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Journal of Physiology (2001), 532.3, pp. 583-593
© Copyright 2001 The Physiological Society
2, 3 and 4, three new Ca2+ channel subunits, in P/Q-type Ca2+ channel expressed in Xenopus oocytes |
ABSTRACT |
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2, the product of the gene mutated in the stargazer mouse, is a homologue of the 1 protein, an accessory subunit of the skeletal muscle L-type Ca2+ channel. 2 is selectively expressed in the brain, and considered to be a putative neuronal Ca2+ channel subunit based mainly on homology to 1. Two new members of the family expressed in the brain have recently been identified: 3 and 4.
2, 3 and 4 subunits with the P/Q-type (CaV2.1) Ca2+ channel and different regulatory subunits (
2-
; 
1, 2, 3 or 4).
subunits confirmed their membrane localization.
subunits on the electrophysiological properties of the channel are, most of the time, minor. However, a fraction of the oocytes expressing subunits displayed an unusual slow-inactivating Ba2+ current. Expression of both and subunits increased the appearance of the slow-inactivating current.
subunit as a brain Ca2+ channel modulatory subunit and suggest that and subunits are involved in a switch between two regulatory modes of the P/Q-type channel inactivation.
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INTRODUCTION |
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Stargazin or 2, a protein selectively expressed in the brain with homology to the skeletal muscle L-type Ca2+ channel 1 subunit (Bosse et al. 1990; Pragnell et al. 1991) was recently discovered as the product of the gene mutated by insertion of a retrotransposon in the stargazer mouse model of petit mal epilepsy and ataxia (Letts et al. 1998).
In skeletal muscle, 1 can be co-immunoprecipitated from muscle extracts with the pore-forming L-type Ca2+ channel subunit 1S (Flockerzi et al. 1986a,b; Sieber et al. 1987). The 1 subunit is a 32 kDa, 222 amino acid, glycosylated transmembrane protein with 10 cysteines that are supposed to form disulfide bridges of major importance for the secondary structure of the native protein (Bosse et al. 1990; Pragnell et al. 1991). Co-expression of 1 with L-type (1C or CaV1.2) or P/Q-type (1A or CaV2.1) Ca2+ channels in heterologous systems has evidenced minor modulatory function including slight changes in the activation and inactivation properties and modification in the peak current amplitude of these Ca2+ channels (Mori et al. 1991; Wei et al. 1991; Singer Lahat et al. 1994; Sipos et al. 1995; Eberst et al. 1997; Ren & Hall, 1997; Freise et al. 2000).
The stargazin protein displays 38 % similarity (25 % identity) with 1 and shares the same gene organization and protein secondary structure including four putative transmembrane segments (Letts et al. 1998; Klugbauer et al. 2000). The stargazer mouse has recessively inherited epilepsy and ataxia characterized by spike wave seizures, characteristic of petit mal or absence epilepsy (Chen et al. 1999). Cerebellar disorders have also been noted, including, most prominently, abnormal migration and maturation and reduced brain-derived neurotrophic factor production of cerebellar granule cells. These features are coincindent with the start of ataxia (Qiao et al. 1996, 1998). Immature or reduced synaptic transmission at parallel fibre-Purkinje cell synapses, Golgi cell-granule cell synapses and mossy fibre-granule cell synapses has also been noticed (Chen et al. 1999; Hashimoto et al. 1999), suggesting a crucial role of the 2/stargazin protein for normal synaptic transmission. The phenotypic similarities between stargazer mice and the other neurological mutant mice, tottering and lethargic, which have defects in genes encoding the 1A and 4 subunits of the P/Q-type Ca2+ channels, together with the homology of gene and protein structures with the 1 subunit, have led to the hypothesis that the 2 subunit is indeed a Ca2+ channel subunit and suggest a channel dysfunction as the basis of the disease.
Three new members of this family (3, 4 and 5) have now been isolated in mice and humans (Black & Lennon, 1999; Klugbauer et al. 2000). Co-expression studies of these four subunits with the L-type (1C) or P/Q-type (1A) Ca2+ channels did not change the kinetics and voltage dependence of activation of the channel (Klugbauer et al. 2000). Analysis of the effects of these subunits on channel inactivation has produced contradictory results, and shifts in the steady-state inactivation curves in both the depolarized and the hyperpolarized direction have been reported, depending on the 1 and subunits (1A, 1C, 1 or 2) used, as well as the cation used as charge carrier (Ba2+ versus Ca2+, Klugbauer et al. 2000). These data suggest that the subunit composition of the channel might be an important determinant of the effects of the subunits on channels properties, and therefore on the physiopathological consequences of its absence in the stargazer mice.
In this work we have analysed the effects of human 2, 3 and 4 subunits and the modulatory roles of the 1, 2, 3 and 4 subunits on P/Q-type Ca2+ channel properties.
