INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Different Ca²⁺ channel auxiliary  subunits have been isolated from mammalian brain and heart. They can potentially be associated with any of the 6 ₁ pore-forming subunits of high-voltage activated Ca²⁺ channel (_1A, _1B, _1C, _1D, _1E, _1F, and _1S) via conserved interaction domains: -interaction domain (AID) on ₁ and -interaction domain (BID) on  (De Waard et al., 1995, 1996; Pragnell et al., 1994; Walker and De Waard, 1998). These  subunits induced generic as well as specific modifications in the expression, electrophysiological properties, and regulation of any of the ₁ subunits. Increased channel expression, activity, and membrane targeting of the ₁ subunit are common modifications induced by all  subunits, whereas changes in the voltage-dependence and kinetics of activation and inactivation are more specific of a given pair of ₁- subunits (Mangoni et al., 1997; Sather et al., 1993; De Waard and Campbell, 1995; Jones et al., 1998; Stea et al., 1994). These effects appeared to be mediated by high affinity interactions between the AID (located on the loop connecting domains I and II of each ₁ subunits) (Pragnell et al., 1994) and the BID (located on the beginning of the second conserved region of the  subunit) (De Waard et al., 1994). Modification of these sites, using site-directed mutagenesis, produced a complete loss of the ₁/ subunits co-localization and co-immunoprecipitation, but only partially reverted the increase in current amplitude and change in electrophysiological properties of ₁ (Gerster et al., 1999; De Waard et al., 1994). These latter results suggest that the  subunit modifies channel targeting and/or current amplitude using molecular determinants distinct from those necessary for the modification of the electrophysiological properties (Gerster et al., 1999). Indeed, functional analysis of mutated and truncated ₁ and  subunits revealed that additional interaction sites of lower affinity (on the N- and C-terminal tails of the ₁ and  subunits) may also participate to these regulations (Birnbaumer et al., 1998; Cens et al., 1998; Olcese et al., 1994; Qin et al., 1996; Walker et al., 1998).

It should be noted that although the AID-BID interaction occurs through high-affinity sites, the recent identification of a G protein binding site overlapping the AID suggests that this interaction can be disrupted in certain circumstances, such as when G proteins are activated (De Waard et al., 1997; Zamponi et al., 1997). Indeed, in an heterologous expression system, the Ca²⁺ channel block by G protein is decreased by expression of an auxiliary  subunit (Bourinet et al., 1996) suggesting competition between overlapping sites and possible disruption of the AID-BID interaction. In normal conditions however, such unbinding has never been observed.

Slowing of inactivation has been reported when the _2a subunit is co-expressed with the neuronal _1A, _{1B, 1C}, and the _1E subunits (Sather et al., 1993; Mangoni et al., 1997, De Waard and Campbell, 1995; Stea et al., 1994; Jones et al., 1998; Cens et al., 1999). The mechanism underlying this effect has recently been proposed to be due to immobilization of the channel inactivation gate by a membrane-anchoring site (Restituito et al., 2000) constituted of two palmitic acid bound to cysteines 3 and 4 of the amino-terminal tail of the _2a subunit (Chien et al., 1996, 1998; Qin et al., 1998).

Here we report a significant heterogeneity in the voltage-dependence and kinetics of inactivation of the _1A P/Q-type Ca²⁺ channel expressed with the _2a or _1b subunits. Variable parameters are the fraction of non-inactivating current (R_in) for _2a and the voltage for half-inactivation (E_in) for _1b. However, expression of _1A and ₂   without  results in more homogeneous inactivation properties. A more complete analysis of voltage-dependent activation and inactivation and G protein regulation was performed using different mutated  subunits at different _1A/_2a cDNA ratios and suggested unbinding of the  subunit from the ₁ subunit. Altogether these results provide evidences for a dynamic association between these two subunits.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Preparation of mutated  subunits

The following calcium channel subunits were used: _1A (Starr et al., 1991), _1b (Pragnell et al., 1991); _2a (Perez-Reyes et al., 1992), and ₂. All these cDNAs were inserted into the pMT2 expression vector (Stea et al., 1994). The chimera CD8-_2aC3,4S construction was produced as described previously (Restituito et al., 2000).

