes, slow inactivation overcame facilitation leading to a maintained Ca2+ entry during the entire train duration (Fig. 4B and C). The behaviour of the 1H channels was more complex and intermediate between 1G and 1I. The 1H channels contributed to a large depolarizing current during the entire train of stimulation (Fig. 4A and C) as a consequence of a robust first interspike interval current and slow inactivation, compared to the 1G channels. This difference between 1H and 1G currents was also found using more negative resting potentials. Similar to the result obtained for a holding potential (HP) of -70 mV (Fig. 4C), 1G currents decreased more rapidly and the ratio Imax last spike : Imax first spike of these currents was ~3 times smaller than the 1H current ratio for a HP of -110 mV (n = 9, not shown). Interestingly, the 1H and 1I currents never inactivated completely during the interspike intervals (Fig. 4A), leading to a residual Ca2+ entry that was still observable at the end of the train of spikes (Fig. 4B). The presence of residual current at the steady state suggests that 1H and 1I currents could participate in sustained pacemaker activities. The second and the seventeenth inward current traces were scaled and superimposed (Fig. 4A, inset) to further demonstrate an adequate voltage control.
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Figure 4. Behaviour of the three T-channel isotypes during tonic firing that occurred in Purkinje neurons The top trace represents spontaneous activity of a Purkinje neuron which was used to perform AP clamp experiments on transfected cells. A, typical recorded currents for the | ||
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Figure 5. Purkinje neuron burst firing clamp The top trace represents burst activity of a hyperpolarized Purkinje neuron which was used to perform AP clamp experiments on transfected cells. Typical | ||
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Figure 6. Thalamocortical relay neuron activity clamp The top trace represents burst activity of a thalamocortical relay neuron which was used to perform AP clamp experiments on transfected cells. A, typical | ||
We next performed AP clamp studies using patterns of burst activities. Figure 5 shows the behaviour of the three channel isotypes during short burst activities. When neurons from the Purkinje cell layer of the cerebellum are maintained hyperpolarized, they exhibit short and fast burst s(100 Hz) of a few APs, three on average as used in our experiments, upon injection of a depolarizing current (Raman & Bean, 1997; Fig. 5). In this case, we found that the 1G and 1H channels produced large inward currents that inactivated rapidly (Fig. 5). In contrast, the 1I channels generated a small inward current during the first spike, which strongly facilitated during the next APs. These experiments demonstrated that at a high frequency (100 Hz) the 1I currents still exhibited facilitation. Moreover, it is important to note that an increase of the 1I current amplitude was elicited during the DAP. We then performed voltage clamp analysis using a typical burst firing of the thalamocortical relay (rTC) neurons generated by the NEURON model (Fig. 6; see Methods). The rTC neurons have a resting membrane potential around -75 mV and respond to depolarizations by generating long-lasting burst activities from membrane potentials around -60 mV. As expected from the previous experiments, the 1G and 1H channels generated larger Ca2+ entry during the first spikes and then inactivated rapidly (Fig. 6A and B). No inward currents were observed after about six spikes for either 1G or 1H channels (Fig. 6C). In contrast, 1I channels produced large Ca2+ entry compared to the 1G and 1H channels (Fig. 6B), due to current facilitation and to a large 'rebound' inward current clearly associated with the DAP transition (Fig. 6A-C). It should be noted, however, that the 1H channels generated a small inward current that occurred during the DAP. Overall, these data suggest that 1I currents contribute to sustained neuronal activity, while 1G and 1H current profiles are more consistent with short burst firing.
