Correspondence to: Bernhard E. Flucher, Department of Physiology, University of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria. Tel:43-512-507-3787 Fax:43-512-507-2836
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Abstract |
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The specific localization of L-type Ca2+ channels in skeletal muscle triads is critical for their normal function in excitationcontraction (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 (16071661) 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, excitationcontraction coupling, immunofluorescence, skeletal muscle
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Introduction |
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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 (
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/
, ß1a, and
1 subunits (
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 (
1 subunit with ß and
2/
increases membrane insertion of functional Ca2+ channels (
2/
and of recombinant ß1a expressed in dysgenic myotubes showed that, without the
1S subunit, both subunits failed to be localized in the junctions (
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 ß 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 (
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.
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Materials and Methods |
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Cell Culture and Transfections
Myotubes of the homozygous dysgenic (mdg/mdg) cell line GLT were cultured as described in
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 (
GFP-1S, GFP-
1A.
Construction of the GFP-tagged 1S (
1A (
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 (
1S.
GFP-1SkI-IIa.
The HindIIIEcoRI fragment of Sk (nt 5' polylinker-1007) and the EcoRI*-SphI A/Sk SOE fusion product (nt A1085Sk1735) 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 SbfISphI fragment (nt 5' polylinker-Sk1735) of this intermediate clone was coligated with the SphIXhoI fragment of Sk (nt 17352654) into the corresponding SbfI/XhoI RE sites of plasmid GFP-1S.
GFP-1SkII-IIIm.
The EcoRIBalI fragment of Sk (nt 10071973) was coligated with the BalINdeI fragment (nt 19822296) from the muscle 1 subunit (M) of Musca domestica (
1S with a cardiac (C) II-III loop (nt C2716Sk2654) (
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 26544488) was ligated into the corresponding XhoI/BglII sites of plasmid GFP-1S.
GFP-1Sa.
The XhoISmaI fragment of Sk (nt 26544038) and the SmaIBglII Sk/A cDNA fusion fragment (nt Sk4038A5891) 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 HindIIIXhoI 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 XhoAccI fragment of A (nt 13954504) was coligated with the A/Sk SOE fusion fragment AccIBglII (nt A4504Sk4488) 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 SalIEcoRI fragment from A (nt 5' polylinker-1567) was coligated with the EcoRIBglII fragment (nt A1567Sk4488) 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 45664991) 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 49844989) were introduced to terminate the reading frame at residue T1661, which is close to the physiological clipping site of the
1S carboxyl terminus (
GFP-1Aas(1524-1591).
The BglII*XbaI* Sk/A cDNA fusion fragment (nt Sk4566A6347) 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 63406345) to terminate the reading frame at residue G2113.
GFP-1Aas(1592-clip).
The BglIIXbaI* A/Sk SOE fusion fragment (nt A5891Sk4991) 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 BglIIXbaI* fragment of A (nt 58916347) 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(15241591).
GFP-1Aas(1607-clip).
The BglIIXbaI* A/Sk SOE fusion fragment (nt A5891Sk4991) 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 (1S subunit at a final concentration of 1:1,000 (
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 24 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 (
Action potentialinduced 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 (
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Results and Discussion |
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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 (
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
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 (
1S expressed in dysgenic myotubes (
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 antiRyR. 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 (
1S form RyR clusters (Fig 1 b), which have previously been shown to correspond to t tubule/SR junctions by electron microscopy (
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Double immunofluorescence labeling of GFP-1S with antiGFP and anti
1S (Fig 1c and Fig d) or with antiGFP and antiRyR (e and f) results in the same clustered distribution pattern in equally large fractions of GFP-
1Sexpressing myotubes (57 and 58%, respectively). Therefore, we continued using antiGFP for the immunolocalization of GFP-
1 constructs, because it recognizes both GFP-
1S and GFP-
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-
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 (
1 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-
1S, was missing and therefore GFP-
1S was retained in the biosynthetic apparatus. However, in addition to myotubes lacking the target for the
1 subunit, retention in the ER/SR system can also be observed in normally differentiated myotubes if they are transfected with
1 constructs lacking the triad targeting signal (see below).
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 antiGFP and antiRyR reveals the colocalization of GFP-
1S and RyR in the junctions (Fig 2, ac), 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.
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To exclude the possibility that the absence of GFP-1A resulted from improper folding or lack of plasma membrane incorporation of the GFP-
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-
1Atransfected 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) (
1A expressed in dysgenic myotubes formed functional channels in the cell membrane. But instead of becoming locally concentrated in the triads, GFP-
1A was distributed diffusely in the plasma membrane at densities below detectability with immunocytochemistry.
