Introduction

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The inhibition of Ca²⁺ influx through presynaptic Ca²⁺ channels by G proteins is an important mechanism for the modulation of synaptic transmission (Wu and Saggau, 1997; Miller, 1998). The Ca²⁺ channels involved in presynaptic Ca²⁺ entry are usually of the N, P/Q, and R-type (Dunlap et al., 1995; Reid et al., 1998; Wu et al., 1998). Cloned N-type (1B) Ca²⁺ channels have generally been found to be more sensitive to inhibition by G proteins than are P/Q- (1A) or R- (1E) type channels (Bourinet et al., 1996; Toth et al., 1996; Yassin et al., 1996; Zhang et al., 1996; Simen and Miller, 1998; but see Meza and Adams, 1998). These differences in sensitivity to inhibition by G proteins may therefore represent one mechanism mediating the efficacy of synaptic depression by presynaptic receptors.

The structural basis of G protein binding to Ca²⁺ channels has been the subject of a number of studies. G subunits have been shown to interact with two distinct regions in the I/II loop of nondihydropyridine (DHP)-sensitive Ca²⁺ channels, and one site also interacts with Ca²⁺  subunits (De Waard et al., 1997; Herlitze et al., 1997; Qin et al., 1997; Zamponi et al., 1997; Hamid et al., 1999). It has also been shown that the C terminus of Ca²⁺ channels can bind G (Qin et al., 1997) as well as G (Furukawa et al., 1998a, 1998b) subunits. Despite a clear role for the I/II loop region in G binding, this region does not seem to be primarily responsible for the differences in G protein sensitivity between the non-DHP sensitive Ca²⁺ channels (Page et al., 1997; Zhang et al., 1996). Similar to the findings of Zhang et al. (1996) and Stephens et al. (1998), we have previously demonstrated that the insertion of domain I from the human 1B Ca²⁺ channel into the human 1E channel yields a construct with significantly increased sensitivity to G proteins (Simen and Miller, 1998) and that the addition of the I/II loop and the N terminus increased inhibition further. The I/II loop region alone did not affect modulation. The involvement of the N terminus has recently been further defined (Page et al., 1998; Canti et al., 1999).

Although these studies suggest an important role of the N-terminal portion of the channel in determining sensitivity to modulation, it remains unclear what portions of this region are involved and how large their individual contributions are to the overall level of modulation of Ca²⁺ channels. We therefore examined the effects of transfer of regions between 1B (highly modulated) and 1E (minimally modulated) to determine sequences responsible for the differences in G protein sensitivity between the two channels. Our findings suggest that the N terminus alone contributes substantially to inhibition, a region extending from the S1/S2 loop to the S3/S4 loop of domain I is involved, and that a fragment of the I/II loop spanning the AID region and a downstream G binding region is also involved in mediating inhibition. The identification of the region of domain I involved is of particular interest because the residues involved are almost entirely extracellular and are therefore unlikely to directly interact with G proteins.