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METHODS |
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tsA-201 cell transfection and immunocytochemistry
tsA-201 cells (human embryonic kidney cell line) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10 % fetal bovine serum and 1 % penicillin-streptomycin at 37 °C in 5 % CO2. Transfections were performed using Superfect (Qiagen, Courtaboeuf, France) according to the manufacturer's protocols, 1 day after plating the cells on poly-L-ornithine-treated 35 mm Petri dishes. Five micrograms of plasmid cDNA(s) were used for each transfection with an incubation time of 2 h. Forty-eight hours later, cells were fixed and permeabilized using PBS supplemented with paraformaldehyde (4 %) and Triton X-100 (0.05 %) applied for 20 and 10 min, respectively. After an incubation of 1 h in PBS + BSA (3 %), cells were incubated for an additional hour with anti-com rabbit polyclonal antibody (cw59), washed three times in PBS and incubated for 1 h with secondary anti-rabbit goat antibody conjugated to Cy3 (Sigma-Aldrich, Saint Quentin Fallavier, France). After three further washes, cells were mounted and viewed on a conventional immunofluorescence microscope.
Xenopus oocyte preparation and injection
Experiments were carried out following national guidelines. Xenopus laevis ovarian lobes were removed from frogs under tricaine methane sulfonate (MS-222, 0.2 %, Sigma-Aldrich) anaesthesia, and placed in a 90 mm Petri dish containing (mM): NaCl, 82.5; KCl, 2; MgCl2, 1; Hepes, 5 (OR-2 solution). Frogs were humanely killed after the final collection. Isolated ovarian lobes were broken into clumps and washed several times with OR-2 solution. They were then incubated at 19 °C under gentle agitation in the collagenase solution (Type 1A, Sigma-Aldrich at 1 mg ml-1 in OR-2 solution) for 1-2 h. After dissociation, oocytes were washed several times in OR-2 solution, and allowed to recover for 1-2 h in medium containing (mM): NaCl, 96; KCl, 2; MgCl2, 2; CaCl2, 1.8; sodium pyruvate, 2.5; Hepes, 5; pH adjusted to 7.4 with NaOH; and supplemented with 50 µg ml-1 gentamicin. Stage V and VI oocytes were selected under a binocular microscope and were usually injected the same day.
The following calcium channel subunits were used: 1A (Starr et al. 1991), 1b (Pragnell et al. 1991), 2a (Perez Reyes et al. 1992), 3, 4 (Castellano et al. 1993a,b) and 2- (Tomlinson et al. 1993). All these cDNAs were inserted into the pMT2 expression vector (Stea et al. 1994). 2, 3 and 4 subunits were subcloned into pc-DNA3.1. Xenopus oocyte injection (5-10 nl of 1 + 2- + ± cDNAs at ~0.3 ng nl-1 each) was performed as described elsewhere (Cens et al. 1996; Restituito et al. 2000). Oocytes were then incubated for 2-7 days at 19 °C under gentle agitation before recording. The incubation medium was renewed daily.
Electrophysiological recordings
Whole-cell Ba2+ currents were recorded under two-electrode voltage clamp using a GeneClamp 500 amplifier (Axon Instruments, Burlingame, CA, USA). Current and voltage electrodes (less than 1 M
) were filled with: CsCl, 2.8 M; Hepes, 10 mM; BAPTA, 10 mM; pH adjusted to 7.2 with CsOH. Ba2+ current recordings were performed after injection of BAPTA (around 50 nl of a solution containing (mM): BAPTA free acid (Sigma), 100; CsOH, 10; Hepes, 10; pH adjusted to 7.2 with CsOH) using the following recording solution (mM): BaOH, 10; TEAOH, 20; NMDG, 50; CsOH, 2; Hepes, 10; pH adjusted to 7.2 with methanesulfonic acid. Currents were filtered and digitized using a DMA-Tecmar labmaster interface, and subsequently stored on an IPC 486 personal computer using version 6.02 of the pCLAMP software (Axon Instruments). Ba2+ currents were recorded during a test pulse from -80 mV to +10 mV of 2.5 s duration. Current amplitudes were measured at the peak of the current (I1) and at the end of the pulse (I2). The percentage inactivation was calculated as the ratio of I2/I1. Isochronal inactivation (2.5 s of conditioning depolarization followed by a 400 ms test pulse to +10 mV) was fitted using the following equations:
I/Imax = Rin + (1 - Rin)/(1 + exp((V - E in)/k in)),
where I is the current amplitude measured during the test pulse at +10 mV for conditioning depolarization varying from -80 to +50 mV, Imax is the current amplitude measured during the test pulse for a conditioning depolarization of -80 mV, Ein is the potential for half-inactivation, V is the conditioning depolarization, kin is a slope factor and Rin is the proportion of non-inactivating current. Current-voltage curves were fitted using the following equation:
I = G (V - E rev)/(1 + exp((V - Vact)/kact)),
where I is the current amplitude measured during depolarizations varying from -80 to +50 mV, G is the maximal macroscopic conductance, E rev is the apparent reversal potential, Vact is the potential for half-activation, V is the value of the depolarization and kact is a slope factor.