Modelling experiments
Since action potential clamp studies do not provide dynamic information on the role of T-channels in shaping the neuronal firing patterns, we have performed simulations using the NEURON model (described in detail by Hines & Carnevale, 1997). This model was adapted for thalamocortical relay cells (Fig. 7) and for reticular cells of the thalamus (Fig. 8), as previously described (McCormick & Huguenard, 1992; Destexhe et al. 1996, 1998). In each simulation protocol, the parameters describing the native T-current were replaced with those of cloned T-channels. Figure 7A shows rTC simulation obtained with the native T-current parameters as described by McCormick & Huguenard (1992). To induce firing, a 100 ms pulse of 180 pA was injected into the virtual soma. This pulse does not trigger APs if T-channels are removed from the model environment (not shown). The parameters of the three cloned T-channels were then sequentially introduced in the model (Fig. 7B-D). The firing patterns predicted by the 1G channel parameters matched well those produced by the native T-channel parameters of the rTC neurons (Fig. 7A and B). Both native and 1G currents are able to depolarize the cells to approximately -60 mV during the 100 ms depolarizing pulse, with few APs (≤ 3) occurring after the stimulation. In addition, these two burst patterns exhibited similar strong frequency adaptation with a rapid decrease in the rate of APs. In both cases, this could be explained by fast current inactivation (see also Fig. 6). The latency to the first spike is nevertheless longer with 1G parameters. This is probably due to the more positive steady-state activation of 1G currents, compared to the native T-currents (V0.5 of -49 and -57 mV, respectively). In contrast, firing patterns predicted by 1H and 1I channel activity are markedly different from those produced by native T-currents. In both cases, there is a slower frequency adaptation and more importantly, 1H and 1I channels are both able to generate firing even after the end of the stimulation (Fig. 7C and D). As best exemplified with the 1I parameters, firing continued up to 200 ms after the end of the stimulation. Current facilitation was again observed with the 1I parameters (see also Fig. 6). In addition, the latency to the first AP obtained with the 1I parameters was the longest, probably due to its positive value for V0.5 of activation, as well as its slow activation kinetics. Overall, these results are in good agreement with the voltage clamp data described earlier and strongly suggest that the 1G channels are the dominant isotypes functionally expressed in the rTC neurons.
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Figure 7. Simulated current clamp recording of thalamocortical relay neurons (rTC) Cloned T-channel parameters ( | ||
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Figure 8. Simulated current clamp recording of the reticular neurons of the thalamus (nRt) Cloned T-channel parameters ( | ||
Figure 8 shows T-channel simulations using a thalamic reticular neuron environment. As previously described, firing was induced using a 100 ms pulse of 180 pA injected into the virtual soma. In contrast with thalamocortical relay neurons, firing was predicted to be persistent in reticular neurons (Fig. 8A). The 1I parameters best accounted for the firing pattern generated using the native T-current of reticular neurons (Fig. 8B; Huguenard & Prince, 1992). Indeed, the native T-current of reticular neurons showed facilitation during the firing activity, as shown for 1I channels both in voltage clamp and modelling experiments. In contrast, the 1G and 1H channels induced firing which stopped at the end of the stimulating pulse (Fig. 8C and D). Overall, simulation experiments indicated that the 1G and 1I currents might differentially participate in the firing pattern of rTC and reticular neurons of the thalamus and further suggest that 1I channels could promote sustained electrical activities.
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DISCUSSION |
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This study demonstrates that the contribution of T-channels to neuronal excitability is related to isotype-specific properties. The properties of the three human T-channel 1 subunits cloned to date (1G, 1H and 1I) were compared and we report significant differences among these isotypes in their biophysical properties, which are highlighted in action potential clamp studies. Modelling experiments further indicate a role of 1I channels in generating pacemaker activity. The functional signatures of the cloned T-channels described here should help both in understanding the diversity of native T-channels and in elucidating their specific physiological roles.
The specific properties of human cloned T-channels
The three T-channel 1 subunits exhibit distinct functional properties. Compared to 1G and 1H currents, 1I currents show a 6 mV positive shift in their steady-state properties. Because the window current component, a background Ca2+ current described in Chemin et al. (2000), results from the overlap of the activation and the inactivation curves, the 1I window current component should be evenly shifted. Such a difference could have major consequences on cell phenotype since the window current component of T-channels occurs near physiological resting potential and regulate basal levels of Ca2+ (Bijlenga et al. 2000; Chemin et al. 2000). In thalamocortical neurons, T-window currents contribute to enhanced firing through an intrinsic bistability-mediated phenomenon (Williams et al. 1997; Hughes et al. 1999). However, there is no evidence to date whether isotype-specific window currents could play distinct roles in neuronal physiology. The three human isotypes also display distinct kinetic properties. Cloned 1G and 1H channels promote fast gated currents typical of native T-currents (Carbone & Lux, 1984; Armstrong & Matteson, 1985; Nowycky et al. 1985; Monteil et al. 2000b), while 1I currents display slow activation and inactivation kinetics but faster deactivation. Nevertheless, 1H currents can be distinguished from 1G currents by their slower activation, inactivation and deactivation kinetics and more importantly by their slow recovery from short inactivation (see also Satin & Cribbs, 2000). These isotype-specific gating properties lead to distinct channel behaviour during neuronal activity. It should be noted, however, that splice variations can tune the properties of each isotype (Chemin et al. 2001). Nevertheless, the primary electrophysiological characteristics are isotype specific and conserved among species since the properties of the human cloned T-channels are in good agreement with those obsered in rat counterparts (Klöckner et al. 1999).
