The Triad Targeting Signal of the Skeletal Muscle Calcium Channel Is Localized in the Cooh Terminus of the α_1S Subunit

Cell Culture and Transfections

Myotubes of the homozygous dysgenic (mdg/mdg) cell line GLT were cultured as described in Powell et al. 1996. At the onset of myoblast fusion (2–4 d after addition of differentiation medium), GLT cultures were transfected using DOTAP or FuGene (Boehringer). Cultures were analyzed 3–4 d after transfection.

Construction of Chimeric α₁ Subunits

The cDNA coding sequences of Ca²⁺ channel α_1S/α_1A subunit chimeras were inserted in-frame 3′ to the coding region of a green fluorescent protein (GFP), which was modified for thermal stability (Grabner et al. 1998) and contained in a proprietary mammalian expression vector (kindly provided by P. Seeburg, Zentrum für Molekulare Biologie, Heidelberg, Germany). Insertion of the cDNAs was accomplished as follows (nucleotide numbers, nt, are given in parentheses; asterisks indicate restriction sites introduced by PCR).

GFP and Immunofluorescence Labeling

Differentiated GLT cultures were fixed and immunostained as previously described (Flucher et al. 1994), using the monoclonal antibody 1A against the DHPR α_1S subunit at a final concentration of 1:1,000 (Morton and Froehner 1987), the affinity purified antibody #162 against the type 1 RyR at a dilution of 1:5,000 (Giannini et al. 1995), and a monoclonal or an affinity purified anti–GFP antibody at a dilution of 1:2,000 and 1:4,000, respectively (Molecular Probes, Inc.). Alexa- and fluorescein-conjugated secondary antibodies were used with the anti–GFP antibodies so that the antibody label and the intrinsic GFP signal were both recorded in the green channel. Texas red–conjugated antibodies were used in double-labeling experiments to achieve a wide separation of the excitation and emission bands. Controls (for example, the omission of primary antibodies and incubation with inappropriate antibodies) were routinely performed. Images were recorded on an Axiophot microscope (Carl Zeiss, Inc.) using a cooled CCD camera and Meta View image processing software (Universal Imaging, Corp.).

Quantitative analysis of the labeling patterns was performed by systematically screening the coverslips for transfected myotubes using a 63× objective. The labeling pattern of positive myotubes with more than two nuclei were classified as either “clustered,” “ER/SR,” or “other” in the case that the labeled compartment was not clearly identifiable. The counts were obtained from several samples of at least three different experiments for each condition.

Patch-Clamp and Fluorescent Ca²⁺ Recording

Whole cell patch-clamp recordings were performed with an Axopatch 200A amplifier controlled by pClamp 8.0 software (Axon Instruments, Inc.). The bath solution contained (mM): 10 CaCl₂ or 3 CaCl₂ plus 7 MgCl₂, 145 tetraethylammonium chloride, and 10 HEPES (pH 7.4 with TEA-OH). Patch pipettes had resistances of 2–4 MΩ when filled with 145 Cs-aspartate, 2 MgCl₂, 10 HEPES, 0.1 Cs-EGTA, 2 Mg-ATP (pH 7.4 with Cs-OH). Leak currents were digitally subtracted by a P/4 prepulse protocol. Recordings were low-pass Bessel filtered at 2 kHz and sampled at 5 kHz. Currents were determined with 200-ms depolarizing steps from a holding potential of −80 mV to test potentials between −40 and +80 mV in 10-mV increments. Test pulses were preceded by a 1-s prepulse to −30 mV to inactivate endogenous T-type Ca²⁺ currents (Adams et al. 1990).

Action potential–induced Ca²⁺ transients were recorded in cultures incubated with 5 μM Fluo-4-AM plus 0.1% Pluronic F-127 (Molecular Probes, Inc.) in HEPES and bicarbonate-buffered DME for 45 min at room temperature, as previously described (Flucher et al. 1994; Powell et al. 1996). Action potentials were elicited by passing 1-ms pulses of 30 V across the 19-mm incubation chamber. 0.5 mM Cd²⁺ and 0.1 mM La³⁺ were added to block Ca²⁺ influx and therefore allow discrimination between Ca²⁺-induced Ca²⁺ release and skeletal-type EC coupling. Application of 6 mM caffeine proved the functionality of SR Ca²⁺ release.