	Materials and Methods

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Chimeric Ca²⁺ channel 1 subunit constructs were created using methods described previously (Simen and Miller, 1998). Final constructs were confirmed by a combination of restriction analysis and DNA sequencing. The native human 1B construct consisted of residues 1 to 2340 of GenBank 2284339 (gift from Dr. R. Harpold, SIBIA Neurosciences, San Diego, CA). The native human 1E construct consisted of residues 1 to 2271 of GenBank 21082919 (gift from Dr. R. Harpold, SIBIA Neurosciences). The negative chimeras were named by appending the amino acids from 1E that were transferred into 1B to the letter "E," using the numbering system shown in Fig. 5. For example, the construct E120-132 consisted of 1B with amino acids 120 to 132 replaced with the corresponding amino acids from 1E. The positive chimeras were named in a similar fashion, by appending the residues from 1B that were transferred into 1E to the letter "B." For example, the construct B1-93 consisted of residues 1 to 93 from 1B in the 1E background. The construct 2037-2087 was created by replacing the cDNA coding for residues 2037 to 2087 of 1B with a HindIII restriction site, coding for the amino acids RL. The construct 1875-2339 was created by deleting the nucleotides coding for amino acids 1875 to 2339 of 1B. The construct BQ1 was created from 1B by site-directed mutagenesis to make the mutations Q383A, E386A, and R387A. The construct BQ2 was created from 1B by site-directed mutagenesis to make the mutations Q383A, Q384A, I385A, E386A, and R387A. tsA-201 cell culture, transfections, solutions for electrophysiological recording, and drug solutions were made as described previously (Simen and Miller, 1998). Cells were transfected with Ca²⁺ 2/, Ca²⁺ 1b, and wild-type or recombinant Ca²⁺ 1 subunits, along with the mouse -opioid receptor and CD8. The mouse -opioid receptor was a gift from Dr. Graeme Bell (Howard Hughes Medical Institute, University of Chicago, Chicago, IL). Currents were elicited by a dual-pulse protocol consisting of two 50-ms depolarizations (pulse 1 and pulse 2) to test potentials varying from 40 to +40 mV from a holding potential of 90 mV, separated by 800 ms at the holding potential, with a 30-ms, 90-mV depolarization ("prepulse") ending 5 ms before the second pulse (Fig. 1A), at an acquisition rate of 10 kHz and filtered with a four-pole Bessel filter at 2 kHz.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Examples of -opioid receptor effects on 1B and 1E Ca2+ currents. Native 1B and 1E Ca2+ channels were expressed in tsA-201 cells along with the 1b and 2/ Ca2+ subunits, the -opioid receptor, and CD8. A, currents were evoked with a dual-pulse protocol with two variable 50-ms test-pulses ranging from 40 to +40 mV separated by a 30-ms pulse to +90 mV ("prepulse"). B, top, normalized current amplitudes from a representative 1B expressing cell are shown. Currents in the presence of U69593 (a OR agonist) were compared with currents in the presence of norBNI (a OR inverse agonist/antagonist) to minimize constitutive receptor activity, as described previously (Simen and Miller, 1998). Currents were normalized to the largest observed current. The area under the current-voltage curves from the first pulse in the presence of norBNI (see legend) and U69593 (see legend), and the second pulse in the presence of norBNI (see legend) and U69593 (see legend) were calculated. Middle, Inhibition was defined in terms of the fractional difference between the normalized areas of the pulse 1 current in the presence of U69593 compared with norBNI. Bottom, facilitation was defined as the ratio of the areas of the pulse 2 currents in the presence of U69593 to the pulse 1 currents in the presence of U69593. Facilitation was adjusted for inactivation as described in Materials and Methods. C, top, representative current-voltage curves from an 1E expressing cell. Middle, integration of inhibition from the current-voltage curves shown in Fig. 1C, top (1E expressing cell). Bottom, integration of facilitation from the current-voltage curves shown in shown in Fig. 1C, top (1E expressing cell).

To facilitate comparison of the different recombinant channel constructs, cumulative integrals (CI) were computed on the current voltage data from each construct, and were defined as

(1)

where I(v) is the observed current amplitude at a particular test potential v. Current amplitudes were estimated from the observed maximum inward current during each 50-ms depolarization, and were scaled to values between 0 and 1 by dividing all four data vectors from each cell [+U69593, prepulse; +U69593, +prepulse; +nor-binaltorphimine (norBNI), prepulse; +norBNI, +prepulse] by the absolute value of the largest observed current. Points between the observed test potentials were interpolated by cubic spline interpolation, and the cumulative integrals (CI) were estimated by integration of the resulting cubic spline functions between 40 and +40 mV. Inhibition was then calculated as [CI(pulse 1, norBNI)  CI(pulse 1, U69593)] / CI(pulse 1, norBNI). Because no outward current was observed between these test potentials, changes in the resulting statistic sensitively reflect changes in inward current induced by G protein activation across the entire current voltage curve. Facilitation (corrected for inactivation) was calculated as [CI(pulse 2, U69593) / CI(pulse 1, U69593)] × [CI(pulse 1, norBNI) / CI(pulse 2, norBNI)]  1. Inactivation by the prepulse was calculated as 1  CI(pulse 2, norBNI)/CI(pulse 1, norBNI).

Facilitation and inhibition were also measured at individual test potentials by calculating an index of inhibition in terms of the simple current amplitudes (I) at each test potential. This index was computed as [I(pulse 1, norBNI)  I(pulse 1, U69593)]/I(pulse 1, norBNI) and an index of facilitation corrected for inactivation was calculated as [I(pulse 2, U69593)/I(pulse 1, U69593)] × [I(pulse 1, norBNI)/I(pulse 2, norBNI)] as described previously (Simen and Miller, 1998). Activation midpoints (V_1/2) for selected constructs were calculated by fitting the function I(v) / I_max = [G(v  V_rev)] / (1 + exp[(V_1/2  v)/K)], where I(v) is the observed inward current maximum at potential v and I_max is the absolute value of the largest observed inward current.

