The Triad Targeting Signal of the Skeletal Muscle Calcium Channel Is L - 《cell》细胞杂志干细胞之家

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发表于 2009-3-5 22:31 |只看该作者 |倒序浏览 |打印

a Department of Biochemical Pharmacology, University of Innsbruck, A-6020 Innsbruck, Austria
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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: j' _; I; k5 {3 u' K5 t$ ^
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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 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

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 (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)1 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.' v) {) m! l& ]: n+ ~9 T

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/, ?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 (Rios 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 ? 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 ?-null mice and both functions can be reconstituted by heterologous expression of ?1a (Gregg et al. 1996 ; Beurg et al. 1997 ). Thus, expression of the ? 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 ?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 ? 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 ? 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.! F2 [( m2 R/ N1 J) p
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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.$ y4 b: c4 q+ F: g9 E1 L% U

Materials and Methods

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.
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Construction of Chimeric 1 Subunits1 H7 p5 }# Y3 ]" D7 M- G' ]
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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./ x" O; m2 u# C

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 ).
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GFP-1SkNa.9 O; A- `, r+ D* V7 C6 \$ ]
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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." C% a6 I$ h# {! ?- P
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GFP-1SkI-IIa.( _6 D7 K5 ]$ J/ K

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.! S0 @! Y0 u4 ]8 `! k$ y- i

GFP-1SkII-IIIm.# x3 D( j7 H% d" M+ v: p4 C, V3 F
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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.
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GFP-1SkIII-IVa.
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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.0 I0 C7 Y5 |8 O2 K$ Y3 Q! W- N8 s9 A% A

GFP-1Sa.2 S' V! B8 X1 L- w! ?3 g

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.2 n3 L5 r& b+ v1 z, @  Q
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GFP-1As.
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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.3 G) }; g. c. s+ N: l/ n8 J

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).
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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.
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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).5 ~8 E" ^: ^8 E9 b0 i4 Z' D0 X- _
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GFP-1Aas(1607-clip).$ D3 f& v2 W5 A6 t9 x1 b3 |3 v; B
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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).) X1 l+ V% r' R- Z
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All cDNA portions modified by PCR were checked for sequence integrity by sequence analysis (sequencing facility of MWG Biotech).( ?) _# L* f* ^5 ^% f
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GFP and Immunofluorescence Labeling
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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.
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Patch-Clamp and Fluorescent Ca2  Recording
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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.9 g+ B" w# ~0 q3 I/ w8 h
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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.
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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 ).  L) _+ j4 J! n8 @5 v

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 ).3 f, U/ U) a) c; ?& m8 R
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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., q4 t) r- d' g4 _
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Double immunofluorescence labeling of GFP-1S with anti–GFP and anti–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-1S–expressing myotubes (57 and 58%, respectively). Therefore, we continued using anti–GFP 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 (Powell et al. 1996 ; Flucher et al. 2000 ). ER/SR distribution of heterologously expressed 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).  d8 C4 `5 c6 l& V
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Differential Targeting of the Skeletal and the Neuronal Ca2  Channel 1 Subunits Expressed in Dysgenic Myotubes
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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.6 ^) I8 u# ?$ l8 [
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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.
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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-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-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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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.
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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.

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.

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).

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 Powell et al. 1996 ; Flucher et al. 2000 ). In contrast, restoration of EC coupling by GFP-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 (Grabner et al. 1998 ). In that study, GFP-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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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 1) 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

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 (Grabner et al. 1994 ) was used for constructing a II-III chimera (GFP-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 anti–GFP and anti–RyR 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 ? subunit (Pragnell et al. 1994 ). Association of the ? 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 1A seems to contain an ER retention signal that is blocked upon association with ? to release the complex from the ER. Since the ? interaction domain in the I-II loop is shared by all known 1 subunits and a ? 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 (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 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 (Takekura et al. 1995 ).5 x7 r5 S0 ~. S/ n5 z/ ~

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.
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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, d–f). 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.+ r# K" f# R! o0 r$ h3 E
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Localization of the Triad Targeting Signal within the COOH Terminus of 1S. E( M+ s; `( p$ k& ^9 C. S

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).3 T; q. F/ I0 e8 j5 U2 ~
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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.& ~# b% c0 `( S( ^
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Comparing this sequence of 1S with the corresponding sequences of the cardiac 1C, which is also targeted to triads (Grabner et al. 1998 ), and with that of the nontargeted 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 (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.( \+ Q$ B: g4 e- H. j
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Effects of Triad Targeting of GFP-1Aas(1592-clip) on Ca2  Currents and EC Coupling4 b* s' B& P; T0 A8 _! b
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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./ R9 B. d; u5 X$ E