Xenopus oocyte preparation and injection

Xenopus oocyte preparation and injection (5-10 nl of a mixture of _{1, 2-}, or  subunits cDNAs at 0.3 ng/nl each) were performed as described elsewhere (Cens et al., 1996). For Fig. 4, starting cDNA concentration was either 1 or 0.1 ng/nl for _1A and _2a subunits, giving a final concentration of 0.3 or 0.03 ng/nl after dilution. Oocytes were selected as expressing slow- or fast-inactivating currents after individual recordings in voltage-clamp. Oocytes from each pool (fast, slow, non-expressing, and non-injected) were then frozen in liquid nitrogen and kept for Western blotting. Each line was loaded with the equivalent of three oocytes expressing the same quantity of currents. µ opioid receptor and _O G protein RNA (1 µg/µl) were injected 1 day after cDNA injection. Oocytes were then incubated for 2 to 7 days at 19° C under gentle agitation before recording.

Western blot

Frozen oocytes were homogenized in 5 µl/oocytes of the following lysis buffer [20 mM Tris (pH 7.5), 50 mM NaCl, 50 mM NaF, 10 mM glycerophosphate, 5 mM Na₄P₂O₇, 5 mM EDTA; 5 mM EGTA, 10 mg/ml PMSF, 0.2 mg/ml pepstatin, 0.2 mg/ml leupeptin, and 0.2 mg/ml aprotinin] and centrifuged for 3 min at 20,000 × g at 4°C. The supernatant was boiled in sodium dodecyl sulfate (SDS) gel loading buffer (Laemmli), electrophoresed on 10% SDS-polyacrylamide gel, and transferred to nitrocellulose filter. Filters were blocked 1 h at room temperature (8% skim milk powder), rinsed in distilled water, incubated overnight at 4°C with the -com rabbit polyclonal primary antibody in 0.1% bovine serum albumin (CW24; Vance et al., 1998), washed 3 times, incubated 1 h with an anti-rabbit antibody, and detected by chemiluminescence (Renaissance NEN, Boston, MA).

Electrophysiological recordings

Whole-cell Ba²⁺ currents (<5 µA) were recorded under two electrodes voltage-clamp using the GeneClamp 500 amplifier (Axon Instruments, Burlingame, CA). Current and voltage electrodes (<1 M) were filled with 2.8 M CsCl and 10 mM BAPTA, pH = 7.2, with CsOH. Ba²⁺ current recordings were performed after injection of BAPTA (around 50 nl of (mM): BAPTA-free acid (Sigma), 100; CsOH, 10; HEPES, 10; pH 7.2 CsOH) using the following solution (in mM): BaOH, 10; TEAOH, 20; NMDG, 50; CsOH, 2; HEPES, 10; pH 7.2 with methanesulfonic acid. Currents were filtered and digitized using a Digidata 1200 interface (Axon Instruments), and subsequently stored on a Pentium II-based personal computer by using the version 6.02 of the pClamp software (Axon Instrument). Ba²⁺ currents were recorded during a test pulse from 80 mV to +10 mV of 2.5 sec duration. Current amplitudes were measured at the peak of the current. Pseudo steady-state inactivation (2.5 s of conditioning depolarization followed by a 400 ms test pulse to +10 mV) was fitted using the following equations

(1)

where I is the current amplitude measured during the test pulse at +10 mV for conditioning depolarization varying from 80 to +50 mV; I_max, the current amplitude measured during the test-pulse for a conditioning depolarization of 80 mV; E_in, the potential for half-inactivation; V, the conditioning depolarization; k, the slope factor; and R_in, the proportion of non-inactivating current. Current to voltage curves were fitted using the following equation

(2)

where I is the current amplitude measured during depolarizations varying from 80 to +50 mV; Imax, the peak current amplitude measured of the current-voltage curve; G, is the normalized macroscopic conductance; E_rev, is the apparent reversal potential, V_act: the potential for half-activation; V, the value of the depolarization; and k, a slope factor.