Statistical analyses were computed on inhibition and facilitation across the entire voltage range using one-way ANOVA followed by the Tukey multiple comparison procedure. These analyses were conducted separately for the three sets of constructs discussed (i.e., positive chimeras, negative chimeras, and chimeras involving G protein binding regions). Each ANOVA also included data from 1B and 1E for comparison.

	Results

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Quantification of OR Effects on Ca²⁺ Currents. We have shown previously (Simen and Miller, 1998) that the -opioid receptor has activity in the absence of agonist (constitutive activity), that the drug norBNI can suppress this activity (i.e., is an inverse agonist), and that norBNI and U69593 can be used to drive the receptor toward minimal activity and maximal activity, respectively, allowing examination of the full range of G protein effects on Ca²⁺ channels. We have also demonstrated that OR effects on 1B currents are completely sensitive to pertussis toxin and insensitive to protein kinase C blockers (Simen and Miller, 1998), are completely N-ethylmaleimide-sensitive, almost totally voltage dependent, and are not mediated by alterations in intracellular Ca²⁺ (A.A. Simen, R.J. Miller, unpublished observations).

View this table: [in this window] [in a new window]   TABLE 1 Inhibition and facilitation calculated across the entire voltage range Inhibition, facilitation, and statistical comparisons were calculated as described under Materials and Methods. Statistical significance with respect to 1B is denoted as B and statistical significance with respect to 1E is denoted as E.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Summary of inhibition (left) and facilitation (right), quantified across the entire current-voltage curve (Fig. 1; see Materials and Methods). Bars represent means, and error bars represent the standard error of the mean. Cartoon depictions of each construct (center) show only the N terminus to I/II loop and C-terminal region of the channel. Values significantly different from 1B are marked with *, and values significantly different from 1E are marked with ^ (4). See Table 1 for numerical values and statistical analyses.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Current-voltage curves of selected constructs. Error bars represent the standard error of the mean. Sample sizes are shown in Table 1. See Fig. 1 for examples of 1B and 1E current-voltage curves for comparison.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   The bottom panel shows sample currents from pulse 1 (before prepulse) at +10 mV from selected constructs (depicted in the top panel) in the presence of U69593 (a) and norBNI (b). Scale bars represent 500 pA and 100 ms.

Negative Chimeras. We have previously determined that a region of 1B extending from the N terminus to the I/II loop could confer high sensitivity to G protein-mediated inhibition when transferred into the 1E background (Simen and Miller, 1998), similar to the results of Stephens et al. (1998). Page et al. (1998) subsequently determined that the N terminus alone was partially responsible for these effects but could not itself bring inhibition up to the level seen in 1B, suggesting an important role for regions within domain I and/or the I/II loop. These findings with regard to the N terminus are consistent with our previous observation that an 1B/1E chimera bBbEeEeEe was modulated to a greater extent than a chimera eBbEeEeEe (Simen and Miller, 1998) lacking the 1B N terminus.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Alignment of the N terminus, domain I, and I/II loop region of 1B and 1E Ca2+ channels. Differences are shown in red, membrane-spanning regions, G binding regions, and G  subunit binding regions are shown enclosed in gray boxes. The ND1 region (see text) is shown in blue. G subunits interact with the AID and GID regions, and Ca2+  subunits interact with the AID region (see text). The majority of the differences between the two channels in the domain I region are in the extracellular loops (S1/S2, S3/S4, and S5/S6). Amino acid positions were numbered according to this alignment throughout the text.

Positive Chimeras. Our findings with respect to single and double negative chimeras suggest that a region in domain I extending from S1/S2 to S3/S4 (ND1 region) is involved in mediating inhibition and that the S3/S4 region may be of particular importance. In addition, our data are consistent with the AID-GID region of the I/II loop also playing an important role. To determine whether these regions are in fact sufficient to mediate 1B-like inhibition, we created a series of positive chimeras by transferring regions from 1B into the 1E background. The N terminus alone increased inhibition when transferred into the 1E background (construct B1-93; Table 1; Figs. 2, 3, and 4), consistent with the results of Page et al. (1998) as well as our own previous findings (Simen and Miller, 1998). The further exchange of the AID-GID region of the I/II loop increased inhibition to a small extent (construct B1-93;382-432; Fig. 2; Table 1). The further addition of the S3/S4 region had relatively little effect on inhibition (construct B1-93;185-190;382-432; Fig. 2; Table 1), suggesting that the S3/S4 region from domain I is not by itself sufficient, in contrast to our results with negative chimeras. However, when the ND1 region (domain I S1/S2 to S3/S4) was exchanged along with the N terminus and AID-GID region (construct B1-93;120-190;382-432) the resulting construct showed inhibition that was similar to that of native 1B (Figs. 2, 3, and 4; Table 1). This triple-positive chimera (construct B1-93;120-190;382-432) showed significantly more inhibition (P < .05) than the N-terminal chimera (construct B1-93), suggesting that 70 amino acids from domain I in the region from S1/S2 to S3/S4 and the AID-GID region of the I/II loop in addition to the N terminus contribute to inhibition when transferred into the 1E background. Because the domain I fragment transferred in this triple chimera lacked sequence from the 1B S4-S4/S5 region, the lack of involvement of the Val/Ile difference at position 215 (as suggested above) is strongly supported. Facilitation generally paralleled inhibition (Fig. 2; Table 1) and is discussed in detail below.