Activities of cloned T-channels during a single action potential
For T-channels, Ca2+ entry occurs mainly during the repolarization phase of the AP. For this reason, the rate of deactivation kinetics is important in tuning the current decay. Due to their slow deactivation kinetics, 1G and 1H channels produce a sustained current typical of that observed in native neurons using similar protocols (McCobb & Beam, 1991; Scroggs & Fox, 1992; Lambert et al. 1998). In contrast, the fast deactivation kinetics of 1I channels yields more transient currents during a single AP. Channel maximum open probability during an AP is also related to activation kinetics. Indeed, 1I channels that display slow activation kinetics are not fully activated during a single AP. Similarly, the reduced maximum open probability of 1H channels compared to 1G channels is likely to be due to slower activation kinetics since no significant difference exists in their steady-state properties. In contrast with the data obtained using square test pulses, our results indicate that 1G and 1H channels produce large and sustained Ca2+ currents during a single AP, while 1I channels generate small and fast inactivating Ca2+ currents. Reciprocally, activation of distinct T-channel populations may differentially influence the shaping of the AP.
Behaviour of cloned T-channels using cerebellum Purkinje neuron activities
The pacemaker activity occurring in Purkinje neurons of the cerebellum is generated by intrinsic properties of these neurons, which abundantly express T-channels (Gruol & Franklin, 1997; Raman & Bean, 1999a; Talley et al. 1999; Monteil et al. 2000a; Raman et al. 2000). Using neuronal activity of these neurons, we showed that 1G channels produce large and sustained currents during the first interspike intervals that rapidly decrease to a negligible Ca2+ entry during prolonged neuronal activity. This fast decrease of the 1G current could be explained by cumulative inactivation resulting from its slow deactivation and its fast inactivation kinetics (Kozlov et al. 1999; Serrano et al. 1999). The 1H channels are also preferentially recruited during short burst activities but generate persistent current during sustained firing, probably due to their slower inactivation kinetics compared to 1G channels. In contrast, the 1I currents that are tiny and transient during the first interspike interval exhibit apparent facilitation and slow inactivation leading to a sustained Ca2+ entry at the steady state. Facilitation that represents cumulative activation, or current summation, could be explained by the slow activation and inactivation kinetics of 1I currents. It is important to note that during fast burst activities, 1I channels generate a large Ca2+ current 'rebound' clearly associated with the DAP. Large current associated with the DAP is highly relevant because it probably favours the triggering of the next AP. The rebound of 1I currents during the DAP could be compared to the 'resurgent' sodium current, although it occurs from reopening of previously inactivated sodium channels, that promotes pacemaker activities in Purkinje neurons (Raman & Bean, 1997, 1999a,b).
Our results suggest that cloned 1G channels are not able to generate pacemaker activities in Purkinje neurons. Similar data were obtained in Purkinje neurons in which native T-current rapidly inactivates during the same train of spikes as used here (Raman & Bean, 1999a) suggesting that Purkinje T-currents are generated by 1G channels. Furthermore, these latter authors reported that the block of T-channels by cobalt or mibefradil does not prevent, but only decreases, the firing rate of Purkinje cells. Nevertheless, T-channels could possibly play a role in pacemaker activity after inhibitory synaptic input-induced hyperpolarization of Purkinje neurons (Raman & Bean, 1999a). In this case, deinactivated T-channels would generate a large current during the first interspike intervals and participate in the Ca2+ entry during short burst activity as observed in our 1G experiments.