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.
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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.
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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 1 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 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).- Q8 v0 S/ J+ r$ _6 ^, E7 i0 y
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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)6 z# ~& w# Q. ]8 x+ e
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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; 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 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 ? 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.
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References

Adams, B.A., Tanabe, T., Mikami, A., Numa, S., and Beam, K.G. 1990. Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs. Nature. 346:569-572.* p( d- J' r$ H/ w
! u/ }7 v5 V/ ^9 X6 L
Adams, B.A., Mori, Y., Kim, M.S., Tanabe, T., and Beam, K.G. 1994. Heterologous expression of BI Ca2  channels in dysgenic skeletal muscle. J. Gen. Physiol 104:985-996.( H( M8 ]) f5 d. \: j

Beam, K.G., Adams, B.A., Niidome, T., Numa, S., and Tanabe, T. 1992. Function of a truncated dihydropyridine receptor as both voltage sensor and calcium channel. Nature 360:169-171.# _8 @* m4 c4 P$ l$ v$ U" x1 l% G

Berridge, M.J. 1998. Neuronal calcium signaling. Neuron. 21:13-26.
' U( i9 d/ [  p9 q" u
Beurg, M., Sukhareva, M., Strube, C., Powers, P.A., Gregg, R.G., and Coronado, R. 1997. Recovery of Ca2  current, charge movements, and Ca2  transients in myotubes deficient in dihydropyridine receptor ?1 subunit transfected with ?1 cDNA. Biophys. J. 73:807-818.9 ?& Y. A4 D' _* }: R: M& P

Bichet, D., Cornet, V., Geib, S., Carlier, E., Volsen, S., Hoshi, T., Mori, Y., and De Waard, M. 2000. The I-II loop of the Ca2  channel 1 subunit contains an endoplasmic reticulum retention signal antagonized by the ? subunit. Neuron. 25:177-190.' C1 O9 B! [$ h1 @

Block, B.A., Imagawa, T., Campbell, K.P., and Franzini-Armstrong, C. 1988. Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle. J. Cell Biol. 107:2587-2600., y. ]# Y0 f, F# u; S+ X7 h
" w9 x% R2 ^- w+ S( |
Brice, N.L., Berrow, N. S., Campbell, V., Page, K.M., Brickley, K., Tedder, I., and Dolphin, A.C. 1997. Importance of the different ? subunits in the membrane expression of the 1A and 2 calcium channel subunits: studies using a depolarization-sensitive 1A antibody. Eur. J. Neurosci. 9:749-759.2 R: {6 V8 H6 H, ~3 A6 |
" F* D9 e8 Y5 B, |6 M% ?, S6 ?6 s
Catterall, W.A. 1996. Molecular properties of sodium and calcium channels. J. Bioenerg. Biomembr. 28:219-230./ A& B# C4 z8 y: `9 U
* y/ p/ c0 L- h! f; H  _% Y$ @' k
Chien, A.J., Zhao, X., Shirokov, R.E., Puri, T.S., Chang, C.F., Sun, D., R赤os, E., and Hosey, M.M. 1995. Roles of a membrane-localized ? subunit in the formation and targeting of functional L-type Ca2  channels. J. Biol. Chem 270:30036-30044.

De Jongh, K.S., Warner, C., Colvin, A.A., and Catterall, W.A. 1991. Characterization of the two size forms of the 1 subunit of skeletal muscle L-type calcium channels. Proc. Natl. Acad. Sci. USA. 88:10778-10782.- t: e& p) u+ a

Flucher, B.E., Morton, M.E., Froehner, S.C., and Daniels, M.P. 1990. Localization of the 1 and 2 subunits of the dihydropyridine receptor and ankyrin in skeletal muscle triads. Neuron. 5:339-351.' t* T9 ~; C- T, Y3 Q

Flucher, B.E., Phillips, J.L., and Powell, J.A. 1991. Dihydropyridine receptor  subunits in normal and dysgenic muscle in vitro: expression of 1 is required for proper targeting and distribution of 2. J. Cell Biol. 115:1345-1356.