Mutations to G-Protein Binding Sites. These data together with a number of other studies (e.g., Page et al., 1997; Simen and Miller, 1998; Stephens et al., 1998) suggest that the differences between 1B and 1E in terms of G protein inhibition do not primarily involve regions thought to directly mediate G protein binding (such as the I/II loop) but are largely determined by regions not thought to mediate direct binding (such as regions in domain I and the N terminus). We were therefore interested in assessing the role of some of the known G protein binding regions in the 1B channel. Mutagenesis of the "QXXER" motif sequence "QQIER" (De Waard et al., 1997; Herlitze et al., 1997) in the AID region of the I/II loop of 1B to "AQIAA" (construct BQ1) or "AAAAA" (construct BQ2) had relatively little effect on inhibition (Fig. 2; Table 1). In fact, both constructs showed somewhat increased facilitation compared with 1B, but this difference was not statistically significant (Fig. 2; Table 1). These results are similar to those of Herlitze et al. (1997), who found that mutagenesis of the "QQIER" sequence of 1A to "AQIAA" increased facilitation to some extent. Deletion of the putative C-terminal G site "CC14" (construct 2037-2087; Qin et al., 1997) or truncation of the C terminus to delete the CC14 site as well as the putative G site (Furukawa et al., 1998b; construct 1875-2339) had relatively little effect on inhibition (Fig. 2; Table 1). These results may partially help to explain our previous observations that exchange of the I/II loop or C-terminal regions of 1B and 1E in isolation had little effect on inhibition (Simen and Miller, 1998). It seems that mutagenesis of these G protein sites in isolation is not sufficient to block inhibition of 1B by G proteins, and exchange of these regions would not be expected, therefore, to affect inhibition by virtue of any one of these particular sites. Because we did not mutate residues within the GID region, we cannot be certain of its role. However, our results are consistent with the notion that the GID region is an important locus of G interaction with the I/II loop. This is consistent with the high affinity of G for the C-terminal end of the I/II loop (De Waard et al., 1997) and the clear effects of phosphorylation (Zamponi et al., 1997; Hamid et al., 1999) and splice variants (Bourinet et al., 1999) in this region.

Voltage Dependence of Inhibition. The voltage dependence of inhibition of various Ca²⁺ channel constructs was examined by determining inhibition at various test potentials (Fig. 6). Two differences were noted between 1B and 1E (Fig. 6A). First, 1B channels showed a larger maximum level of inhibition than 1E channels. Second, both channels showed decreasing levels of inhibition at increasingly positive test potentials, but inhibition declined more rapidly for 1E channels than for 1B channels (Fig. 6A). When chimeric constructs were compared in this manner (Fig. 6B), two patterns emerged. The transfer of the N terminus of 1B into 1E (construct B1-93) caused little change in the maximum level of inhibition compared with 1E, but inhibition did not decline with increasing test potential. These results suggest that the N terminus may play a role in determining the voltage dependence of inhibition, and the domain I and I/II loop region may play a role in determining the maximum extent of inhibition. Consistent with this notion, transfer of the N terminus as well as regions from domain I and the I/II loop (ND1 and the AID-GID region) into 1E (construct B1-93;120-190;382-432) resulted in a construct with a maximum extent of inhibition similar to 1B as well as a slow decline of inhibition with increasing test potentials (Fig. 6B).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Inhibition was calculated at each test pulse potential as the fractional difference between the pulse 1 current in the presence of U69593 compared with the pulse 1 current in the presence of norBNI (see Materials and Methods). A, inhibition of 1B (see legend) compared with 1E (see legend) at individual test potentials. B, comparison of inhibition of constructs B1-93, B1-93;120-190;382-432, and 1E (see legend) at individual test potentials.