All values are presented as means ± S.E.M. Student's t test was used at the 0.05 confidence level to determine the significance of the difference between two means.
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RESULTS |
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Several works have reported that the 2- subunit does not alter the effects of subunits on either P/Q- or L-type Ca2+ channels (Klugbauer et al. 2000). We have therefore expressed the 1A (CaV2.1) subunit with the 2- subunit and each of the possible and / combinations (see Fig. 1 for the combinations and nomenclature used).
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Figure 1. Schematic representation of all the combinations of subunits used in this study Between 5 and 35 oocytes were recorded for each combination (except
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Membrane expression of subunits
In a first set of experiments, we checked the correct expression and membrane targeting of the three neuronal subunits 2, 3 and 4. It has been recently shown that cytoplasmic subunits can be localized at the membrane by association with the 1 pore-forming subunit of the channel (Neuhuber et al. 1998). Therefore, to confirm the transmembrane nature of the subunit, we expressed it without co-expression of any other calcium channel subunits. Two days after transfection, expression and localization of each of the subunits was analysed by immunocytochemistry using an anti-com antibody that recognized all three subunits and a secondary antibody coupled to Cy3. In all three cases, a strong expression was noticed. The clear membrane localization of the three subunits seen in Fig. 2 confirms that their putative secondary structure involves transmembrane segments.
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Figure 2. Membrane expression of the Human
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Effects of subunits on Ca2+ channel activation and isochronal inactivation
We then characterized the functional effects of the 2 and 3 subunits on the voltage-dependent properties of the P/Q-type Ca2+ channels expressed in oocytes after injection of cDNA for 1A and 2- subunits (without expression of any subunits). The new neuronal subunits had minor effects on the activation properties, with slight, but significant, hyperpolarizing changes in the voltage dependence of activation (Vact; see Methods) and a decrease in the slope factor kact (see Fig. 3A and column labelled 'No ' in Table 1). 2 and 3 subunits also hyperpolarized significantly (P < 0.05) the potential for half-inactivation (E in) by 3-5 mV, increased the inactivation slope factor (k in) and decreased the fraction of non-inactivating current (Rin) in the steady-state inactivation curve (see Fig. 3A and column labelled 'No ' in Table 2). Therefore the 2 and 3 subunits hyperpolarized channel activation and increased inactivation.
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Figure 3. Modulation of the The different combinations of subunits used are indicated above the graphs, which show a comparison of current-voltage curves and steady-state inactivation curves in the absence and presence of
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These parameters, however, were differentially affected when each of the three neuronal subunits was co-expressed with different subunits. Voltage-dependent activation and inactivation parameters were not modified by expression of 2, 3 and 4 subunits, when the 1b subunit was expressed with the 1A/2- subunits (except a small but significant depolarization of Vact recorded with 1/4; see Fig. 3 and Tables 1 and 2). Similarly, activation of 3-containing channels was not affected by any of the subunits while inactivation was significantly hyperpolarized (E in decreased by ~5 mV for 3/2 and 3/4) and increased (see Rin for 3/2).
Expression of the 2a subunit induced varying effects on Vact, with no effect (2/3) or depolarizing (2/2) or hyperpolarizing (2/4) shifts. These shifts were accompanied by a significant increase in the slope factor, and, in the case of /2, a depolarizing shift in the inactivation curve. Interestingly, all three subunits induced a decrease in inactivation when 2a was expressed (see Rin in Table 2).
With 1A + 2- + 4 subunit combinations, no effects on channel activation and inactivation parameters (Vact, kact, E in, k in and Rin; see Tables 1 and 2) were recorded when 3 or 4 subunits were co-expressed (4/3, 4/4). The expression of the 2 subunits, however, induced a significant depolarizing shift in activation and inactivation (4/2).
Therefore, the effects of the different subunits are specific to subunits and pleiotropic, in the sense that a reduction or increase in the percentage of non-inactivating current and hyperpolarization or depolarization of the potential for half-inactivation can be recorded. Interestingly, except for a significant increase in Rin with the 2/3 combination, the 3 subunit did not seem to have any significant effect on either activation or inactivation of the channel when a subunit was expressed.
Effects of subunits on Ca2+ channel inactivation kinetics
The changes in the steady-state inactivation properties suggest that normal gating kinetics of the Ca2+ channel might be affected by co-expression of the subunits. Although a fine analysis of Ca2+ channel activation kinetics cannot be achieved using the Xenopus oocyte system, a perfectly good analysis of the inactivation kinetics of these different combinations of subunits can be made. This was done by recording Ba2+ currents during long depolarizing pulses (2.5 s at +10 mV; see Fig. 4), and by a subsequent fit of these traces by the sum of two exponential functions. I2/I1, the ratio of the non-inactivating current at the end of the pulse to the peak current, was used as an index of the speed of inactivation, while the fast and slow time constants of inactivation, and relative importance of the slow component (
1, 2 and % 2, respectively) allowed a more precise analysis.