Reconstitution of Triad Targeting in Dysgenic Myotubes Transfected with the Skeletal Muscle Ca²⁺ Channel α_1S Subunit

Normal skeletal muscle in culture forms junctions between the SR and t tubules (triads and diads) and between the SR and the plasma membrane (peripheral couplings). These types of junctions are equivalent in function in that they support skeletal muscle type EC coupling, in molecular composition in that they contain RyRs in the SR and DHPRs in t tubules and plasma membrane, and in structure in that the two types of Ca²⁺ channels are organized in characteristic arrays of feet and tetrads, respectively. Therefore, we will henceforth use the terms “triad” and “triad targeting” in a generic sense to include all these types of junctions.

Dysgenic muscle lacks the α_1S subunit of the L-type Ca²⁺ channel, but still forms regular junctions containing RyRs (Powell et al. 1996). Transient transfection of myotubes of the dysgenic cell line GLT with an expression plasmid encoding a fusion protein of the GFP and the α_1S subunit (GFP-α_1S) restores expression of the α_1S subunit in the triads, L-type Ca²⁺ currents, and skeletal muscle EC coupling (present study, and Grabner et al. 1998). Reconstitution of dysgenic myotubes with α_1S is well established and the properties observed here with the NH₂-terminal GFP-fusion protein are similar to those previously reported with a COOH-terminal GFP-fusion protein (Flucher et al. 2000) or to wild-type α_1S expressed in dysgenic myotubes (Tanabe et al. 1988).

The triad localization of GFP-α_1S can be detected in double immunofluorescence labeling experiments with antibodies against the α_1S subunit and against the RyR (Fig. 1, a and b). Immunolabeling results in a punctate distribution pattern of anti–α_1S that is colocalized with similar clusters of anti–RyR. This clustered distribution pattern is characteristic of triad proteins in developing myotubes, and the coexistence of the t-tubule protein α_1S with the SR protein, the RyR, is indicative of their localization in junctions between the two membrane systems (Flucher et al. 1994). Myotubes not expressing GFP-α_1S form RyR clusters (Fig. 1 b), which have previously been shown to correspond to t tubule/SR junctions by electron microscopy (Powell et al. 1996).

Double immunofluorescence labeling of GFP-α_1S with anti–GFP and anti–α_1S (Fig. 1c and Fig. d) or with anti–GFP and anti–RyR (e and f) results in the same clustered distribution pattern in equally large fractions of GFP-α_1S–expressing myotubes (57 and 58%, respectively). Therefore, we continued using anti–GFP for the immunolocalization of GFP-α₁ constructs, because it recognizes both GFP-α_1S and GFP-α_1A, allowing the direct comparison of the labeling patterns of all chimeras used in this study. Fig. 1 e also shows a myotube in which the GFP-α_1S is expressed but its labeling pattern is not clustered. Instead it is distributed throughout a tubular membrane system that is very dense in the perinuclear region and has previously been identified as the ER/SR (Powell et al. 1996; Flucher et al. 2000). ER/SR distribution of heterologously expressed α₁ subunits can be observed with all constructs and occurs preferentially in poorly differentiated cells; i.e., myotubes in which triads are not formed. The myotube shown in Fig. 1e and Fig. f, for example, lacks RyR clusters, indicating that triad junctions, and thus the target for GFP-α_1S, was missing and therefore GFP-α_1S was retained in the biosynthetic apparatus. However, in addition to myotubes lacking the target for the α₁ subunit, retention in the ER/SR system can also be observed in normally differentiated myotubes if they are transfected with α₁ constructs lacking the triad targeting signal (see below).