Facilitation. The degree to which a depolarizing prepulse to +90 mV relieved inhibition (i.e., caused facilitation) for a particular construct generally paralleled inhibition (Fig. 2; Table 1). In fact, the two parameters were highly correlated in an approximately linear fashion (Fig. 7A). However, some constructs showed more or less facilitation than expected given the degree of inhibition obtained and could be identified as "outliers" with respect to the regression relationship between the two parameters (Fig. 7A). One construct showed a disproportionately large amount of facilitation (construct 1875-2339), and other constructs showed disproportionately small amounts of facilitation (constructs B1-93;120-190, B1-93;120-190;382-432, and E250-377; Fig. 7A). These differences in facilitation are not caused by differences in current expression levels, because current levels did not differ significantly between the various constructs (p > .05). Although sequences from the N terminus, domain I, and the I/II loop effectively accounted for the differences in inhibition between 1B and 1E, the differences between these two Ca²⁺ channels in terms of facilitation were not adequately accounted for by these sequences in our experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   A, average inhibition and facilitation values from the constructs described in this study are shown with a linear regression fit. B, to assess the potential role of voltage dependent inactivation in "masking" facilitation, facilitation was compared with inactivation across the entire voltage range (see Materials and Methods). Averages and bivariate error bars representing standard errors are shown for constructs with high levels of inhibition (Fig. 3; Table 1) with very different levels of facilitation. C, sample currents before a prepulse (a) and after a prepulse (b) from selected constructs in the presence of norBNI are shown to illustrate differences in inactivation. Scale bars represent 500 pA and 100 ms.

	Discussion

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The results of the present study further define the region in the N-terminal portion of the Ca²⁺ channel responsible for the differences in inhibition between 1B and 1E Ca²⁺ channels to the N terminus, 70 amino acids from the N-terminal portion of domain I between S1/S2 and S3/S4, and 50 amino acids from the I/II loop. The 1B and 1E channels differ in the region from S1/S2 to S3/S4 at only 14 positions, and all but four of these positions are extracellular. Three of the four remaining differences are in transmembrane regions, and the one position localized cytoplasmically is a conservative Val/Ile difference in the S2/S3 loop (Fig. 5). The S2/S3 loop is only 11 amino acids long and therefore seems unlikely to be involved in G binding, although we cannot rule this out on the basis of our data. These results confirm that domain I plays an important role in determining sensitivity to inhibition by G proteins and that a relatively small region of domain I is sufficient to mediate these effects with regard to the differences between 1B and 1E. It is important to note that the chimera approach used in these studies is limited to identifying structures involved in G protein regulation that differ between the two Ca²⁺ channel isoforms and cannot identify regions that are homologous between the two channels. It is possible, therefore, that a larger portion of the Ca²⁺ channel plays a role in G protein regulation than we can resolve in these experiments.

Our findings with respect to the S3/S4 segment of domain I are complex. Negative chimeras suggested that the S3/S4 segment was sufficient to reduce inhibition significantly only in the "context" of changes to the I/II loop. However, positive chimeras suggested that this region was not sufficient to enhance inhibition. The S3 and S3/S4 segments of domain I have been shown to mediate the differences in activation kinetics between cardiac and skeletal muscle L-type Ca²⁺ channels (Nakai et al., 1994). The S3/S4 segment has also been shown to affect the activation kinetics of Shaker potassium channels (Mathur et al., 1997). It is possible therefore to speculate that this region could play some role in mediating the slowing of activation seen during G protein inhibition, although our data indicate that this region is not sufficient in itself to do so. Interestingly, there is a strong correlation between the length of the S3/S4 segment in domain I and sensitivity to G protein inhibition among Ca²⁺ channels [1A (8) = 1B (8)] > 1E (12) > 1C (24).

Our results with respect to the N terminus are consistent with those of Page et al. (1998), who have observed that exchange of the N terminus from 1B into 1E increases the inhibition of the resulting construct to a level intermediate between 1B and 1E. These results are also consistent with our previous observations that the exchange of the N terminus of 1B increased the modulation of a chimeric Ca²⁺ channel over and above that seen when domain I and the I/II loop were transferred alone (Simen and Miller, 1998). It is possible that the N terminus physically binds G proteins. However, Qin et al. (1997) observed that the N terminus of 1E channels does not bind G in vitro, suggesting that the N terminus may not be involved in direct G protein binding. It has recently been demonstrated that the N terminus of 1A has a low affinity Ca²⁺ 4 subunit binding site (Walker et al., 1999). It is possible therefore that the N terminus plays a role in the kinetic changes produced by G protein binding, perhaps by virtue of differential  subunit interactions.