Flucher, B.E., Andrews, S.B., and Daniels, M.P. 1994. Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation–contraction coupling in skeletal muscle. Mol. Biol. Cell 5:1105-1118." f6 T1 {* C; ?$ g' B- r* |. r
+ R4 M8 x9 P2 h; P& C, }6 B  E$ n
Flucher, B.E., Kasielke, N., Gerster, U., Neuhuber, B., and Grabner, M. 2000. Insertion of the full-length calcium channel 1S subunit into triads of skeletal muscle in vitro. FEBS Lett 474:93-98.8 t& m5 b9 E, p5 m/ n' ]

Franzini-Armstrong, C., and Jorgensen, A.O. 1994. Structure and development of E-C coupling units in skeletal muscle. Annu. Rev. Physiol 56:509-534.

Freise, D., Held, B., Wissenbach, U., Pfeifer, A., Trost, C., Himmerkus, N., Schweig, U., Freichel, M., Biel, M., and Hofmann, F. et al. 2000. Absence of the  subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2  currents and alters channel inactivation properties. J. Biol. Chem. 275:14476-14481.8 ]4 a  [+ K% Z' O9 D2 s
3 v$ m' h$ Z4 F: n' J1 o1 P' ?
Gao, T., Buenemann, M., Gerhardstein, B.L., Ma, H., and Hosey, M.M. 2000. Role of the C-terminus of the 1C (CaV1.2) subunit in membrane targeting of cardiac L-type calcium channels. J. Biol Chem. 275:25436-25444.
5 _. M, N+ Z* w- E+ M+ z
Giannini, G., Conti, A., Mammarella, S., Scrobogna, M., and Sorrentino, V. 1995. The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues. J. Cell Biol. 128:893-904.

Grabner, M., Bachmann, A., Rosenthal, F., Striessnig, J., Schulz, C., Tautz, D., and Glossmann, H. 1994. Insect calcium channels: molecular cloning of an 1 subunit from housefly (Musca domestica) muscle. FEBS Lett. 339:189-194.

Grabner, M., Dirksen, R.T., and Beam, K.G. 1998. Tagging with green fluorescent protein reveals a distinct subcellular distribution of L-type and non–L-type Ca2  channels expressed in dysgenic myotubes. Proc. Natl. Acad. Sci. USA. 95:1903-1908.

Grabner, M., Dirksen, R.T., Suda, N., and Beam, K.G. 1999. The II-III loop of the skeletal muscle dihydropyridine receptor is responsible for the bi-directional coupling with the ryanodine receptor. J. Biol. Chem. 274:21913-21919.

Gregg, R.G., Messing, A., Strube, C., Beurg, M., Moss, R., Behan, M., Sukhareva, M., Haynes, S., Powell, J.A., Coronado, R., and Powers, P.A. 1996. Absence of the ? subunit (cch?1) of the skeletal muscle dihydropyridine receptor alters expression of the 1 subunit and eliminates excitation–contraction coupling. Proc. Natl. Acad. Sci. USA. 93:13961-13966.

Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., and Pease, L.R. 1989. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 77:61-68.* v& x% {3 I4 ?* h! J  j
" x  f; S2 Y( N3 H" x! d
Jaggar, J.H., VA.Porter, W.J., Lederer,, and Nelson, M.T. 2000. Calcium sparks in smooth muscle. Am. J. Physiol. Cell Physiol 278:C235-C256.

Jorgensen, A.O., Shen, A.C.-Y., Arnold, W., Leung, A.T., and Campbell, K.P. 1989. Subcellular distribution of the 1,4-dihydropyridine receptor in rabbit skeletal muscle in situ: an immunofluorescence and immunocolloidal gold-labeling study. J. Cell Biol. 109:135-147.
% H3 ]6 a- {# S9 k8 M- j
Lim, S.T., Antonucci, D.E., Scannevin, R.H., and Trimmer, J.S. 2000. A novel targeting signal for proximal clustering of the Kv2.1 K  channel in hippocampal neurons. Neuron 25:385-397.

Melzer, W., Herrmann-Frank, A., and Luttgau, H.C. 1995. The role of Ca2  ions in excitation–contraction coupling of skeletal muscle fibres. Biochim. Biophys. Acta 1241:59-116.

Mori, Y., Friedrich, T., Kim, M.-S., Mikami, A., Nakai, J., Ruth, P., Bosse, E., Hofmann, F., Flockerzi, V., and Furuichi, T. et al. 1991. Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature. 350:398-402.

Morton, M.E., and Froehner, S.C. 1987. Monoclonal antibody identifies a 200-kDa subunit of the dihydropyridine-sensitive calcium channel. J. Biol. Chem 262:11904-11907.

