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© The Rockefeller University Press, 0021-9525/2000//467 $5.00
The Journal of Cell Biology, Volume 151, Number 2, , 2000 467-478

The Triad Targeting Signal of the Skeletal Muscle Calcium Channel Is Localized in the Cooh Terminus of the _1S Subunit

^a Department of Biochemical Pharmacology, University of Innsbruck, A-6020 Innsbruck, Austria
Department of Physiology, University of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria.43-512-507-283643-512-507-3787

The specific localization of L-type Ca²⁺ channels in skeletal muscle triads is critical for their normal function in excitation–contraction (EC) coupling. Reconstitution of dysgenic myotubes with the skeletal muscle Ca²⁺ channel _1S subunit restores Ca²⁺ currents, EC coupling, and the normal localization of _1S in the triads. In contrast, expression of the neuronal _1A subunit gives rise to robust Ca²⁺ currents but not to triad localization. To identify regions in the primary structure of _1S involved in the targeting of the Ca²⁺ channel into the triads, chimeras of _1S and _1A were constructed, expressed in dysgenic myotubes, and their subcellular distribution was analyzed with double immunofluorescence labeling of the _1S/_1A chimeras and the ryanodine receptor. Whereas chimeras containing the COOH terminus of _1A were not incorporated into triads, chimeras containing the COOH terminus of _1S were correctly targeted. Mapping of the COOH terminus revealed a triad-targeting signal contained in the 55 amino-acid sequence (1607–1661) proximal to the putative clipping site of _1S. Transferring this triad targeting signal to _1A was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca²⁺-dependent EC coupling.

Key Words: calcium channel • dihydropyridine receptor • excitation–contraction coupling • immunofluorescence • skeletal muscle

© 2000 The Rockefeller University Press

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The precise localization of Ca²⁺ channels in specialized membrane domains is essential for their specific actions in multiple functions of excitable cells. In neurons, for example, voltage-gated Ca²⁺ channels located in the nerve terminal trigger neurotransmitter release and distinct populations of pre- and postsynaptic Ca²⁺ channels participate in different forms of synaptic plasticity (Berridge 1998). In muscle cells, voltage-gated Ca²⁺ channels are specifically located in the intracellular junctions between the Ca²⁺ stores of the sarcoplasmic reticulum (SR) and either the transverse tubules (t tubules) or the plasma membrane, called triads and peripheral couplings, respectively (Franzini-Armstrong and Jorgensen 1994), in which the Ca²⁺ channel initiates excitation–contraction (EC) coupling (Melzer et al. 1995). Even though voltage-gated Ca²⁺ channels play such important roles in vital cell functions, the signals and mechanisms that direct and immobilize the different voltage-gated Ca²⁺ channel isoforms into their specific membrane domains are not known.

The skeletal muscle L-type Ca²⁺ channel (also called dihydropyridine receptor, DHPR) is composed of four subunits, the pore-forming _1S and the accessory ₂/, β_1a, and ₁ subunits (Catterall 1996). The channel is concentrated in the junctional membranes of the triads (Jorgensen et al. 1989; Flucher et al. 1990), where it is in close contact with the Ca²⁺ release channels (ryanodine receptors, RyR) of the SR Ca²⁺ stores. Freeze-fracture analysis showed that in the triad junctions the DHPRs are arranged in square groups of four integral membrane particles called the tetrads and that their distribution pattern corresponds to that of the RyR arrays in the opposite SR membrane (Block et al. 1988). It is believed that the skeletal muscle DHPR functions as the voltage sensor for the gating of the SR Ca²⁺ release channel by a mechanism that is independent of Ca²⁺ influx through the L-type channel (Ríos et al. 1992). Thus, the highly orderly arrangement of DHPRs and RyRs in the triads is the structural basis for the depolarization-induced SR Ca²⁺ release in skeletal muscle EC coupling.