Activation of G protein-coupled µ opioid receptor was performed by perfusion of 10 µmol of the specific agonist Tyr-D-Ala-Gly-N-Methyl-Phe-Gly-ol (DAMGO; Sigma-Aldrich, Saint Quentin Fallavier, France). % block induced by DAMGO was calculated by dividing the current amplitude in control conditions by the current amplitude recorded at the steady state effect of 10 µM DAMGO during train of 50-ms depolarizations at 0 mV. All values are presented as mean ± SEM.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Xenopus oocytes were injected with cDNA encoding the _1A + ₂   and either the _1b or _2a subunit, and Ba²⁺ currents were recorded 2-5 days later. As seen in Fig. 1, A and B (left), and as already shown by others (De Waard and Campbell, 1995; Stea et al., 1994), these two combinations of subunits induced the expression of P/Q type Ba²⁺ currents with fast and slow inactivation, respectively. The voltage protocol applied to the oocytes (shown on top of Fig. 1) allows for the same recording to calculate current-voltage and inactivation curves as well as inactivation kinetics. The slowing of inactivation by the _2a subunit was clear for depolarized (>20 mV) potentials, and the inactivation curves allow to calculate the potential for half-inactivation (E_in) and the proportion of non-inactivating current (R_in) for each combination of subunits. Averaged values gave an E_in of 28.2 ± 9.8 mV and 12.8 ± 5 mV and an R_in of 0.098 ± 0.002 and 0.48 ± 0.004 for _1b (n = 28) and _2a (n = 56). Comparison with the values obtained in the absence of expressed  subunit (_1A + ₂  ; E_in = 12.2 ± 2.2mV and R_in = 0.08 ± 0.002, n = 16) demonstrate that the major effect of _1b subunit was to decrease the availability of the channel by hyperpolarizing E_in, whereas the major effect of _2a was to prevent inactivation by increasing R_in. However, when typical individual inactivation curves recorded for each combination were plotted on the same graph (Fig. 1, right), a large variation in the E_in and R_in values can be seen for each subunit combination. This variation was better seen in Fig. 2 A, in which individual R_in values are plotted against their corresponding E_in value for all combinations of subunits tested (n > 200 oocytes). From such a plot, 3 groups can be identified. The control _1A group (open square), corresponds to oocytes expressing the _1A + ₂ subunits without  subunit. For this group, the inactivation parameters (E_in and R_in) are well centered around their average values, as expected for an homogeneous population. The ₂ group (triangle) represents oocytes injected with _1A + ₂ and the _2a subunit cDNAs. This group displays low variability in their E_in values (which are almost similar to E_in of the _1A group), but large variations in R_in ranging from 0.1 to 0.9. In this group, smaller values of R_in are close to the average R_in value of the _1A group. In the case of the ₁ group (oocytes injected with _1A + ₂   + _1b cDNA; Fig. 2 A, open circle) the variations are more pronounced for E_in (starting from 10 mV, the average value of the control _1A group, and decreasing up to 55 mV), whereas R_in appeared almost unchanged when compared to _1A group. Hyperpolarization of the inactivation curve and reduction of inactivation, the two fundamental effects of the _1b and _2a subunits, respectively, are therefore subject to large variations in vivo despite the known high-affinity interaction between the _1A and the  subunits. Such variations has also been found in chromaffin cells and tsA201 cells transfected with the _1B, or _1A and _2a subunits (Cahill et al., 2000; Hurley et al., 2000).