Differential Targeting of the Skeletal and the Neuronal Ca²⁺ Channel α₁ Subunits Expressed in Dysgenic Myotubes

Fig. 2 shows a direct comparison of the labeling patterns of the skeletal muscle GFP-α_1S and the neuronal GFP-α_1A expressed in dysgenic myotubes. Whereas double immunolabeling with anti–GFP and anti–RyR reveals the colocalization of GFP-α_1S and RyR in the junctions (Fig. 2, a–c), GFP-α_1A is localized exclusively in the ER/SR network (d), even though the presence of RyR clusters (e) indicates the normal differentiation of junctions. The merged color images of GFP-α₁ in green and RyR in red further emphasizes the differential targeting of the skeletal and neuronal channels. Here, colocalization of GFP-α_1S with RyR shows up as yellow clusters (c), whereas the distinct localization of GFP-α_1A in the ER/SR and RyR in clusters is seen as separate green and red label, respectively (f). Quantification of the labeling patterns in at least six independent experiments showed that, while the overall expression of both constructs was the same, clusters were observed in 58% (n = 967) of the myotubes transfected with GFP-α_1S, but never in myotubes expressing GFP-α_1A (n = 418). Thus, GFP-α_1A fails to be incorporated into triad junctions.

To exclude the possibility that the absence of GFP-α_1A resulted from improper folding or lack of plasma membrane incorporation of the GFP-α_1A construct rather than lack of a triad targeting signal, we performed patch-clamp recordings of myotubes expressing this construct. Even though a plasma membrane stain was not detected with immunocytochemistry in GFP-α_1A–transfected myotubes, the whole-cell recordings showed large Ca²⁺ currents with the macroscopic properties of class-A Ca²⁺ channels expressed in heterologous mammalian expression systems (example shown in Fig. 6, below) (Adams et al. 1994). Thus, GFP-α_1A expressed in dysgenic myotubes formed functional channels in the cell membrane. But instead of becoming locally concentrated in the triads, GFP-α_1A was distributed diffusely in the plasma membrane at densities below detectability with immunocytochemistry.

Recordings of cytoplasmic Ca²⁺ transients in response to electrical field stimulation showed that GFP-α_1S regularly restored skeletal muscle EC coupling in dysgenic myotubes (see Fig. 7, and Powell et al. 1996; Flucher et al. 2000). In contrast, restoration of EC coupling by GFP-α_1A was only rarely observed (see below). Thus, both the skeletal muscle GFP-α_1S isoform and the neuronal GFP-α_1A isoform were functionally expressed in dysgenic myotubes, but only GFP-α_1S was targeted into the triad junctions and efficiently restored EC coupling. The differential distribution of GFP-α_1S and GFP-α_1A as well as their functional differences are in general agreement with observations from a previous study comparing the expression of GFP-α_1S, GFP-α_1A, and a cardiac GFP-α_1C construct in primary cultured dysgenic myotubes (Grabner et al. 1998). In that study, GFP-α_1A differed from the muscle isoforms in that its distribution patterns were restricted to near the injection site and that only GFP-α_1A failed to respond in a contraction assay. Together, these data support our hypothesis that the skeletal muscle α_1S contains a signal for its targeting and selective incorporation into triads, but that such a triad targeting signal is missing from the neuronal α_1A subunit isoform.

Localization of the Triad Targeting Site to the COOH Terminus of the α_1S Subunit

With a properly targeted GFP-α_1S and a nontargeted GFP-α_1A in our hands, we decided to start screening for the location of the targeting signal by replacing the prominent cytoplasmic portions of GFP-α_1S with the corresponding sequences of α_1A. GFP-α_1S/α_1A chimeras were generated with the following portions of α_1A (Fig. 3): the NH₂ terminus (GFP-α₁SkNa), the cytoplasmic loop connecting repeats I and II (GFP-α₁SkI-IIa) or that connecting repeats III and IV (GFP-α₁SkIII-IVa), and the COOH terminus (GFP-α₁Sa). Because the II-III loop of α_1A is more than three times the size of that of α_1S, we were concerned that it might impede appropriate incorporation of a chimera, not because of lacking a triad targeting signal, but because of sterical hindrance. Instead, the II-III loop of the house fly (M. domestica) α₁ subunit (Grabner et al. 1994) was used for constructing a II-III chimera (GFP-α₁SkII-IIIm). Its II-III loop has similar size as that of the rabbit skeletal muscle α_1S but shows very little sequence homology to α_1S and, like α_1A, the Musca α₁ subunit failed to be targeted into the junctions. Double immunolabeling with anti–GFP and anti–RyR and subsequent analysis of the targeting properties revealed that all of these chimeras with the exception of GFP-α₁Sa were clustered together with the RyR. The clustering efficiencies were between 50 and 65% (n = 200) for each construct, which was similar to that of GFP-α_1S (Fig. 3). Thus, neither the NH₂ terminus nor any of the major cytoplasmic loops of α_1S are essential for triad targeting.