But what are the mechanisms by which the Ca²⁺ channels are specifically targeted into the triad junction and by which they achieve their characteristic organization? In skeletal muscle of the dysgenic mouse, which lacks the ₁ subunit of skeletal muscle DHPR, triads form and RyRs are normally incorporated in these junctions in the absence of _1S (Powell et al. 1996). Conversely, in myotubes of a skeletal RyR knock-out mouse, triads are also formed and DHPRs aggregate in these junctions, despite the absence of the RyR (Takekura et al. 1995); however, their arrangement in tetrads fails (Protasi et al. 1998). This suggests that triad targeting and tetrad formation are two independent processes and that interactions with the RyR are not necessary for the targeting of the DHPR into the junctional membrane domain of the triad. Evidence from studies in heterologous expression systems showing that coexpression of the DHPR ₁ subunit with β and ₂/ increases membrane insertion of functional Ca²⁺ channels (Chien et al. 1995; Brice et al. 1997; Neuhuber et al. 1998a; Walker et al. 1998) suggests a role of the accessory Ca²⁺ channel subunits in the targeting process. Moreover, Ca²⁺ currents and EC coupling are deficient in skeletal myotubes of β-null mice and both functions can be reconstituted by heterologous expression of β_1a (Gregg et al. 1996; Beurg et al. 1997). Thus, expression of the β subunit is important for the efficient expression and functional insertion of the Ca²⁺ channel in the membrane, but not necessarily for its targeting into triads. Immunolocalization of ₂/ and of recombinant β_1a expressed in dysgenic myotubes showed that, without the _1S subunit, both subunits failed to be localized in the junctions (Flucher et al. 1991; Neuhuber et al. 1998b). Instead, the β and ₂/ subunits required coexpression of _1S for their own incorporation into the triads, suggesting that their own triad targeting is secondary to that of the _1S subunit and that β and ₂/ do not posses an independent targeting signal. The role of the subunit of the skeletal muscle Ca²⁺ channel is still poorly understood. However, the fact that EC coupling is not perturbed in skeletal muscle of a knock-out mouse also suggests that this subunit is not required for a function as important as the targeting of the Ca²⁺ channel complex into the triad (Freise et al. 2000). Thus, considering that the RyR and the accessory DHPR subunits either play no role in triad targeting of the DHPR or depend on the _1S subunit for their own targeting into the junctions, the signal for triad targeting is likely to be contained within the _1S subunit itself.

Here, we used heterologous expression of different ₁ subunit isoforms and isoform chimeras in dysgenic myotubes to identify the targeting signal in the skeletal muscle DHPR. Taking advantage of differential targeting properties of the skeletal muscle _1S and the neuronal _1A subunit, we generated a series of _1S/_1A chimeras and used them to localize the triad targeting signal in the COOH terminus of _1S. The 55 amino acid sequence of _1S, which is sufficient to confer triad targeting properties to _1A, is the first description of a targeting signal of a voltage-gated Ca²⁺ channel to its native membrane domain.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
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 ₁ Subunits
The cDNA coding sequences of Ca²⁺ channel _1S/_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-_1S, GFP-_1A.
Construction of the GFP-tagged _1S (Tanabe et al. 1987) and _1A (Mori et al. 1991) subunits that were used as maternal clones for chimerization are described elsewhere (Grabner et al. 1998).

GFP-₁SkNa.
The _1A (A) cDNA coding for the NH₂ terminus was fused to the _1S (Sk) cDNA at position (nt A294/Sk154) using the "gene SOEing" technique (Horton et al. 1989). The SalI*-SacI fragment (nt 5' polylinker-Sk651) of the cDNA product generated by the fusion PCR was coligated with the SacI–BglII fragment of Sk (nt 651–4488) into the corresponding SalI*/BglII restriction enzyme (RE) sites of plasmid GFP-_1S.

GFP-₁SkI-IIa.
The HindIII–EcoRI fragment of Sk (nt 5' polylinker-1007) and the EcoRI*-SphI A/Sk SOE fusion product (nt A1085–Sk1735) with the A/Sk transition at position (nt A1461/Sk1297) were coligated into the HindIII/SphI-cleaved polylinker of plasmid pSV-Sport1 (Life Technologies.). The SbfI–SphI fragment (nt 5' polylinker-Sk1735) of this intermediate clone was coligated with the SphI–XhoI fragment of Sk (nt 1735–2654) into the corresponding SbfI/XhoI RE sites of plasmid GFP-_1S.