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Figure 4. Modulation of inactivation kinetics by Left panel in A, time course of normalized Ba2+ currents recorded from cells expressing different subunit combinations evoked by step deplarizations of 2.5 s duration to +10 mV. Right panel in A, non-inactivating current at 2.5 s (I2/I1) calculated for oocytes expressing different subunits during a depolarization to +10 mv (holding potential -80 mV). B, the inactivating phase of the current was adjusted to a bi-exponential decay.
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As shown on Fig. 4A, when no subunit was expressed, both 2 and 3 induced a significant acceleration of inactivation (see I2/I1). This acceleration was due to a decrease in both 1 and 2, together with a decrease in the proportion of 2 (% 2; see Fig. 4B).
2, 3 and 4 did not produce any changes, on average, in the kinetics of inactivation (see I2/I1, 1, 2 and % 2; Fig. 4), when 3 or 4 was expressed with the 1A and 2- subunits. 3 was also without effect (P > 0.05) on channels containing the 1 or 2a subunits. However, expression of the / subunit combinations 1/2, 2/2 and 2/4 produced a significant increase in I2/I1. This increase was due to an increase in both the slow time constant of inactivation and the relative amplitude of this time constant (2 and % 2; see Fig. 4). In all cases the fast time constant of inactivation was either not affected or reduced (Fig. 4; combinations no /2, 1/2, 1/3, 1/4, 2/2 and 3/4).
Unexpected slow inactivation preferentially recorded with subunits
In view of these results, it appears that the regulation of the P/Q-type Ca2+ channel by the neuronal subunits is complex, with small, but specific, modifications of the activation and inactivation properties. However, a statistical comparison of the averaged parameters of these different combinations gave only a partial picture of the variability of our results. Indeed, when looking at individual traces, we noticed that some of our recordings displayed unusual slow inactivation. This slow inactivation was recorded particularly in oocytes expressing a subunit, and can be seen in Fig. 5A, where 20 inactivation curves, recorded from 10 different oocytes expressing 3/no or 3/3 subunits are displayed. We thus decided to analyse separately the fast- and slow-inactivating currents. Rin > 20 % was chosen as the criterion for defining slow-inactivating currents for combinations containing no subunits, or 1, 3 or 4 subunits. This criterion was increased to Rin > 50 %, when the 2 subunit was expressed. Slow inactivation was never seen when no subunit was expressed, whether a subunit was expressed or not (see Fig. 5B; n = 16, 15 and 15 for no , no /2 and no /3, respectively). When subunits were expressed, but without , it was only rarely seen (1, 2/22; 2, 0/16; 3, 1/28; 4, 0/5). However, the probability of having slow-inactivating currents was clearly increased by expression of a given subunit. This probability was larger with 2 in the combination 1/2 and 4/2 , and with 3 and 4 in the combinations 2/3, 2/4, 3/3 and 3/4. Although the variable sizes of these populations did not allow a precise comparison of these probabilities, the effects of the subunits were clear.
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Figure 5. Two types of inactivation favoured by expression of the A, steady-state inactivation curves recorded from 20 different oocytes expressing the
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We thus performed a separate analysis of fast- and slow-inactivating currents for the 3-containing combinations of subunits. In control conditions (see control (3/no ) in Tables 3 and 4), the fast- and slow-inactivating currents can be separated by only one distinctive steady-state property, the fraction of non-inactivating current, Rin. Expression of 2, 3 or 4 subunits did not change the voltage dependence of activation of both fast- and slow-inactivating currents, but significantly reduced both voltage dependence of inactivation (E in) and Rin, for fast inactivating currents (see Table 3). These effects were similar to those seen without subunits expressed. Statistical analysis of the slow current was not performed due to the small size of the population, but activation and inactivation did not appear to be strongly modified (see Table 4).
Kinetic properties of fast and slow currents recorded with 3 were also analysed. As seen in Fig. 6, for fast 3 currents, 1 and 2 (fast and slow time constants of inactivation, respectively) were significantly reduced by expression of 3 or 4, while %2 remained unchanged. These reductions accounted for the increase in inactivation recorded upon expression of the subunit in the fast population. In the slow population, 2 was significantly increased with all subunits, and other parameters were not modified.
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Figure 6. Separate analysis of fast- and slow-inactivating currents for Averaged
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Lack of direct / interaction
The possibility of direct interaction between the 1 subunit and 2, 3 or 4 subunits was tested by immunocytochemistry. The 1 subunit has a cytoplasmic localization when expressed alone. Due to direct protein-protein interaction, this localization is redistributed to the membrane when the 1A subunit is co-expressed (Gao et al. 1999). Direct / interaction would thus be expected to relocalize the 1 subunit to the membrane in a / co-transfection experiment. We performed this experiment in tsA-201 cells by co-transfection of the 1 and 2, 3 or 4 subunits. Immunostaining with an anti- subunit antibody (Vance et al. 1998) revealed that expression of subunits (2, 3 or 4; see Fig. 7) did not change the cytoplasmic localization of 1, suggesting that there was no direct interaction between the two subunits.