Contribution of cloned T-channels to thalamic neuron excitability: action potential clamp and modelling studies
In thalamocortical relay (rTC) and thalamic reticular neurons (nRt), firing activity is proposed to be largely due to the interaction of two currents, the hyperpolarization-activated current, Ih, and the T-current (Jahnsen & Llinas, 1984; McCormick & Pape, 1990). T-current is thought to mediate APs and to control the frequency and time course of repetitive firing. The rTC neurons have a resting potential around -75 mV and respond to depolarization by rhythmic activity generated from a membrane potential around -60 mV (Contreras et al. 1993). This phenomenon, called bistability-mediated activity, is related to the T-window current component which maintains the membrane potential near -60 mV (Williams et al. 1997; Hughes et al. 1999). The voltage clamp data obtained using thalamic activity indicates that 1G and 1H channels produce currents that inactivate rapidly, while 1I currents facilitate and can be recorded during the entire stimulus. It should be noted that sustained 1I current, mainly associated with the DAP, persists even during the slower frequency phase of the burst. These results strongly suggest that 1I current is able to participate and to generate pacemaker activity in thalamic neurons.
The influence of each cloned T-channel on firing patterns was estimated in simulation experiments using the NEURON model adapted for rTC and nRt neurons (McCormick & Huguenard, 1992; Destexhe et al. 1996, 1998; Hines & Carnevale, 1997). The modelling experiments are in good agreement with the voltage clamp data. The 1G and 1H currents rapidly inactivate, while 1I currents display facilitation and slowly inactivate during the burst activity. More importantly, modelling experiments demonstrate that 1G currents induce firing activity of short duration while that generated by 1I currents (and to a lesser extent by 1H currents) is significantly more prolonged. Both firing pattern and Ca2+ entry induced by simulated 1G currents are similar to those induced by the native T-currents of rTC cells, while 1I currents better account for the native T-currents of nRt neurons. Interestingly, a recent report describing that 1I channels could be modulated by the 
2 subunit in HEK-293 cells (Green et al. 2001) indicates that further studies should investigate the physiological relevance of such a modulation in nRT neurons.
Concluding remarks
The data presented here allow us to ascribe distinct roles in firing activities to each T-channel isotype and complement well the previous observations made for native T-channels in a variety of neurons. Indeed, the critical role of 1G currents in generating burst firing in rTC neurons has just been demonstrated using mice lacking 1G channels (Kim et al. 2001). Using neuronal activity as waveforms and modelling studies, we illustrate that 1I channel properties are compatible with a role in sustained neuronal firing and in promoting pacemaker activities. The existence of native T-currents that resemble 1I currents is still a matter of debate. Zhuravleva et al. (2001) have reported that 'slow' and 'fast' components of T-currents can be recorded from thalamic neurons. These data are in good agreement with previous findings from nRt neurons in which slow T-currents participate in the generation of long-duration calcium-dependent spike bursts (Huguenard & Prince, 1992). The behavioural properties of cloned 1I channels described here can account for the T-channel properties in nRt, including long burst firing. Overall, these functional conclusions corroborate well in situ hybridization experiments that indicated that the 1G mRNA is predominantly expressed in the rTC nucleus and in cerebellum, while 1I mRNA is strongly expressed in the nRt nucleus, along with 1H mRNA (Talley et al. 1999). All together, the finding that T-channel isotypes differentially contribute to neuronal excitability strongly suggests that brain region specific expression of these channels plays an important role in the regulation of information processing in the nervous system.