GFP-₁SkII-IIIm.
The EcoRI–BalI fragment of Sk (nt 1007–1973) was coligated with the BalI–NdeI fragment (nt 1982–2296) from the muscle ₁ subunit (M) of Musca domestica (Grabner et al. 1994) into plasmid pSP72 (Promega) using the internal NdeI site (plasmid nt 2379) and the EcoRI site of the polylinker. The NdeI/EcoRI RE sites of pSP72 were also used to coligate two cDNA fragments, the NdeI*/XhoI fragment that was PCR generated from clone SkLC, a GFP-_1S with a cardiac (C) II-III loop (nt C2716–Sk2654) (Grabner et al. 1999) plus the XhoI/BglII fragment of Sk (nt 2654–4488). The NdeI* primer was designed to introduce downstream of the NdeI* site additional Musca residues, A907G and S908T. In a subsequent step fragments EcoRI–NdeI (nt Sk1007–M2297) and NdeI*–BglII (C2716–Sk4488) were isolated from the pSP72 subclones and coligated into the EcoRI/BglII-cleaved pSP72 vector. Finally, the SalI–EcoRI fragment of Sk (nt 5' polylinker-1007) was coligated with the EcoRI–BglII fragment (nt Sk1007–Sk4488) from the last pSP72 subclone into the SalI/BglII sites of plasmid GFP-_1S.

GFP-₁SkIII-IVa.
The III-IV loop of the A cDNA was inserted into the corresponding Sk cDNA by a three-fragment SOE fusion PCR, thereby generating the transitions Sk/A (nt Sk3195/A4561) and A/Sk (nt A4725/Sk3355). The final PCR product was cleaved at its peripheral Sk XhoI/BglII RE sites and the resulting fragment (nt 2654–4488) was ligated into the corresponding XhoI/BglII sites of plasmid GFP-_1S.

GFP-₁Sa.
The XhoI–SmaI fragment of Sk (nt 2654–4038) and the SmaI–BglII Sk/A cDNA fusion fragment (nt Sk4038–A5891) with the Sk/A transition (nt Sk4143/A5461) created by SOE PCR were coligated into the XhoI/BglII RE sites of plasmid GFP-_1A (nt 1395/5891). Note that the XhoI sites are not corresponding RE sites and were used for subcloning only. Finally, the HindIII–XhoI fragment of Sk (nt 5' polylinker-2654) was inserted into this HindIII/XhoI (nt 5' polylinker/A1395, Sk2654) opened subclone to yield plasmid GFP-₁Sa.

GFP-₁As.
The Xho–AccI fragment of A (nt 1395–4504) was coligated with the A/Sk SOE fusion fragment AccI–BglII (nt A4504–Sk4488) carrying its A/Sk transition at nt A5460/Sk4144, into the XhoI/BglII (nt 2654/4488) cleaved plasmid GFP-_1S. Again, the A and Sk XhoI sites are not corresponding RE sites and were only used for subcloning. To yield GFP-₁As, the SalI–EcoRI fragment from A (nt 5' polylinker-1567) was coligated with the EcoRI–BglII fragment (nt A1567–Sk4488) after isolation from the subclone into the SalI/BglII (nt 5' polylinker/4488) cleaved plasmid GFP-_1S.

GFP-₁Aas.
The PCR generated BglII*–XbaI* fragment of Sk (nt 4566–4991) was inserted into the corresponding BglII/XbaI RE sites of plasmid GFP-_1A (nt 5891/3' polylinker). Upstream from the artificial XbaI* site of the Sk fragment, two stop codons (nt 4984–4989) were introduced to terminate the reading frame at residue T1661, which is close to the physiological clipping site of the _1S carboxyl terminus (De Jongh et al. 1991).

GFP-₁Aas(1524-1591).
The BglII*–XbaI* Sk/A cDNA fusion fragment (nt Sk4566–A6347) with the Sk/A transition (nt Sk4773/A6118) generated by SOE PCR was ligated into the corresponding BglII/XbaI RE sites of plasmid GFP-_1A (nt 5891/3' polylinker). Again, two stop codons were introduced upstream of the artificial XbaI* site of the A portion of the fusion product (nt 6340–6345) to terminate the reading frame at residue G2113.

GFP-₁Aas(1592-clip).
The BglII–XbaI* A/Sk SOE fusion fragment (nt A5891–Sk4991) with the A/Sk transition at nt A6117/Sk4774 was ligated into the corresponding BglII/XbaI RE sites of plasmid GFP-_1A (nt 5891/3' polylinker).

GFP-₁A-clip.
The BglII–XbaI* fragment of A (nt 5891–6347) was ligated into the corresponding BglII/XbaI RE sites of plasmid GFP-_1A (nt 5891/3' polylinker). Stop codons were introduced as in plasmid GFP-₁Aas(1524–1591).

GFP-₁Aas(1607-clip).
The BglII–XbaI* A/Sk SOE fusion fragment (nt A5891–Sk4991) with the A/Sk transition at nt A6165/Sk4819 was ligated into the corresponding BglII/XbaI RE sites of plasmid GFP-_1A (nt 5891/3' polylinker).