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Figure 7. Lack of direct Cells were transfected with the
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DISCUSSION |
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We show in this study that coexpression of the 2, 3 and 4 subunits with the 1A, 2- and subunits modified channel properties in a subunit-specific manner in two different ways. (1) When no subunits were expressed, 2 and 3 induced a small negative shift of the inactivation curve and an acceleration of the inactivation kinetics. (2) When subunits were expressed, these basic effects were still present but, in some oocytes, Ca2+ channels displayed an unusual slow inactivation, never seen in the absence of subunits (0/46). subunits increased the proportion of oocytes expressing these slow-inactivating Ca2+ channels.
The intrinsic effects of the subunits (when the slow-inactivating currents are removed from the analysis) are an acceleration of inactivation and a small hyperpolarizing shift in the inactivation curves. The global effects (taking the slow-inactivating current into consideration) depend largely on the proportion of these slow currents and on the specific subunit expressed.
subunit-induced shift in inactivation
The basic effects of (2, 3 and 4) subunits on 1A, 1G and 1C subunits have recently been reported by Letts et al. (1998) and Klugbauer et al. (2000). These effects are qualitatively similar to those of skeletal muscle 1 subunits on the 1S, 1C or 1A subunits (Singer et al. 1991; Mori et al. 1991; Wei et al. 1991; Sipos et al. 1995; Eberst et al. 1997). Studies performed using oocytes or transfected cells, and 1b or 2a subunits gave essentially the same results, suggesting that these effects are independent of the expression system used and subunits expressed. The only exception is the depolarizing shift recorded with the 2/2 combination (see Table 2). A positive shift for this combination was also seen by Klugbauer et al. (2000), but only using Ba2+ as the charge carrier, with a negative shift being recorded when Ca2+ replaced Ba2+. Negative charges are present in the first extracellular loop of all subunits. These charges have been proposed to participate, by modification of the local surface potential, in the observed shift in inactivation properties (Singer et al. 1991). Such modifications are sensitive to the concentrations and types of cations used in the external solutions, and their impact on channel properties is influenced by the subunit composition of the channel (Mangoni et al. 1997; Cens et al. 1998). Although the overall primary sequence in the skeletal muscle 1 subunit is poorly conserved in 2, 3 and 4 subunits (25 % identity), preservation of the extracellular negative charge and glycosylation sites and the non-specific nature of these surface potential effects may explain the lack of subunit specificity. However, varying effects with different subunits may arise from their specific influence on the sensing of the surface charges by the channel gating machinery (Cens et al. 1998).
The unexpected slow inactivation
The recording of unusual slow-inactivating currents is more puzzling. This type of current kinetics was recorded in the absence of subunits in a very small proportion of oocytes (3/72), but required the expression of a subunit (no recordings of slow inactivation without subunit expression were obtained, n = 46). These data suggest that the mechanism and the molecular determinants underlying this type of inactivation are present in the 1/ subunit complex, although we did not try to analyse the requirement for an 2- subunit. The subunit specificity of this type of inactivation has not been tested directly because of its very low frequency of appearance, but our recordings made with subunits suggest that slow inactivation can be recorded with all four subunit types.
Recently, it has been shown that P/Q-type Ca2+ channels expressed in tsA-201 cells (1A + 2- + 2) display large variations in their kinetics and voltage dependence of inactivation (Hurley et al. 2000). Similar variations were also recorded in bovine chromaffin cells, in more physiological conditions, and were attributed to dynamic palmitoylation of two cysteines of crucial importance for the 2a-induced slowing of inactivation (Hurley et al. 2000). Under our conditions, the fact that slow inactivation was recorded with and without 2a subunits makes the participation of subunit palmitoylation very unlikely and rather suggests another regulatory mechanism for reducing channel inactivation. This mechanism, however, may act in concert with the 2a subunit to induce, for the combinations of subunits investigated by us in oocytes, a very slow inactivation.
Molecular events of the slow inactivation
Interestingly, when subunits were expressed, subunits increased the frequency of this slow inactivation although no strong direct / interaction could be revealed by co-expression in tsA-201 cells (see Fig. 7). Three important questions thus arise. (1) What are the molecular events involved in the slow inactivation? (2) What type of modulation inhibits this slow inactivation in most of the oocytes? (3) What is the role for the subunit in the slow inactivation?