View larger version (21K): [in this window] [in a new window]   FIGURE 1   Heterogeneity of voltage-dependent inactivation. Voltage-dependent inactivation curves recorded from oocytes expressing the 1A and 2   Ca2+ channel subunits with either the 1b (A) or 2a (B) auxiliary subunits. (Left) Typical current traces recorded on oocytes expressing 1A + 2   + 1b or 1A + 2   + 2a subunit during the voltage protocol shown on top. The conditioning depolarisation had a duration of 2.5s, test-pulse (+10 mV) duration was 0.4 s. Scale bar, 0.5 µA. (Right) Example of 8 typical steady-state inactivation curves recorded on different oocytes for each of the two combinations of subunits. Note the variability in the voltage for half-inactivation (Ein) and in the proportion of the non-inactivating current (Rin).

View larger version (28K): [in this window] [in a new window]   FIGURE 2   1 and 2-induced regulation of inactivation. (A and B) Scatter plot of Rin versus Ein for different batches of oocytes (n>200) injected with the 1A and 2   subunits alone or in combination with 1b, 2a (A), CD8-2C3,4S or 2C3,4S (B; see Results and Discussion). Note that these oocytes can be divided into 3 groups. The control 1A group included oocytes expressing the 1A + 2   subunits and show limited variability in either Ein and Rin. The 2 group, displays the same Ein than 1A + 2   but with higher Rin (includes oocytes expressing 1A+ 2   + 2a, or CD8-2C3, 4S). Currents recorded from oocyte belonging to the 1 group have the same Rin than 1A + 2  , but more hyperpolarized Ein (group of oocytes expressing 1A + 2   + 1b or 2C3, 4S). For the 1b group, variability is more pronounced for Ein, while for the 2 group, Rin was the most variable parameter. (C) Scatter plot of Ein versus Vact, the potential of half activation for the oocytes shown on A. The two groups of oocytes induced an hyperpolarization of the IV curves. This hyperpolarization was correlated with a negative shift of Ein for the 1 group that was less pronouned for the 2 group. (D) Scatter plot of Rin versus Vact, the potential of half activation, for the currents recorded from the same oocytes shown on A. Hyperpolarizing shift of the IV curve (Vact) was correlated with an increase in Rin for the 2, but not the 1 group. Lines are drawn to show tendencies. Symbols in C and D are the same as in A and B.

The slowing of inactivation by _2a has been attributed to palmitoylation of cysteines at position 3 and 4 in the _2a subunit (Qin et al., 1998). Modulation of this post-translational modification can thus be a reason for variations in the voltage-dependence and kinetics of inactivation (Chien et al., 1998; Hurley et al., 2000; Qin et al., 1998). To assess this, we have injected cDNA of the _2a subunits mutated at cysteines 3,4 (₂C3, 4S subunit) with the _1A and ₂   subunits and analyzed the inactivation parameters of the expressed channels. Mutation of Cys3,4 to Ser decreases R_in to values normally recorded with the _1A+ ₂   subunits expressed alone or with _1b (Fig. 2 B, diamond). The mutation also shifts E_in toward hyperpolarized values to varying degrees. Thus, the effect of the mutation on the _2a subunits seems to transfer the variability in R_in associated to _2a subunit to variability in E_in normally recorded with the _1b subunit, suggesting that variations of these two parameters arise from a common mechanism. Further analysis of the role of Cys3,4 was done by expressing a chimera where a transmembrane CD8 domain was added at the N terminus of this ₂C3,4S mutated subunit (CD8-₂C3, 4S subunit). This subunit is able to slow _1A channel inactivation without being palmitoylated (Restituito et al., 2000). Interestingly, this CD8-₂C3,4S subunit expressed with the _1A + ₂   subunits gave slowly inactivating channels that behaved exactly like the _2a subunit, i.e., displaying large variations in their R_in values but low variability in E_in (Fig. 2 B, inverted triangle). Such results clearly argue against any participation of the palmitoylation of the _2a subunit in the variability recorded with R_in as can be seen in other systems (Hurley et al., 2000).