All cDNA portions modified by PCR were checked for sequence integrity by sequence analysis (sequencing facility of MWG Biotech).

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 _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 63x 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 M 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.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Reconstitution of Triad Targeting in Dysgenic Myotubes Transfected with the Skeletal Muscle Ca²⁺ Channel _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 _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 _1S subunit (GFP-_1S) restores expression of the _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 _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 _1S expressed in dysgenic myotubes (Tanabe et al. 1988).

The triad localization of GFP-_1S can be detected in double immunofluorescence labeling experiments with antibodies against the _1S subunit and against the RyR (Fig. 1, a and b). Immunolabeling results in a punctate distribution pattern of anti–_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 _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-_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).

View larger version (111K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Normal incorporation of heterologously expressed GFP-1S in t tubule/SR junctions of dysgenic myotubes. (a and b) Double immunofluorescence labeling of 1S subunits (a) and RyRs (b) shows that GFP-1S is colocalized with RyRs in clusters (examples indicated by arrows), presumably representing triad junctions and peripheral couplings. A nontransfected myotube in the same field (#) shows RyR clusters but no 1S labeling. (c and d) Double staining of GFP-1S with antibodies against 1S and against GFP shows that the fusion protein can be localized using either one of the antibodies. (e and f) As with anti–1S (a), clusters labeled with anti–GFP (e) are colocalized with RyR clusters (examples indicated by arrows). In poorly differentiated myotubes that lack RyR clusters (*), GFP-1S accumulates in a reticular membrane system with densities in the perinuclear region, presumably the ER/SR network. N, nuclei. Bar, 10 µm.

Differential Targeting of the Skeletal and the Neuronal Ca²⁺ Channel ₁ Subunits Expressed in Dysgenic Myotubes
Fig. 2 shows a direct comparison of the labeling patterns of the skeletal muscle GFP-_1S and the neuronal GFP-_1A expressed in dysgenic myotubes. Whereas double immunolabeling with anti–GFP and anti–RyR reveals the colocalization of GFP-_1S and RyR in the junctions (Fig. 2, a–c), GFP-_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-₁ in green and RyR in red further emphasizes the differential targeting of the skeletal and neuronal channels. Here, colocalization of GFP-_1S with RyR shows up as yellow clusters (c), whereas the distinct localization of GFP-_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-_1S, but never in myotubes expressing GFP-_1A (n = 418). Thus, GFP-_1A fails to be incorporated into triad junctions.

View larger version (91K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Differential targeting of GFP-1S and GFP-1A in transiently transfected dysgenic myotubes. Double immunofluorescence labeling of the skeletal GFP-1S and the neuronal GFP-1A (using anti–GFP for both) and of the RyR shows that only GFP-1S is colocalized with RyR clusters (a and b; examples indicated by arrows). GFP-1A is not colocalized with RyR clusters (d and e), but is retained in a reticular cytoplasmic membrane system, the ER/SR (d). (c and f) The differential subcellular distribution of GFP-1S and GFP-1A is highlighted in a color overlay of the images of a and b, and d and e, respectively (insets at twofold magnification). Colocalization of GFP-1S (green) with RyRs (red) results in yellow clusters. In contrast, separate green GFP-1A and red RyR labeling indicates the lack of colocalization of GFP-1A and RyRs. Bar, 10 µm.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Targeting properties and current properties of wild-type 1 subunit isoforms and COOH-terminal chimeras. (a) Isoform sequence composition of COOH termini in the studied chimeras, with sequences of 1S in gray and 1A in black. Bar graph indicates the percentages of transfected myotubes showing triad targeting in immunofluorescence analysis. Alignment of the 55 1S amino acids containing the targeting signal with the corresponding sequences of 1C and 1A. (b) Representative current traces recorded from dysgenic myotubes transfected with wild-type GFP-1S (in 10 mM Ca2+), with GFP-1A or with the targeted chimera GFP-1As (both in 3 mM Ca2+). Currents from GFP-1Sa (in 10 mM Ca2+) were too small for systematic analysis; see frequency distribution of current densities. (c) Representative current trace of the targeted chimera GFP-1Aas(1592-clip) and comparison of peak current densities recorded from GFP-1A, GFP-1Aas(1592-clip), and GFP-1Aas(1524-1591). Substituting COOH-terminal 1A sequence with the skeletal sequence 1592–1661 results in a twofold increase of current density compared with GFP-1A, whereas substituting for skeletal sequence 1524–1591 reduces the current densities to near the detection level and could be analyzed only after increasing the Ca2+ concentration to 10 mM (n = 7–17).