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REFERENCES |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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| ARMSTRONG, C. M. & MATTESON, D. R. (1985). Two distinct populations of calcium channels in a clonal line of pituitary cells. Science 227, 65-67 | |
| BIJLENGA, P., LIU, J. H., ESPINOS, E., HAENGGELI, C. A., FISCHER-LOUGHEED, J., BADER, C. R. & BERNHEIM, L. (2000). T-type alpha 1H Ca2+ channels are involved in Ca2+ signaling during terminal differentiation (fusion) of human myoblasts. Proceedings of the National Academy of Sciences of the USA 97, 7627-7632 | [Abstract/Full Text] |
| BRIDEAU, A. D., BANFIELD, B. W. & ENQUIST, L. W. (1998). The Us9 gene product of pseudorabies virus, an alphaherpes virus, is a phosphorylated, tail-anchored type II membrane protein. Journal of Virology 72, 4560-4570 | [Abstract/Full Text] |
| CARBONE, E. & LUX, H. D. (1984). A low voltage-activated, fully inactivating Ca channel in vertebrate sensory neurones. Nature 310, 501-502 | [Medline] |
| CHEMIN, J., MONTEIL, A., BOURINET, E., NARGEOT, J. & LORY, P. (2001). Alternatively spliced alpha1G (CaV3. 1) intracellular loops promote specific T-type Ca2+ channel gating properties. Biophysical Journal 80, 1238-1250 | [Abstract/Full Text] |
| CHEMIN, J., MONTEIL, A., BRIQUAIRE, C., RICHARD, S., PEREZ-REYES, E., NARGEOT, J. & LORY, P. (2000). Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation. FEBS Letters 478, 166-172 | [Medline] |
| CONNORS, B. W. & GUTNICK, M. J. (1990). Intrinsic firing patterns of diverse neocortical neurons. Trends in Neurosciences 13, 99-104 | [Medline] |
| CONTRERAS, D., CURRO-DOSSI, R. & STERIADE, M. (1993). Electrophysiological properties of cat reticular thalamic neurones in vivo. Journal of Physiology 470, 273-294 | [Abstract] |
| CRIBBS, L. L., LEE, J. H., YANG, J., SATIN, J., ZHANG, Y., DAUD, A., BARCLAY, J., WILLIAMSON, M. P., FOX, M., REES, M. & PEREZ-REYES, E. (1998). Cloning and characterization of alpha1H from human heart, a member of the T-type Ca2+ channel gene family. Circulation Research 83, 103-109 | [Abstract/Full Text] |
| DESTEXHE, A., CONTRERAS, D., STERIADE, M., SEJNOWSKI, T. J. & HUGUENARD, J. R. (1996). In vivo, in vitro, and computational analysis of dendritic calcium currents in thalamic reticular neurons. Journal of Neuroscience 16, 169-185 | [Abstract] |
| DESTEXHE, A., NEUBIG, M., ULRICH, D. & HUGUENARD, J. (1998). Dendritic low-threshold calcium currents in thalamic relay cells. Journal of Neuroscience 18, 3574-3588 | [Abstract/Full Text] |
| GREEN, P. J., WARRE, R., HAYES, P. D., MCNAUGHTON, N. C. L., MEDHURST, A. D., PANGALOS, M., DUCKWORTH, D. M. & RANDALL, A. D. (2001). Kinetic modification of the 1I subunit-mediated T-type Ca2+ channel by a human neuronal Ca2+ channel subunit. Journal of Physiology 533, 467-478 |
[Abstract/Full Text] |
| GRUOL, D. L. & FRANKLIN, C. L. (1987). Morphological and physiological differentiation of Purkinje neurons in cultures of rat cerebellum. Journal of Neuroscience 7, 1271-1293 | [Abstract] |
| HINES, M. L. & CARNEVALE, N. T. (1997). The NEURON simulation environment. Neural Computation 9, 1179-1209 | [Abstract] |
| HUGHES, S. W., COPE, D. W., TOTH, T. I., WILLIAMS, S. R. & CRUNELLI, V. (1999). All thalamocortical neurones possess a T-type Ca2+ 'window' current that enables the expression of bistability-mediated activities. Journal of Physiology 517, 805-815 | [Abstract/Full Text] |
| HUGUENARD, J. R. (1996). Low-threshold calcium currents in central nervous system neurons. Annual Review of Physiology 58, 329-348 | [Abstract] |
| HUGUENARD, J. R. (1999). Neuronal circuitry of thalamocortical epilepsy and mechanisms of antiabsence drug action. Advances in Neurology 79, 991-999 | [Medline] |