Inspection of the ability of these subunits to modulate the activation properties of the _1A subunit (Fig. 2 C) has been done by plotting E_in versus the potential for half-activation (V_act) for each oocyte. As seen in Fig. 2 C, all subunit combinations are capable of hyperpolarizing the voltage-dependence of activation of the channel. In the case of the _1b group, however, this shift was graded with the hyperpolarizing shift in E_in. In the case of the ₂ group, almost no change in E_in could be recorded. Similarly, when R_in was plotted versus V_act (Fig. 2 D), the same type of relation appeared, i.e., the hyperpolarizing shift in V_act was graded with the increase in R_in value for the ₂ group, but not for the ₁ group. The _1A group (open square) still displays a low degree of variability.

One obvious explanation for these large variations in E_in and R_in is the variable degree of expression of auxiliary  subunit between different oocytes. In this case, since the primary effect of  subunits is to increase channel expression, the magnitude of the effect (shift in E_in or increase in R_in) is expected to be correlated to the amplitude of the current, because this latter parameter is proportional to the number of functional channels expressed. Plots of E_in and R_in values against current amplitude (Fig. 3, A and B) for these different mutations clearly show that this was not the case, and the lack of clear relation between changes in E_in and R_in and current amplitude therefore argues against a deficit in  subunit expression as a possible mechanisms to account for the observed effects. For the same reasons, any technical artifacts due to a poor voltage-control also appears very unlikely in these oocytes. Moreover, the fact that small R_in values in the ₂ group have E_in comparable to the _1A group (Fig. 2 A), suggest that these variations are not due to over-expression of an endogenous ₃ subunits (Tareilus et al., 1997) which one would expect to also hyperpolarize E_in.

View larger version (34K): [in this window] [in a new window]   FIGURE 3   (A and B) Scatter plots of Ein (A) and Rin (B) versus the maximum amplitude of the corresponding current. In each case variations in Ein (for the 1 group, A), or Rin (for the 2 group, B) were not related to the amplitude of the current suggesting that deficient expression of the channel auxiliary subunits was not the cause of the variability. (C and D) Individual and averaged Ba2+ current amplitude (C) and inactivation (Rin, D) recorded on oocytes expressing different ratios of 1A/2 subunits cDNA. Ten nl of a v/v mixture of 1A, 2 (at a concentration of 0, 0.1, or 1 µg/µl, as indicated) and 2   (1 µg/µl) subunit cDNAs were injected into oocytes, and currents were recorded 3-4 days later. (E) Lack of correlation between current amplitude and Rin for oocytes injected with ratios of 1A/2a cDNA giving the largest variability in inactivation (1/1, inverted triangle, and 1/0.1, diamond).

Indeed, the slow inactivation induced by the _2a subunit was never observed when _2a cDNA was not injected. In such conditions, expression was low (0.2-1 µA, see ₁/ ₌ 0.1/0 and 1/0, Fig. 3 C) and inactivation was fast (low R_in values, Fig. 3 D). Similar values are obtained for two concentrations of _1A subunit cDNA injected (0.1 and 1ng/nl, p > 0.05), suggesting that functional expression of this subunit was already saturating at the lowest cDNA concentration. Co-injection of _2a subunit cDNA, either at low (0.1) or high (1) concentration induced a significant increase in current amplitude (Fig. 3 C) and a slowing of inactivation (p < 0.05; Fig. 3 D). Both effects were recorded for the two _1A concentrations (₁/₌ 0.1/1 and 1/1, Fig. 3 C). However, whereas variability in current amplitude was recorded at all the concentrations of the _1A and _2a cDNA tested (maximum and minimum amplitude values were almost identical in all cases, ₁/ ₌ 0.1/1; 1/1 and 1/0.1), variability in inactivation (R_in) was more prominent for the highest _1A/_2a subunit ratio (1/0.1), and only slow-inactivating currents were recorded at ₁/ ₌ 0.1/1. The fact that different degree of regulation of inactivation by the _2a subunit can be recorded at two concentrations of _1A, (0.1 and 1, both saturating for expression of functional channels), and that this regulation did not correlate with the expressed current amplitude using two different  subunit concentrations (see 1/0.1, inverted triangle; and 1/1, diamond; n = 67 and 119, respectively, Fig. 3 E) suggested that the individual variation was not due to inter-oocyte variability in the level of expression of the _2a subunit.