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Restoration of EC coupling by targeted and nontargeted Ca2+ channel isoforms. Action potential–induced Ca2+ transients were recorded in transfected dysgenic myotubes loaded with the fluorescent Ca2+ indicator Fluo4-AM, using tetanic electrical stimulation (left, 20 Hz, 2 s, bracket) or low frequency stimulation (center, 0.3–0.5 Hz as marked). 0.5 mM Cd2+/0.1 mM La3+ (gray bar) was applied to block the Ca2+ influx during the low-frequency stimulation protocol. Ca2+ release from the SR could be triggered by the application of 6 mM caffeine (gray bar) to the bath after current block. Myotubes transfected with the skeletal GFP-1S responded to electrical stimulation with Ca2+ transients independently of Ca2+ influx. The cardiac GFP-1C also reconstituted EC coupling in dysgenic myotubes; however, Ca2+ transients stopped when the Ca2+ influx was blocked. Cardiac-type Ca2+ transients in response to electrical stimulation were rarely observed in dysgenic myotubes transfected with GFP-1A (see Table ) and about nine times more often with the targeted GFP-1Aas(1592-clip). Example traces for each construct were recorded from the same myotubes in sequential order.

Localization of the Triad Targeting Site to the COOH Terminus of the _1S Subunit

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Screening for the molecular location of the triad targeting signal in 1S/1A chimeras. Schematic representation of the membrane topology of the 1 subunit isoforms and chimeras with 1S sequences in gray and 1A sequences in black. Large cytoplasmic portions of 1S were systematically replaced by the corresponding sequences of 1A (or in the case of GFP-1SkII-IIIm of the Musca 1 sequence). The bar graph at the right shows the percentage of transfected myotubes in which the expressed 1 subunit isoform/chimera achieved a clustered distribution indicative of correct triad targeting.

Replacing the COOH terminus of GFP-_1S with that of _1A (GFP-₁Sa) disrupted triad targeting (Fig. 4, a–c). Similar to the distribution pattern described above for GFP-_1A, GFP-₁Sa was found in the ER/SR system, but never in clusters together with the RyR. Ca²⁺ currents in GFP-₁Sa-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 _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 _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-₁Sa and the associated lack of interactions with the RyR would result in the attenuation of Ca²⁺ currents.

View larger version (116K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Exchange of the targeting properties between GFP-1S and GFP-1A by swapping their COOH-terminal tails. (a and b) Expression of an 1S chimera with the COOH terminus of 1A (GFP-1Sa) in dysgenic myotubes results in a loss of triad targeting; instead, GFP-1Sa was consistently localized in the ER/SR system. (d and e) The converse chimera, GFP-1As, has gained the ability to become coclustered with RyRs in the junctions (examples indicated by arrows). (c and f) The color overlays show the lack of colocalization of GFP-1Sa and RyR clusters, but a high degree of colocalization of clustered GFP-1As and RyRs (insets at twofold magnification). Red and green clusters in f are mostly due to differences in labeling intensities and not due to differential distribution of GFP-1As and RyR (compare d and e). N, nuclei. Bar, 10 µm.

Localization of the Triad Targeting Signal within the COOH Terminus of _1S
The skeletal muscle _1S subunit isolated from muscle preparations exists in two size forms, the minor fraction corresponding to the full-length _1S sequence and the major fraction corresponding to a COOH-terminally truncated _1S, lacking the sequences distal to approximately residue 1661 (De Jongh et al. 1991). Although truncation occurs after incorporation of _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 ₁ 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 _1S. We created one chimera (GFP-₁Aas) with the skeletal sequence 1524–1661 substituted for the corresponding region of _1A, and two daughter chimeras, each containing one half of that region from _1S (see Fig. 6 a). GFP-₁Aas_(1524-1591), which contains the proximal half of this region from _1S (1524–1591) and the distal half from _1A, ends at an arbitrary site of _1A corresponding to the location of the clipping site in _1S, because _1A does not contain this putative clipping site itself. GFP-₁Aas_(1592-clip) is neuronal up to residue 2039 of _1A, with the distal part of _1S, from residue 1592 to the putative clipping site (1661).