Theoretically, G protein inhibition must involve an interplay between sequences that physically interact with G proteins (binding) and regions that are involved with producing the conformational changes associated with inhibition (efficacy). Residues in domain I may play some role in the immobilization of gating charge that follows G protein binding. Interestingly, the C-terminal end of the ND1 region (S3/S4 segment) is attached to the S4 voltage-sensing region of domain I. Therefore, this segment of domain I is positioned in such a way that it could directly affect voltage sensing of the channel, although other models are also possible. Garcia et al. (1997) showed that neutralization of single positive charges in the S4 segment of domains I and III but not domains II or IV of L-type Ca²⁺ channels altered activation kinetics. Although these findings have not yet been extended to non-DHP sensitive Ca²⁺ channels, they may suggest that domain I and III of Ca²⁺ channels play a particularly important role in determining channel activation. We therefore propose that regions in domain I may play a role in "efficacy" rather than in directly binding G proteins. Efficacy can be incorporated into models of G protein modulation by positing that Ca²⁺ channels bind G proteins but are not modulated until a distinct conformational change takes place. A recent article on the interpretation of mutagenesis data with respect to this binding/efficacy distinction has appeared (Colquhoun, 1998) and emphasizes the difficulties in distinguishing between effects on binding and effects on efficacy experimentally.

The apparent increase in facilitation caused by alanine mutagenesis of the AID region is consistent with previous findings (Herlitze et al., 1997). It has been demonstrated that alanine mutagenesis of the Q or R residues in the QXXER motif to A blocks binding of G to AID in vitro (De Waard et al., 1997). The small increase in facilitation resulting from alanine substitutions in the QXXER motif may suggest that G dissociation from this region is one step in the pathway leading to Ca²⁺ channel facilitation. The overall lack of effect of mutations to certain G protein binding regions with respect to inhibition is consistent with multiple regions on Ca²⁺ channels interacting with G proteins such that disruption of one site does not block inhibition or binding to the channel as a whole. Alternatively, these data can be interpreted as evidence for the notion that the GID region of the I/II loop is primarily responsible for G binding.

Because we carried out these experiments with the 1b subunit exclusively, we cannot be certain of the importance of the  subunit isoform in determining inhibition in our system. A number of studies have suggested that  subunits affect inhibition by G proteins (e.g., Campbell et al., 1995; Roche et al., 1995; Bourinet et al., 1996; Roche and Treistman, 1998a, 1998b). In addition, it has been shown that different  subunits support voltage-dependent inactivation to varying extents, with the 2a subunit supporting significantly less inactivation than other  subunits (e.g., Olcese et al., 1994; Qin et al., 1996). It is likely that the 1b subunit used in our study contributed to the degree of inactivation that we observed for 1E and some of our chimeric constructs. This inactivation probably exacerbated the degree of "masking" of facilitation that we observed (discussed above), as suggested by Meza and Adams (1998). It has been shown previously that a region extending from the S5/S6 loop of domain I to the proximal portion of the I/II loop contributes to the differences in inactivation between the ray 1E channel and the rabbit 1A channel (Zhang et al., 1994). We have shown previously that this also seems to be the case for human 1B and 1E Ca²⁺ channels (Simen and Miller, 1998). The enhanced inactivation of the E250-377 construct compared with 1B (Fig. 7C) provides further support for the involvement of this region in determining the differences in inactivation between 1B and 1E. Because our positive chimeras did not contain 1B sequence in this region, it is likely that they showed enhanced inactivation compared with native 1B as a consequence. This may account for the fact that we were unable to reconstitute facilitation in our positive chimeras. We cannot be certain whether the determinants of facilitation and inhibition are identical on the basis of our data.

The differences in sensitivity to inhibition by G proteins between 1B and 1E Ca²⁺ channels seem to involve contributions from the N terminus, a region of domain I extending from S1/S2 to S3/S4, and a region of the I/II loop containing Ca²⁺  and G binding regions. The differences in sensitivity to G protein inhibition between 1B and 1E channels therefore involves regions involved in G protein binding (the I/II loop) as well as regions that probably do not bind G proteins (N-terminal portion of domain I and the N terminus) that may play a role in determining conformation change as a consequence of G protein binding to other portions of the channel.