This hypothesis was tested directly by coupling the electrophysiological measurement with the immunochemical evaluation of the _2a subunit expression. Oocytes were injected with a _1A/_2a subunit:cDNA ratio of 1:1, and Ba²⁺ currents were recorded 2-3 days later. Current amplitude and inactivation (R_in) were then estimated and allowed the selection of the oocytes into two groups (fast and slowly inactivating). Oocytes were then homogenized, and analyzed by SDS-page electrophoresis and Western blot using a anti- antibody. Bottom of Fig. 4 A shows averaged current amplitude and inactivation of these two groups (n = 5 for each group). Despite differences in inactivation, the amplitude of each group was very similar. Moreover Western blot analysis (3 oocytes/line, representing ~5 µA of current) showed that the level of expression of the _2a subunit was almost identical between the fast and slowly inactivating oocytes, and clearly distinct from the non-expressing or non-injected oocytes. Therefore, neither defect in palmitoylation, nor deficient expression of the _2a subunit, could explained the observed changes in inactivation.

View larger version (23K): [in this window] [in a new window]   FIGURE 4   (A) Oocytes with currents showing fast or slow inactivation expressed the same level of 2a subunit. Oocytes were injected with 10 nl of cDNA with a 1A/2a ratio of 1/0.1. Three days later, oocytes were selected in 3 batches according to the properties of their Ba2+ currents (n.e., no expression; slow, current with an Rin > 0.5; fast, current with Rin < 0.5). For each batch, averaged current amplitude and inactivation were recorded (see histogram). Oocytes were then homogenized, centrifuged and processed for Western blotting using an anti-com antibody (3 oocytes/line corresponding to ~5 µA; n.i, non-injected). The mean current amplitude and Western blot suggest similar levels of expression of the 1A and 2a subunit in the fast and slow oocytes. (B) Typical current traces recorded from oocytes expressing 1A + 2   or 1A + 2   + 2a subunits before and at the steady state effects of DAMGO (*, 10 µM). Holding potential was 80 mV. The second test pulse (0 mV, see voltage protocol at top) is preceded by a prepulse at +100 mV that allow partial relieve of the inhibition induced by DAMGO. Note the differences in inactivation kinetics and block by DAMGO between 1A + 2 and 1A + 2   + 2a oocytes. Scale bar, 0.5 µA. (C) Scatter plots of the percentage of inhibition of the Ba2+ currents recorded during the perfusion of the µ opioid agonist DAMGO (10 µM), and the inactivation properties of the corresponding current. Inactivation was quantified by calculating Rin as above. Two different combinations of subunits were studied 1A + 2   and 1A + 2   + 2a. In the case of 1A + 2   + 2a, note the correlation between Rin and the magnitude of the inhibition by DAMGO.