Fast, voltage-dependent, inactivation of the 1A subunit is the result of molecular rearrangements involving the I-II loop and the C-terminus of the 1 subunit (Zhang et al. 1994; Cens et al. 1999; Bourinet et al. 1999). The 2a subunit induces a reduction of this type of inactivation by immobilization of the I-II loop, supposed to act as a pore blocker in a similar way to the ball-and-chain model proposed for Shaker K+ channel inactivation. Both a single valine insertion and a point mutation in the I-II loop are known to reduce inactivation drastically without disturbing, qualitatively, regulation of inactivation by subunits (Herlitze et al. 1997; Bourinet et al. 1999). Such amino acid changes modify the I-II loop organization and could therefore decrease the affinity of this pore blocker for its binding site. The 'natural' slow inactivation recorded under our conditions could result from such structural changes occurring without the necessity of mutation, by natural modification of either the pore-blocker particle or its binding site. Possible triggering events for this type of modification are phosphorylation, or oxido-reduction changes, bringing new charges or new disulfide bonds into the target sequences. Indeed, multiple reports have already shown in Shaker K+ channels that these types of modifications could influence inactivation kinetics and protein kinase A phosphorylation is known to modulate L-type Ca2+ channel inactivation. subunits could possibly act as catalysts of such reactions, optimizing channel secondary/tertiary structures for modification. A direct effect of the subunit on channel inactivation cannot be discarded, but, as is the case for the 2a subunit, would require a direct 1/ interaction, which has yet to be demonstrated. It should be noted that the small proportion of oocytes expressing the slow inactivation will complicate future analysis of these mechanisms.
The presence of the slow-inactivating current suggests an equilibrium between two gating modes of the P/Q-type Ca2+ channels, such as those described for L-type Ca2+ channels (Hess et al. 1984). Bay-K8644, a dihydropyridine agonist of L-type Ca2+ channels, favours preferentially the slow gating mode of the channel (mode II), whose presence is otherwise only very rarely observed. Similar gating modes have also been proposed for P/Q-type Ca2+ channels, but the physiological agents which facilitate the switch between these two modes remain to be identified. However, the slow inactivation should be undoubtedly of primary importance for Ca2+ homeostasis in the particular situations where it is induced. It may be related to the episodic nature of the absence seizures found in mice and underlines the regulatory role the subunits in this phenomenon and the physiopathology of the stargazer mouse. Based on the present data, chronic (basic) and phasic effects acting in opposite directions can be expected to result from suppression of subunits.
In pathological situations, the loss of subunit expression should cause Ca2+ channels to inactivate more slowly and at more depolarized potentials, thus increasing the number of active channels and the Ca2+ influx at a given resting potential. Such modifications, together with the change in the AMPA receptor expression and targeting (Chen et al. 2000), could produce alteration of transmitter release and neuronal excitability. In normal, physiological conditions, when the subunits are expressed, it is clear that conditions where unexpected slow inactivation is favoured will also have important consequences on neuronal Ca2+ homeostasis. Further experiments are clearly required to understand the physiological significance and molecular pathway of this new regulation.
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REFERENCES |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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| BLACK J. L. III & LENNON, V. A. (1999). Identification and cloning of putative human neuronal voltage-gated calcium channel gamma-2 and gamma-3 subunits: neurologic implications. Mayo Clinic Proceedings 74, 357-361 | [Medline] |
| BOSSE E., REGULLA, S., BIEL, M., RUTH, P., MEYER, H. E., FLOCKERZI, V. & HOFMANN, F. (1990). The cDNA and deduced amino acid sequence of the gamma subunit of the L-type calcium channel from rabbit skeletal muscle. FEBS Letters 267, 153-156 | [Medline] |
| BOURINET E., SOONG, T. W., SUTTON, K., SLAYMAKER, S., MATHEWS, E., MONTEIL, A., ZAMPONI, G. W., NARGEOT, J. & SNUTCH, T. P. (1999). Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels. Nature Neuroscience 2, 407-415 | [Medline] |