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Recordings of cytoplasmic Ca2+ transients in response to electrical field stimulation showed that GFP-1S regularly restored skeletal muscle EC coupling in dysgenic myotubes (see Fig 7, and
1A was only rarely observed (see below). Thus, both the skeletal muscle GFP-
1S isoform and the neuronal GFP-
1A isoform were functionally expressed in dysgenic myotubes, but only GFP-
1S was targeted into the triad junctions and efficiently restored EC coupling. The differential distribution of GFP-
1S and GFP-
1A as well as their functional differences are in general agreement with observations from a previous study comparing the expression of GFP-
1S, GFP-
1A, and a cardiac GFP-
1C construct in primary cultured dysgenic myotubes (
1A differed from the muscle isoforms in that its distribution patterns were restricted to near the injection site and that only GFP-
1A failed to respond in a contraction assay. Together, these data support our hypothesis that the skeletal muscle
1S contains a signal for its targeting and selective incorporation into triads, but that such a triad targeting signal is missing from the neuronal
1A subunit isoform.
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Localization of the Triad Targeting Site to the COOH Terminus of the 1S Subunit
With a properly targeted GFP-1S and a nontargeted GFP-
1A in our hands, we decided to start screening for the location of the targeting signal by replacing the prominent cytoplasmic portions of GFP-
1S with the corresponding sequences of
1A. GFP-
1S/
1A chimeras were generated with the following portions of
1A (Fig 3): the NH2 terminus (GFP-
1SkNa), the cytoplasmic loop connecting repeats I and II (GFP-
1SkI-IIa) or that connecting repeats III and IV (GFP-
1SkIII-IVa), and the COOH terminus (GFP-
1Sa). Because the II-III loop of
1A is more than three times the size of that of
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)
1 subunit (
1SkII-IIIm). Its II-III loop has similar size as that of the rabbit skeletal muscle
1S but shows very little sequence homology to
1S and, like
1A, the Musca
1 subunit failed to be targeted into the junctions. Double immunolabeling with antiGFP and antiRyR and subsequent analysis of the targeting properties revealed that all of these chimeras with the exception of GFP-
1Sa 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-
1S (Fig 3). Thus, neither the NH2 terminus nor any of the major cytoplasmic loops of
1S are essential for triad targeting.
The I-II loop contains the interaction domain for the ß subunit (1A seems to contain an ER retention signal that is blocked upon association with ß to release the complex from the ER. Since the ß interaction domain in the I-II loop is shared by all known
1 subunits and a ß 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-
1SkI-IIa chimera. The II-III loop of
1S contains the sequence important for the interaction with the RyR. It specifies the tissue-specific mode of EC coupling (
1S II-III loop by a loop as different as that of Musca's
1 subunit had no adverse effect on triad targeting of chimera GFP-
1SkII-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,
1S clusters in the junctions despite the lack of RyR1 (
Replacing the COOH terminus of GFP-1S with that of
1A (GFP-
1Sa) disrupted triad targeting (Fig 4, ac). 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.
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.
The failure of triad targeting in the GFP-1Sa chimera suggests that the triad targeting signal may be contained within the COOH terminus of
1S. To verify this interpretation, the corresponding reverse chimera, GFP-
1A with the COOH terminus of
1S (GFP-
1As) was constructed. When expressed in dysgenic myotubes, GFP-
1As was found coclustered with the RyR (Fig 4, df). The efficiency of clustering was somewhat reduced compared with wild-type GFP-
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
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-
1A were expressed (see Fig 6 b), indicating that this channel chimera was functional. Since swapping the COOH termini of
1S and
1A conferred triad targeting properties to the neuronal isoform (GFP-
1As) and disrupted triad targeting in the skeletal muscle Ca2+ channel isoform (GFP-
1Sa), it was evident that the signal responsible for this specific localization must be contained in the COOH terminus of
1S.
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 (
1S into the junctions (
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 15241661 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 (15241591) 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, ac, 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, df). In contrast, its sister chimera GFP-
1Aas(1592-clip) was efficiently targeted to the junctions (Fig 5, gi). 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.
Comparing this sequence of 1S with the corresponding sequences of the cardiac
1C, which is also targeted to triads (
1A reveals surprisingly few residues that are conserved between
1S and
1C but distinct from
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
1S is made up of more than those residues conserved between
1S and
1C, or the signal that is contained in this sequence stretch of
1S is located outside the corresponding region of
1C (see below). To distinguish between these possibilities, the corresponding targeting signal in
1C and other
1 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 (1S contains only one known consensus protein binding site. The sequence SPV in position 16401642 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 (
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 proteinprotein 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 (
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 (15241591) 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 15921661 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 (
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 potentialinduced Ca2+ transients based on their all-or-none characteristics (
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 (
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 potentialinduced Ca2+ transients in GFP-
1Atransfected myotubes (Table 1 and Fig 7). While >25 responsive myotubes were found in almost all of GFP-
1S and GFP-
1Ctransfected 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 potentialinduced Ca2+ transients were observed in 60% of the cultures (Table 1). 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).
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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 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;
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 proteinprotein 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 ß 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 16071661 of the skeletal muscle
1S subunit is necessary and sufficient to confer this targeting property to a neuronal
1 subunit.
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Footnotes |
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1 Abbreviations used in this paper: DHPR, dihydropyridine receptor; EC, excitationcontraction; GFP, green fluorescent protein; nt, nucleotides; RE, restriction enzyme; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; t tubule, transverse tubule.
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Acknowledgements |
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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
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References |
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