The I-II loop contains the interaction domain for the β subunit (Pragnell et al. 1994). Association of the β subunit with this loop has been implicated in an important early step in membrane insertion of Ca²⁺ channels (Bichet et al. 2000). The I-II loop at least of α_1A seems to contain an ER retention signal that is blocked upon association with β to release the complex from the ER. Since the β interaction domain in the I-II loop is shared by all known α₁ subunits and a β subunit is endogenously expressed in dysgenic myotubes, a negative effect on triad targeting due to this mechanism was not to be expected with the GFP-α₁SkI-IIa chimera. The II-III loop of α_1S contains the sequence important for the interaction with the RyR. It specifies the tissue-specific mode of EC coupling (Tanabe et al. 1990a) and the amplification of Ca²⁺ currents by association with the skeletal type 1 RyR (Nakai et al. 1996; Grabner et al. 1999). Therefore, it is quite remarkable that replacement of the α_1S II-III loop by a loop as different as that of Musca's α₁ subunit had no adverse effect on triad targeting of chimera GFP-α₁SkII-IIIm. On the other hand, the finding that the molecular domain responsible for DHPR-RyR interactions is not essential for triad targeting is consistent with the observations showing that, in the RyR1 knock-out mouse, α_1S clusters in the junctions despite the lack of RyR1 (Takekura et al. 1995).

Replacing the COOH terminus of GFP-α_1S with that of α_1A (GFP-α₁Sa) disrupted triad targeting (Fig. 4, a–c). Similar to the distribution pattern described above for GFP-α_1A, GFP-α₁Sa was found in the ER/SR system, but never in clusters together with the RyR. Ca²⁺ currents in GFP-α₁Sa-transfected cells were very small and frequently below detectability. This is not surprising for skeletal Ca²⁺ channels not localized in the triad junctions. Nakai et al. 1996 and Grabner et al. 1999 have shown that skeletal muscle α_1S requires the specific interaction with type 1 RyR in the junctions for the expression of normal current densities. Both the absence of RyR1 in dyspedic myotubes and the interruption of the interaction between α_1S and the RyR resulted in a considerable attenuation of skeletal Ca²⁺ currents. Similarly, it is to be expected that in addition to decreased membrane insertion, the failure of triad targeting of GFP-α₁Sa and the associated lack of interactions with the RyR would result in the attenuation of Ca²⁺ currents.

The failure of triad targeting in the GFP-α₁Sa chimera suggests that the triad targeting signal may be contained within the COOH terminus of α_1S. To verify this interpretation, the corresponding reverse chimera, GFP-α_1A with the COOH terminus of α_1S (GFP-α₁As) was constructed. When expressed in dysgenic myotubes, GFP-α₁As was found coclustered with the RyR (Fig. 4, d–f). The efficiency of clustering was somewhat reduced compared with wild-type GFP-α_1S (28% of 1,479 transfected myotubes from eight separate experiments; Fig. 3); however, it was clear that by replacing its COOH terminus with that of α_1S, this otherwise class-A channel gained the ability to be targeted into the triad junctions. Ca²⁺ currents with properties similar to those of GFP-α_1A were expressed (see Fig. 6 b), indicating that this channel chimera was functional. Since swapping the COOH termini of α_1S and α_1A conferred triad targeting properties to the neuronal isoform (GFP-α₁As) and disrupted triad targeting in the skeletal muscle Ca²⁺ channel isoform (GFP-α₁Sa), it was evident that the signal responsible for this specific localization must be contained in the COOH terminus of α_1S.