| CASTELLANO A., WEI, X., BIRNBAUMER, L. & PEREZ REYES, E. (1993a). Cloning and expression of a third calcium channel beta subunit. Journal of Biological Chemistry 268, 3450-3455 | [Abstract] |
| CASTELLANO A., WEI, X., BIRNBAUMER, L. & PEREZ REYES, E. (1993b). Cloning and expression of a neuronal calcium channel beta subunit. Journal of Biological Chemistry 268, 12359-12366 | [Abstract] |
| CENS T., DALLE, C. & CHARNET, P. (1998). Expression of beta subunit modulates surface potential sensing by calcium channels. Pflügers Archiv 435, 865-867 | [Medline] |
| CENS T., MANGONI, M. E., RICHARD, S., NARGEOT, J. & CHARNET, P. (1996). Coexpression of the beta2 subunit does not induce voltage- dependent facilitation of the class C L-type Ca channel. Pflügers Archiv 431, 771-774 | [Medline] |
| CENS T., RESTITUITO, S., GALAS, S. & CHARNET, P. (1999). Voltage and calcium use the same molecular determinants to inactivate calcium channels. Journal of Biological Chemistry 274, 5483-5490 | [Abstract/Full Text] |
| CHEN L., BAO, S., QIAO, X. & THOMPSON, R. F. (1999). Impaired cerebellar synapse maturation in waggler, a mutant mouse with a disrupted neuronal calcium channel gamma subunit. Proceedings of the National Academy of Sciences of the USA 96, 12132-12137 | [Abstract/Full Text] |
| CHEN L., CHETKOVICH, D. M., PETRALIA, R. S., SWEENEY, N. T., KAWASAKI, Y., WENTHOLD, R. J., BREDT, D. S. & NICOLL, R. A. (2000). Stargazing regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408, 936-943 | [Medline] |
| EBERST R., DAI, S., KLUGBAUER, N. & HOFMANN, F. (1997). Identification and functional characterization of a calcium channel gamma subunit. Pflügers Archiv 433, 633-637 | [Medline] |
| FLOCKERZI V., OEKEN, H. J. & HOFMANN, F. (1986a). Purification of a functional receptor for calcium-channel blockers from rabbit skeletal-muscle microsomes. European Journal of Biochemistry 161, 217-224 | [Abstract] |
| FLOCKERZI V., OEKEN, H. J., HOFMANN, F., PELZER, D., CAVALIE, A. & TRAUTWEIN, W. (1986b). Purified dihydropyridine-binding site from skeletal muscle t-tubules is a functional calcium channel. Nature 323, 66-68 | [Medline] |
| FREISE D., HELD, B., WISSENBACH, U., PFEIFER, A., TROST, C., HIMMERKUS, N., SCHWEIG, U., FREICHEL, M., BIEL, M., HOFMANN, F., HOTH, M. & FLOCKERZI, V. (2000). Absence of the gamma subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties. Journal of Biological Chemistry 275, 14476-14481 | [Abstract/Full Text] |
| GAO T., CHIEN, A. J. & HOSEY, M. M. (1999). Complexes of the alpha1C and beta subunits generate the necessary signal for membrane targeting of class C L-type calcium channels. Journal of Biological Chemistry 274, 2137-2144 | [Abstract/Full Text] |
| HASHIMOTO K., FUKAYA, M., QIAO, X., SAKIMURA, K., WATANABE, M. & KANO, M. (1999). Impairment of AMPA receptor function in cerebellar granule cells of ataxic mutant mouse stargazer. Journal of Neurosciences 19, 6027-6036 | |
| HERLITZE S., HOCKERMAN, G.H., SCHEUER, T. & CATTERALL, W. A. (1997). Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel alpha1A subunit. Proceedings of the National Academy of Sciences of the USA 94, 1512-1516 | [Abstract/Full Text] |
| HESS P., LANSMAN, J. B. & TSIEN, R. W. (1984). Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists. Nature 311, 538-544 | [Medline] |
| HURLEY J. H., CAHILL, A. L., CURRIE, K. P. & FOX, A. P. (2000). The role of dynamic palmitoylation in Ca2+ channel inactivation. Proceedings of the National Academy of Sciences of the USA 97, 9293-9298 | [Abstract/Full Text] |
| KLUGBAUER N., DAI, S., SPECHT, V., LACINOVA, L., MARAIS, E., BOHN, G. & HOFMANN, F. (2000). A family of gamma-like calcium channel subunits. FEBS Letters 470, 189-197 | [Medline] |
| LETTS V. A., FELIX, R., BIDDLECOME, G. H., ARIKKATH, J., MAHAFFEY, C. L., VALENZUELA, A., BARTLETT, F. S., MORI, Y., CAMPBELL, K. P. & FRANKEL, W. N. (1998). The mouse stargazer gene encodes a neuronal Ca2+-channel gamma subunit. Nature Genetics 19, 340-347 | [Medline] |
| MANGONI M. E., CENS, T., DALLE, C., NARGEOT, J. & CHARNET, P. (1997). Characterisation of alpha1A Ba2+, Sr2+ and Ca2+ currents recorded with ancillary beta1-4 subunits. Receptors and Channels 5, 1-14 | [Medline] |
| MORI Y., FRIEDRICH, T., KIM, M. S., MIKAMI, A., NAKAI, J., RUTH, P., BOSSE, E., HOFMANN, F., FLOCKERZI, V., FURUICHI, T. et al. (1991). Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature 350, 398-402 | [Medline] |