Dissociation of a variable proportion of the auxiliary  from _1A subunits, after targeting of the pore-forming subunit to the membrane, may also cause these effects. Indeed, these two different functions of the  subunits have recently been shown to be due to different and independent interaction sites (Gerster et al., 1999) and reversible 1/ interaction has been proposed (Bichet et al., 2000; Gerster et al., 1999). This "unbinding" hypothesis can be tested by analyzing the regulation of the Ba²⁺ currents by G proteins. G protein-induced inhibition of the _1A Ca²⁺ channel has been shown to be greatly reduced by co-expression of  subunits (Bourinet et al., 1996), and can therefore be used as an index of association between the _1A and the  subunits. Oocytes were co-injected with RNA encoding the µ opioid receptor and cDNA of _1A and ₂ subunits alone or with the _2a subunit. G protein activation was achieved by superfusion of 10 µM DAMGO, a specific µ opioid agonist, and both the level of inactivation of the Ba²⁺ current (determined as R_in) and the extend of G protein block of the current were calculated. Fig. 4 B shows that perfusion of DAMGO on oocytes expressing _1A+₂ subunits induced a pronounced inhibition (70%) of the rapidly inactivating Ba²⁺ current. This inhibition was voltage-dependent, as it could be partially reversed by applying a 50-ms prepulse to positive voltages (+100 mV). When the same experiment was performed on oocytes expressing the _1A + ₂   + _2a subunits, current inhibition induced by DAMGO was greatly reduced, especially on those oocytes that have the slowest inactivation kinetics (see bottom of Fig. 4 B). A scatter plot of this inhibition versus the inactivation parameter R_in (see Materials and Methods) for different oocytes is shown on Fig. 4 C. Both, the fast inactivation kinetics and the marked inhibition by DAMGO recorded with _1A + ₂   were systematically recorded on these batches of oocytes, and therefore values for R_in and percentage of inhibition by DAMGO display a low dispersion (_1A group, Fig. 4 C). However, on oocytes injected with _1A + ₂   + _2a cDNA, values for both inactivation (Fig. 1) and percentage of inhibition by DAMGO were more dispersed (₂ group, Fig. 4 C). In this group, the decrease in the inhibition by DAMGO was correlated to a slowing of inactivation. Again, these effects were not related to the absolute amplitude of the current, since G protein-sensitive or G protein-resistant currents of similar amplitude could be recorded from oocytes expressing the _1A + ₂   + _2a subunits (data not shown).

The simplest explanation for these results is to propose an unbinding of the auxiliary  subunit from the _1A. Such a dissociation removes the modulatory role of the  subunit on inactivation, as well as blockade of G protein modulation. Thus, for each oocyte, a mixed population of channels with or wihtout  subunit in variable proportions, could co-exist on the oocyte membrane. The magnitude of the  subunit effect may therefore result from the proportion of bound  subunits on the AID of channel. Because no clear relation can be found between the magnitude of this effect and current amplitude, we propose that the  subunit can unbind the channel after membrane targeting of the 1 subunits. Moreover, considering that fast or slow inactivating kinetics could be recorded for current of similar amplitudes in the same batch of _1A + ₂   + _2a-injected oocytes, lead us to suggest that this unbinding of auxiliary subunit does not affect the probability of opening of the channel, but only the transition to the inactivated state. Unbinding can occur for the ₁ or the ₂ subunits (as well as the different mutants presented) and is best revealed by the modulation of the inhibition by G proteins. Because the presence of a  subunit is an important determinant for channel regulation by G proteins, dissociation occurring in physiological conditions, therefore, represents a new pathway for modulating channel properties and regulation. Preliminary experiments using cAMP derivative and kinase inhibitors (W7, H89) suggest that PKA phosphorylation is not directly involved in this pathway. Recently, based on the different membrane distribution of the ₁ subunit when expressed alone or with the  subunit, it has been hypothesized that ₁/₂ interaction may results in secondary interactions with other cellular proteins (such as PDZ-domain-containing proteins; Gao et al., 1999) and stabilization of the complex at specific membrane location. Differential expression of such proteins between oocytes of a same batch may therefore constitute an interesting direction for future experiments. Whether  subunits remain bound to the ₁ subunit, using secondary interaction sites of minor importance regarding inactivation and G protein regulations, remains also to be determined.