Localization of the Triad Targeting Signal within the COOH Terminus of α_1S

The skeletal muscle α_1S subunit isolated from muscle preparations exists in two size forms, the minor fraction corresponding to the full-length α_1S sequence and the major fraction corresponding to a COOH-terminally truncated α_1S, lacking the sequences distal to approximately residue 1661 (De Jongh et al. 1991). Although truncation occurs after incorporation of α_1S into the junctions (Flucher et al. 2000), the truncated form by itself is sufficient to restore skeletal muscle EC coupling in dysgenic myotubes (Beam et al. 1992), suggesting that it is correctly inserted into the junctions. Therefore, it seemed unlikely that the triad targeting signal resides in the part of the COOH terminus distal to the putative clipping site. Sequence comparison of different α₁ subunit isoforms showed that the first 140 residues of the COOH terminus are highly homologous, followed by a stretch of similar length with much lower sequence homology. Thus, we concentrated our search for the triad targeting signal on the stretch in between the highly homologous region and the putative clipping site of α_1S. We created one chimera (GFP-α₁Aas) with the skeletal sequence 1524–1661 substituted for the corresponding region of α_1A, and two daughter chimeras, each containing one half of that region from α_1S (see Fig. 6 a). GFP-α₁Aas_(1524-1591), which contains the proximal half of this region from α_1S (1524–1591) and the distal half from α_1A, ends at an arbitrary site of α_1A corresponding to the location of the clipping site in α_1S, because α_1A does not contain this putative clipping site itself. GFP-α₁Aas_(1592-clip) is neuronal up to residue 2039 of α_1A, with the distal part of α_1S, from residue 1592 to the putative clipping site (1661).

Fig. 5, a–c, shows that GFP-α₁Aas was correctly targeted into the triad junctions. 38% (n = 1,602) of the transfected myotubes showed a clustered distribution of GFP-α₁Aas colocalized with RyR immunolabel. This confirmed that the region beyond residue 1661, which can be subject to truncation, is not necessary for triad targeting. Rather, the triad targeting signal is contained in the sequence between residues 1524 and 1661 of α_1S. Within this region, the proximal half did not confer triad targeting properties to α_1A. Not a single myotube out of 887 GFP-α₁Aas_(1524-1591)–transfected myotubes showed a clustered distribution pattern of this α₁ chimera; instead, GFP-α₁Aas_(1524-1591) was regularly found in the ER/SR (Fig. 5, d–f). In contrast, its sister chimera GFP-α₁Aas_(1592-clip) was efficiently targeted to the junctions (Fig. 5, g–i). 41% (n = 1,076) of the transfected myotubes showed a clustered distribution of GFP-α₁Aas_(1592-clip) colocalized with the RyR, indicating that this 70 amino acid sequence contains the triad targeting signal. To further restrict the region containing the triad targeting signal, one more chimera was created with only the last 55 residues proximal to the putative clipping site from α_1S (GFP-α₁Aas_(1607-clip)). This construct was also found in clusters in 47% (n = 603) of the transfected myotubes (Fig. 6 a; immunofluorescence image not shown), demonstrating that the 55 amino acid segment between residues 1607 and 1661 of the skeletal muscle α_1S is sufficient to confer triad targeting properties to the neuronal α_1A subunit.

Comparing this sequence of α_1S with the corresponding sequences of the cardiac α_1C, which is also targeted to triads (Grabner et al. 1998), and with that of the nontargeted α_1A reveals surprisingly few residues that are conserved between α_1S and α_1C but distinct from α_1A (Fig. 6 a). However, replacing individual of these residues with alanin did so far not result in a loss of targeting properties (data not shown). Either a targeting motif within this 55 amino acid stretch of α_1S is made up of more than those residues conserved between α_1S and α_1C, or the signal that is contained in this sequence stretch of α_1S is located outside the corresponding region of α_1C (see below). To distinguish between these possibilities, the corresponding targeting signal in α_1C and other α₁ isoforms needs to be localized and extensive single and combinatorial amino acid mapping needs to be performed.