| NEUHUBER B., GERSTER, U., MITTERDORFER, J., GLOSSMANN, H. & FLUCHER, B. E. (1998). Differential effects of Ca2+ channel beta1a and beta2a subunits on complex formation with alpha1S and on current expression in tsA201 cells. Journal of Biological Chemistry 273, 9110-9118 | [Abstract/Full Text] |
| PEREZ REYES E., CASTELLANO, A., KIM, H. S., BERTRAND, P., BAGGSTROM, E., LACERDA, A. E., WEI, X. Y. & BIRNBAUMER, L. (1992). Cloning and expression of a cardiac/brain beta subunit of the L-type calcium channel. Journal of Biological Chemistry 267, 1792-1797 | [Abstract] |
| PRAGNELL M., SAKAMOTO, J., JAY, S. D. & CAMPBELL, K. P. (1991). Cloning and tissue-specific expression of the brain calcium channel beta-subunit. FEBS Letters 291, 253-258 | [Medline] |
| QIAO X., CHEN, L., GAO, H., BAO, S., HEFTI, F., THOMPSON, R. F. & KNUSEL, B. (1998). Cerebellar brain-derived neurotrophic factor-TrkB defect associated with impairment of eyeblink conditioning in Stargazer mutant mice. Journal of Neurosciences 18, 6990-6999 | |
| QIAO X., HEFTI, F., KNUSEL, B. & NOEBELS, J. L. (1996). Selective failure of brain-derived neurotrophic factor mRNA expression in the cerebellum of stargazer, a mutant mouse with ataxia. Journal of Neurosciences. 16, 640-648 | |
| REN D. & HALL, L. M. (1997). Functional expression and characterization of skeletal muscle dihydropyridine receptors in Xenopus oocytes. Journal of Biological Chemistry 272, 22393-22396 | [Abstract/Full Text] |
| RESTITUITO S., CENS, T., BARRERE, C., GEIB, S., GALAS, S., DE WAARD, M. & CHARNET, P. (2000). The 2a subunit is a molecular room for the Ca2+ channel inactivation gate. Journal of Neurosciences 20, 9046-9052 |
|
| SIEBER M., NASTAINCZYK, W., ZUBOR, V., WERNET, W. & HOFMANN, F. (1987). The 165-kDa peptide of the purified skeletal muscle dihydropyridine receptor contains the known regulatory sites of the calcium channel. European Journal of Biochemistry 167, 117-122 | [Abstract] |
| SINGER D., BIEL, M., LOTAN, I., FLOCKERZI, V., HOFMANN, F. & DASCAL, N. (1991). The roles of the subunits in the function of the calcium channel. Science 253, 1553-1557 | [Medline] |
| SINGER LAHAT D., LOTAN, I., BIEL, M., FLOCKERZI, V., HOFMANN, F. & DASCAL, N. (1994). Cardiac calcium channels expressed in Xenopus oocytes are modulated by dephosphorylation but not by cAMP-dependent phosphorylation. Receptors and Channels 2, 215-226 | [Medline] |
| SIPOS I., JURKAT-ROTT, K., HARASZTOSI, C., FONTAINE, B., KOVACS, L., MELZER, W. & LEHMANN-HORN, F. (1995). Skeletal muscle DHP receptor mutations alter calcium currents in human hypokalaemic periodic paralysis myotubes. Journal of Physiology 483, 299-306 | [Abstract] |
| STARR T. V., PRYSTAY, W. & SNUTCH, T. P. (1991). Primary structure of a calcium channel that is highly expressed in the rat cerebellum. Proceedings of the National Academy of Sciences of the USA 88, 5621-5625 | [Abstract] |
| STEA A., TOMLINSON, W. J., SOONG, T. W., BOURINET, E., DUBEL, S. J., VINCENT, S. R. & SNUTCH, T. P. (1994). Localization and functional properties of a rat brain alpha 1A calcium channel reflect similarities to neuronal Q- and P-type channels. Proceedings of the National Academy of Sciences of the USA 91, 10576-10580 | [Medline] |
| TOMLINSON W. J., STEA, A., BOURINET, E., CHARNET, P., NARGEOT, J. & SNUTCH, T. P. (1993). Functional properties of a neuronal class C L-type calcium channel. Neuropharmacology 32, 1117-1126 | [Medline] |
| VANCE C. L., BEGG, C. M., LEE, W. L., HAASE, H., COPELAND, T. D. & MCENERY, M. W. (1998). Differential expression and association of calcium channel alpha1B and beta subunits during rat brain ontogeny. Journal of Biological Chemistry 273, 14495-14502 | [Abstract/Full Text] |
| WEI X. Y., PEREZ-REYES, E., LACERDA, A. E., SCHUSTER, G., BROWN, A. M. & BIRNBAUMER, L. (1991). Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels. Journal of Biological Chemistry 266, 21943-21947 | [Abstract] |
| ZHANG J. F., ELLINOR, P. T., ALDRICH, R. W. & TSIEN, R. W. (1994). Molecular determinants of voltage-dependent inactivation in calcium channels. Nature 372, 97-100 | [Medline] |
Acknowledgements
This work was supported by MENESR, FRM and GRRC (financial support to M.R., T.C. and S.R.), Ligue Régionale contre le Cancer (Pyrénées Orientales), Association pour le Recherche contre le Cancer and Association Française contre les Myopathies.
Corresponding author
P. Charnet: CRBM, CNRS UPR 1086, UFR 24, 1919 Route de Mende, 34293 Montpellier Cedex 05, France.
Email: charnet{at}crbm.cnrs-mop.fr
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