| HUGUENARD, J. R. & MCCORMICK, D. A. (1992). Simulation of the currents involved in rhythmic oscillations in thalamic relay neurons. Journal of Neurophysiology 68, 1373-1383 | [Medline] |
| HUGUENARD, J. R. & PRINCE, D. A. (1992). A novel T-type current underlies prolonged Ca2+-dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus. Journal of Neuroscience 12, 3804-3817 | [Abstract] |
| JAHNSEN, H. & LLINAS, R. (1984). Ionic basis for the electro-responsiveness and oscillatory properties of guinea-pig thalamic neurones in vitro. Journal of Physiology 349, 227-247 | [Abstract] |
| KIM, D., SONG, I., KEUM, S., LEE, T., JEONG, M. J., KIM, S. S., MCENERY, M. W. & SHI, H. S. (2001). Lack of burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking a1G T-type Ca2+ channels. Neuron 31, 35-45 | [Medline] |
| KLÖCKNER, U., LEE, J. H., CRIBBS, L. L., DAUD, A., HESCHELER, J., PEREVERZEV, A., PEREZ-REYES, E. & SCHNEIDER, T. (1999). Comparison of the Ca2+ currents induced by expression of three cloned alpha1 subunits, alpha1G, alpha1H and alpha1I, of low-voltage-activated T-type Ca2+ channels. European Journal of Neuroscience 11, 4171-4178 | [Medline] |
| KLUGBAUER, N., MARAIS, E., LACINOVA, L. & HOFMANN, F. (1999). A T-type calcium channel from mouse brain. Pflügers Archiv 437, 710-715 | [Medline] |
| KOZLOV, A. S., MCKENNA, F., LEE, J. H., CRIBBS, L. L., PEREZ-REYES, E., FELTZ, A. & LAMBERT, R. C. (1999). Distinct kinetics of cloned T-type Ca2+ channels lead to differential Ca2+ entry and frequency-dependence during mock action potentials. European Journal of Neuroscience 11, 4149-4158 | [Medline] |
| LACINOVA, L., KLUGBAUER, N. & HOFMANN, F. (2000). Low voltage activated calcium channels: from genes to function. General Physiology and Biophysics 19, 121-136 | [Medline] |
| LAMBERT, R. C., MCKENNA, F., MAULET, Y., TALLEY, E. M., BAYLISS, D. A., CRIBBS, L. L., LEE, J. H., PEREZ-REYES, E. & FELTZ, A. (1998). Low-voltage-activated Ca2+ currents are generated by members of the CavT subunit family (alpha1G/H) in rat primary sensory neurons. Journal of Neuroscience 18, 8605-8613 | [Abstract/Full Text] |
| LEE, J. H., DAUD, A. N., CRIBBS, L. L., LACERDA, A. E., PEREVERZEV, A., KLÖACKNER, U., SCHNEIDER, T. & PEREZ-REYES, E. (1999). Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family. Journal of Neuroscience 19, 1912-1921 | [Abstract/Full Text] |
| LLINAS, R. & JAHNSEN, H. (1982). Electrophysiology of mammalian thalamic neurones in vitro. Nature 297, 406-408 | [Medline] |
| LLINAS, R. R. (1988). The intrinsic electrophysiological properties of mammalian neurons: insights into central nervous system function. Science 242, 1654-1664 | |
| MCCOBB, D. P. & BEAM, K. G. (1991). Action potential waveform voltage-clamp commands reveal striking differences in calcium entry via low and high voltage-activated calcium channels. Neuron 7, 119-127 | [Medline] |
| MCCORMICK, D. A. & BAL, T. (1997). Sleep and arousal: thalamocortical mechanisms. Annual Review of Neuroscience 20, 185-215 | [Abstract/Full Text] |
| MCCORMICK, D. A. & HUGUENARD, J. R. (1992). A model of the electrophysiological properties of thalamocortical relay neurons. Journal of Neurophysiology 68, 1384-1400 | [Medline] |
| MCCORMICK, D. A. & PAPE, H. C. (1990). Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation in thalamic relay neurones. Journal of Physiology 431, 291-318 | [Abstract] |
| MCRORY, J. E., SANTI, C. M., HAMMING, K. S. C., MEZEYOVA, J., SUTTON, K. G., BAILLIE, D. L., STEA, A. & SNUTCH, T. P. (2001). Molecular and functional characterization of a family of rat brain T-type calcium channels. Journal of Biological Chemistry 276, 3999-4011 | [Abstract/Full Text] |
| MONTEIL, A., CHEMIN, J., BOURINET, E., MENNESSIER, G., LORY, P. & NARGEOT, J. (2000a). Molecular and functional properties of the human alpha1G subunit that forms T-type calcium channels. Journal of Biological Chemistry 275, 6090-6100 | [Abstract/Full Text] |