Nakai, J., Dirksen, R.T., Nguyen, H.T., Pessah, I.N., Beam, K.G, and Allen, P.D. 1996. Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor. Nature. 380:72-75.9 c. Q8 E  C* Y, i& T
- D; q1 p- F1 c3 ~) W+ [
Neuhuber, B., Gerster, U., Mitterdorfer, J., Glossmann, H., and Flucher, B.E. 1998a. Differential effects of Ca2  channel ?1a and ?2a subunits on complex formation with 1S and on current expression in tsA201 cells. J. Biol. Chem. 273:9110-9118.
$ {' U7 X( G( N3 q
Neuhuber, B., Gerster, U., Doring, F., Glossmann, H., Tanabe, T., and Flucher, B.E. 1998b. Association of calcium channel 1S and ?1a subunits is required for the targeting of ?1a but not of 1S into skeletal muscle triads. Proc. Natl. Acad. Sci. USA. 95:5015-5020.$ b0 E: w0 s5 h3 a
* a8 f4 m' _1 @1 ]% v
Powell, J.A., Petherbridge, L., and Flucher, B.E. 1996. Formation of triads without the dihydropyridine receptor  subunits in cell lines from dysgenic skeletal muscle. J. Cell Biol. 134:375-387.
4 `8 Z9 Y( I3 R' g( L- K# P$ I
Pragnell, M., De Waard, M., Mori, Y., Tanabe, T., Snutch, T.P., and Campbell, K.P. 1994. Calcium channel ?-subunit binds to a conserved motif in the I-II cytoplasmic linker of the 1-subunit. Nature. 368:67-70.
* }; {- @% b9 u+ M
Proenza, C., Wilkens, C., Lorenzon, N.M., and Beam, K.G. 2000. A carboxyl-terminal region important for the expression and targeting of the skeletal muscle dihydropyridine receptor. J. Biol. Chem. 275:23169-23174.7 \6 F/ y8 N& b1 J3 ~) n- W
! v: a+ b0 G. [% x' q& d4 W
Protasi, F., Franzini-Armstrong, C., and Allen, P.D. 1998. Role of ryanodine receptors in the assembly of calcium release units in skeletal muscle. J. Cell Biol 140:831-842.7 Q, t$ a' H5 ]0 y* P4 m" }! a

R赤os, E., Pizarro, G., and Stefani, E. 1992. Charge movement and the nature of signal transduction in skeletal muscle excitation–contraction coupling. Annu. Rev. Physiol 54:109-133.. |& h. G6 n6 m. \* F- i
/ u! W7 h5 h* ~+ E: n
Sheng, M. 1996. PDZs and receptor/channel clustering: rounding up the latest suspects. Cell. 17:575-578.

Sheng, M., and Pak, D.T. 1999. Glutamate receptor anchoring proteins and the molecular organization of excitatory synapses. Ann. NY Acad. Sci. 868:483-493.

Takekura, H., Nishi, M., Noda, T., Takeshima, H., and Franzini-Armstrong, C. 1995. Abnormal junctions between surface membrane and sarcoplasmic reticulum in skeletal muscle with a mutation targeted to the ryanodine receptor. Proc. Natl. Acad. Sci. USA 92:3381-3385.
& [5 ]' y/ M& n* c5 m2 E
Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., and Numa, S. 1987. Primary structure of the receptor for calcium channel blocker from skeletal muscle. Nature. 328:313-318.( }7 w" D6 _" v
% Q. j7 L: |) u* N
Tanabe, T., Beam, K.G., Powell, J.A., and Numa, S. 1988. Restoration of excitation–contraction coupling and slow Ca2  current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature. 336:134-139.& A# N( h& R, u) y
6 g- X5 _& b6 |" ]1 `
Tanabe, T., Beam, K.G., Adams, B.A., Niidome, T., and Numa, S. 1990a. Regions of the skeletal muscle dihydropyridine receptor critical for excitation–contraction coupling. Nature 346:567-569.2 N5 g7 b, y$ m: Z6 T- \& K

Tanabe, T., Mikami, A., Numa, S., and Beam, K.G. 1990b. Cardiac-type excitation–contraction coupling in dysgenic skeletal muscle injected with cardiac dihydropyridine receptor cDNA. Nature. 344:451-453.
  M0 Z5 |9 V2 s, ~- F% n
Walker, D., Bichet, D., Campbell, K.P., and De Waard, M. 1998. A ?4 isoform-specific interaction site in the carboxyl-terminal region of the voltage-dependent Ca2  channel 1A subunit. J. Biol. Chem 173:1361-1367.(Bernhard E. Fluchera, Nicole Kasielkea, )

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