Fig. 5, a–c, shows that GFP-₁Aas was correctly targeted into the triad junctions. 38% (n = 1,602) of the transfected myotubes showed a clustered distribution of GFP-₁Aas 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 _1S. Within this region, the proximal half did not confer triad targeting properties to _1A. Not a single myotube out of 887 GFP-₁Aas_(1524-1591)–transfected myotubes showed a clustered distribution pattern of this ₁ chimera; instead, GFP-₁Aas_(1524-1591) was regularly found in the ER/SR (Fig. 5, d–f). In contrast, its sister chimera GFP-₁Aas_(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-₁Aas_(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 _1S (GFP-₁Aas_(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 _1S is sufficient to confer triad targeting properties to the neuronal _1A subunit.

View larger version (106K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Localization of the triad targeting signal within the COOH-terminal tail of 1S. (a–c) Chimera GFP-1Aas consisting of the body plus the first 145 COOH-terminal residues of 1A and the remaining COOH terminus of 1S ending at the putative clipping site at position 1661 is readily targeted into the junctions when expressed in dysgenic myotubes (example indicated by arrows). (d–f) Chimera GFP-1Aas(1524-1591) containing only the proximal half of this COOH-terminal 1S sequence fails to be targeted into the junctions. (g–i) However, chimera GFP-1Aas(1592-clip) containing the distal half of this COOH-terminal 1S sequence is efficiently targeted into junctions of t tubules and plasma membrane with the SR (example indicated by arrows). (c, f, and i) Color overlays of images shown above; insets at twofold magnification. N, nuclei. Bar, 10 µm.

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 _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 _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 _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 _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 _1C in tsA201 cells (Gao et al. 2000). However, the region identified in that study to be important for functional membrane expression of _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 _1S and _1A, to the complex process that culminates in triad targeting.

Effects of Triad Targeting of GFP-₁Aas_(1592-clip) on Ca²⁺ Currents and EC Coupling
Comparison of the current properties of GFP-_1A and GFP-₁Aas_(1524-1591), both of which are not targeted into the triad, with that of the targeted GFP-₁Aas_(1592-clip) provides additional evidence for the existence of distinct mechanisms for membrane and triad targeting (Fig. 6b and Fig. c). Compared with GFP-_1A, GFP-₁Aas_(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 _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 _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-_1A and expressed it in the dysgenic myotubes (data not shown). Current expression of this GFP-_1A-clip was also low, suggesting that the distal COOH terminus of _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 _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 _1A, but that current densities of the targeted GFP-₁Aas_(1592-clip) were increased by approximately twofold over those of the wild-type _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, _1S and _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 _1S and the cardiac _1C activate SR Ca²⁺ release in dysgenic myotubes differs. In _1S, it functions independently of Ca²⁺ influx, whereas _1C does require Ca²⁺ influx for the activation of EC coupling. In Fig. 7, we show that dysgenic myotubes transfected with GFP-_1S or GFP-_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-_1S, these Ca²⁺ transients continued after blocking the Ca²⁺ currents by the addition of Cd²⁺/La³⁺ to the bath solution, whereas with GFP-_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 _1A failed to display (Grabner et al. 1998), or only very rarely displayed, evoked contractions (Adams et al. 1994), despite the fact that _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-_1A–transfected myotubes (Table and Fig. 7). While >25 responsive myotubes were found in almost all of GFP-_1S– and GFP-_1C–transfected cultures, on average only every fifth culture transfected with GFP-_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-₁Aas_(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-₁Aas_(1592-clip) restored EC coupling was Ca²⁺-induced Ca²⁺ release (Fig. 7).

View this table: [in this window] [in a new window]   Table 1 Restoration of Action Potential-induced Ca2+ Transients in Dysgenic Myotubes Expressing Different 1 Subunit Isoforms and the Targeted Chimera GFP-1Aas(1592-clip)

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 _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 _1S subunit that structurally sets it apart from the cardiac _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 _1S subunit is necessary and sufficient to confer this targeting property to a neuronal ₁ subunit.

	Acknowledgments

 
We thank Dr. J. Hoflacher and E. Emberger for their excellent experimental help, and Dr. H. Glossmann for generously providing support for the pursuit of this project.

This work was supported in part by the Fonds zur Förderung der wissenschaftlichen Forschung, Austria, grants P12653-MED (B.E. Flucher), and P13831-GEN (M. Grabner), and by the European Commission's Training and Mobility of Researchers Network grant ERBFMRXCT960032 (B.E. Flucher).

Submitted: 1 August 2000

Revised: 6 September 2000

Accepted: 7 September 2000

Abbreviations used in this paper: DHPR, dihydropyridine receptor; EC, excitation–contraction; GFP, green fluorescent protein; nt, nucleotides; RE, restriction enzyme; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; t tubule, transverse tubule.

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