| MONTEIL, A., CHEMIN, J., LEURANGUER, V., ALTIER, C., MENNESSIER, G., BOURINET, E., LORY, P. & NARGEOT, J. (2000b). Specific properties of T-type calcium channels generated by the human alpha 1I subunit. Journal of Biological Chemistry 275, 16 530-16 535 | [Abstract/Full Text] |
| NOWYCKY, M. C., FOX, A. P. & TSIEN, R. W. (1985). Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature 316, 440-443 | [Medline] |
| PEREZ-REYES, E., CRIBBS, L. L., DAUD, A., LACERDA, A. E., BARCLAY, J., WILLIAMSON, M. P., FOX, M., REES, M. & LEE, J. H. (1998). Molecular characterization of a neuronal low-voltage-activated T-type calcium channel. Nature 391, 896-900 | [Medline] |
| RAMAN, I. M. & BEAN, B. P. (1997). Resurgent sodium current and action potential formation in dissociated cerebellar Purkinje neurons. Journal of Neuroscience 17, 4517-4126 | [Abstract/Full Text] |
| RAMAN, I. M. & BEAN, B. P. (1999a). Ionic currents underlying spontaneous action potentials in isolated cerebellar Purkinje neurons. Journal of Neuroscience 19, 1663-1674 | [Abstract/Full Text] |
| RAMAN, I. M. & BEAN, B. P. (1999b). Properties of sodium currents and action potential firing in isolated cerebellar Purkinje neurons. Annals of the New York Academy of Sciences 868, 93-96 | [Medline] |
| RAMAN, I. M., GUSTAFSON, A. E. & PADGETT, D. (2000). Ionic currents and spontaneous firing in neurons isolated from the cerebellar nuclei. Journal of Neuroscience 20, 9004-9016 | [Abstract/Full Text] |
| SATIN, J. & CRIBBS, L. L. (2000). Identification of a T-type Ca2+ channel isoform in murine atrial myocytes (AT-1 cells). Circulation Research 86, 636-642 | [Abstract/Full Text] |
| SCROGGS, R. S. & FOX, A. P. (1992). Multiple Ca2+ currents elicited by action potential waveforms in acutely isolated adult rat dorsal root ganglion neurons. Journal of Neuroscience 12, 1789-1801 | [Abstract] |
| SERRANO, J. R., PEREZ-REYES, E. & JONES, S. W. (1999). State-dependent inactivation of the alpha1G T-type calcium channel. Journal of General Physiology 114, 185-201 | [Abstract/Full Text] |
| STERIADE, M., MCCORMICK, D. A. & SEJNOWSKI, T. J. (1993). Thalamocortical oscillations in the sleeping and aroused brain. Science 262, 679-685 | |
| TALLEY, E. M., CRIBBS, L. L., LEE, J. H., DAUD, A., PEREZ-REYES, E. & BAYLISS, D. A. (1999). Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. Journal of Neuroscience 19, 1895-1911 | [Abstract/Full Text] |
| TSAKIRIDOU, E., BERTOLLINI, L., DECURTIS, M., AVANZINI, G. & PAPE, H. C. (1995). Selective increase in T-type calcium conductance of reticular thalamic neurons in a rat model of absence epilepsy. Journal of Neuroscience 15, 3110-3117 | [Abstract] |
| WILLIAMS, M. E., WASHBURN, M. S., HANS, M., URRUTIA, A., BRUST, P. F., PRODANOVICH, P., HARPOLD, M. M. & STAUDERMAN, K. A. (1999). Structure and functional characterization of a novel human low-voltage activated calcium channel. Journal of Neurochemistry 72, 791-799 | [Abstract/Full Text] |
| WILLIAMS, S. R., TOTH, T. I., TURNER, J. P., HUGHES, S. W. & CRUNELLI, V. (1997). The 'window' component of the low threshold Ca2+ current produces input signal amplification and bistability in cat and rat thalamocortical neurones. Journal of Physiology 505, 689-705 | [Abstract] |
| ZHURAVLEVA, S. O., KOSTYUK, P. G. & SHUBA, Y. M. (2001). Subtypes of low voltage-activated Ca2+ channels in laterodorsal thalamic neurons: possible localization and physiological roles. Pflügers Archiv 441, 832-839 | [Medline] |
Acknowledgements
This work was supported in part by CNRS, the Association pour la Recherche contre le Cancer (ARC), Association Française contre les myopathies (AFM). We thank M. Mangoni, S. Dubel, P. Fontanaud and V. Chevaleyre for helpful discussions and for critical reading of the manuscript and C. Barrére for help with cell cultures.
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