The importance of COOH-terminal sequences in targeting and immobilization in specialized neuronal membrane domains has been demonstrated for ligand- and voltage-gated ion channels (Sheng and Pak 1999; Lim et al. 2000). To our knowledge, the sequence between 1607 and 1661 of α_1S contains only one known consensus protein binding site. The sequence SPV in position 1640–1642 corresponds to the PDZ-binding motif S/TXV; however, it is not positioned at the very COOH terminus as is the case in the majority of reported PDZ-binding proteins (Sheng 1996). Using a yeast–two-hybrid assay, Proenza et al. 2000 recently observed that this motif is part of a highly reactive region in the COOH terminus of α_1S and that substitution of the valine within this motif by aspartate abolished the high reactivity. This makes the PDZ-binding motif within the region demonstrated to contain the triad targeting signal in the present study a good candidate for protein–protein interactions that may contribute to triad targeting. However, the corresponding sequence in α_1C lacks the critical valine of this motif. Thus, if binding to PDZ proteins were the mechanism of triad targeting, other PDZ-binding motifs located elsewhere in the channel had to be responsible for the same function in the cardiac isoform. Two such motifs exist in the COOH terminus of α_1C, however, immediately distally to the putative clipping site. Other evidence for the importance of the COOH terminus in membrane targeting comes from heterologous expression of the cardiac α_1C in tsA201 cells (Gao et al. 2000). However, the region identified in that study to be important for functional membrane expression of α_1C is in the proximal, highly homologous part of the COOH terminus, ending 69 amino acids upstream of the region corresponding to the triad targeting signal identified in our study using a gain-of-function approach. Assuming that the loss of function in response to COOH-terminal truncations and deletions arose from specific effects on the targeting process rather than from nonspecific damage to the expressed channel, this domain is most likely not involved in the highly specific insertion of the channel into triad junctions, but may be important for a more general aspect like the export from the ER or membrane incorporation. Our present results do not exclude the possible contribution of other signals, shared by α_1S and α_1A, to the complex process that culminates in triad targeting.

Effects of Triad Targeting of GFP-α₁Aas_(1592-clip) on Ca²⁺ Currents and EC Coupling

Comparison of the current properties of GFP-α_1A and GFP-α₁Aas_(1524-1591), both of which are not targeted into the triad, with that of the targeted GFP-α₁Aas_(1592-clip) provides additional evidence for the existence of distinct mechanisms for membrane and triad targeting (Fig. 6b and Fig. c). Compared with GFP-α_1A, GFP-α₁Aas_(1524-1591) exhibited strongly reduced current densities, even though the immunolabeling experiments gave no indication that the transfection efficiency or the amount of protein expressed had been decreased. It was necessary to increase the concentration of the charge carrier from 3 to 10 mM Ca²⁺ (Fig. 6 c) to show that this chimera did in fact express functional channels in the membrane; however, at strongly reduced levels. This suggested that in this chimera a distinct signal important for membrane expression of α_1A had been abolished, but that this loss had not been compensated by the addition of the skeletal muscle triad targeting signal. To find out whether this putative membrane insertion signal of α_1A resides in the region replaced by the skeletal sequence (1524–1591) or whether it is contained in the distal portion of the COOH terminus that had been truncated in this chimera, we generated a truncated GFP-α_1A and expressed it in the dysgenic myotubes (data not shown). Current expression of this GFP-α_1A-clip was also low, suggesting that the distal COOH terminus of α_1A contains a separate signal that is important for its efficient membrane expression, but is not sufficient for triad targeting.

Considering the fact that our final triad-targeting chimeras lack this α_1A membrane-targeting signal, it is even more astonishing that the skeletal residues 1592–1661 not only fully compensated the loss of current expression caused by the truncation of α_1A, but that current densities of the targeted GFP-α₁Aas_(1592-clip) were increased by approximately twofold over those of the wild-type α_1A (Fig. 6 c). Among several possible causes for this effect, an increase in current density accompanying triad targeting is consistent with a model by which the specific incorporation into the triadic complex stabilizes the channel in the membrane, thus leading to an increased total number of functional channels.

Finally, to the question of how the localization of a Ca²⁺ channel in skeletal muscle affects EC coupling. The two muscle isoforms, α_1S and α_1C, which are both targeted into triads, have repeatedly been shown to rescue EC coupling in dysgenic myotubes (Tanabe et al. 1988 and Tanabe et al. 1990b; Grabner et al. 1998; Neuhuber et al. 1998b; Flucher et al. 2000). However, the mechanism by which the skeletal muscle α_1S and the cardiac α_1C activate SR Ca²⁺ release in dysgenic myotubes differs. In α_1S, it functions independently of Ca²⁺ influx, whereas α_1C does require Ca²⁺ influx for the activation of EC coupling. In Fig. 7, we show that dysgenic myotubes transfected with GFP-α_1S or GFP-α_1C respond to electrical stimulation with strong Ca²⁺ transients, which have previously been described as action potential–induced Ca²⁺ transients based on their all-or-none characteristics (Flucher et al. 1994; Powell et al. 1996). With GFP-α_1S, these Ca²⁺ transients continued after blocking the Ca²⁺ currents by the addition of Cd²⁺/La³⁺ to the bath solution, whereas with GFP-α_1C the Ca²⁺ transients ceased, confirming that this SR Ca²⁺ release was Ca²⁺-induced. Caffeine induced strong Ca²⁺ transients even after the Cd²⁺/La³⁺ block, indicating that the cessation of Ca²⁺ transients was not due to depletion of the SR Ca²⁺ stores or damage to the release mechanism.

Previous attempts to rescue EC coupling in dysgenic myotubes by expressing the neuronal α_1A failed to display (Grabner et al. 1998), or only very rarely displayed, evoked contractions (Adams et al. 1994), despite the fact that α_1A expressed sizable Ca²⁺ currents. Using a more direct method to monitor SR Ca²⁺ release with the fluorescent Ca²⁺ indicator fluo-4, we did observe rare action potential–induced Ca²⁺ transients in GFP-α_1A–transfected myotubes (Table and Fig. 7). While >25 responsive myotubes were found in almost all of GFP-α_1S– and GFP-α_1C–transfected cultures, on average only every fifth culture transfected with GFP-α_1A contained one or two responsive myotubes. The degree of restoration of EC coupling increased significantly (P = 0.002) when the class-A channel was targeted into the triad. In myotubes transfected with GFP-α₁Aas_(1592-clip), action potential–induced Ca²⁺ transients were observed in 60% of the cultures (Table ). As expected, these Ca²⁺ transients could be blocked with Cd²⁺/La³⁺, indicating that the mechanism by which GFP-α₁Aas_(1592-clip) restored EC coupling was Ca²⁺-induced Ca²⁺ release (Fig. 7).

The enhanced restoration of EC coupling by the targeting of a class-A channel into the triads shows the importance of the correct localization of the Ca²⁺ channel in close proximity to the SR Ca²⁺ release channel. Apparently, the large Ca²⁺ influx through α_1A distributed throughout the plasma membrane was not sufficient to induce Ca²⁺-induced Ca²⁺ release except in a few myotubes. However, concentrating the Ca²⁺ current to the restricted spaces of the triadic compartments strongly improved the chance of reaching the threshold for Ca²⁺-induced Ca²⁺ release. This interpretation is consistent with other cellular processes where the close proximity of a Ca²⁺ source and a Ca²⁺ target is required for normal function (e.g., the association of the RyR and the Ca²⁺-activated potassium channel in smooth muscle cells; Jaggar et al. 2000) and highlights the importance of specific targeting mechanisms for Ca²⁺ channels.

The more we learn about Ca²⁺ channels in their native environments, the more it appears to be the rule rather than the exception that they are specifically localized in functional domains. The signal contained in the 55 amino acid sequence of the COOH terminus of α_1S is the first description of a targeting signal of a voltage-gated Ca²⁺ channel for a specific membrane domain. It may share its anchoring mechanism with other ion channels that have been shown to interact with proteins containing PDZ domains, but for which the importance of this protein–protein interaction in the targeting process has yet to be shown. The complex passage of the skeletal muscle Ca²⁺ channel from the biosynthetic apparatus into the triad junction involves multiple steps. At the beginning of the journey, the interaction of the β subunit with the I-II loop and with the COOH terminus seems to play an important role in the export of the channel from the ER and for its functional expression in the plasma membrane. At the end of the journey, the specific interaction with the RyR determines the tetradic organization of the α_1S subunit that structurally sets it apart from the cardiac α_1C. But between these two events occurs the essential targeting of the Ca²⁺ channel into the junctional domain of the triad, and the signal contained within residues 1607–1661 of the skeletal muscle α_1S subunit is necessary and sufficient to confer this targeting property to a neuronal α₁ subunit.