Originally published In Press as doi:10.1074/jbc.M509566200 on November 28, 2005
J. Biol. Chem., Vol. 281, Issue 6, 3521-3527, February 10, 2006
Organization of Calcium Channel 
1a Subunits in Triad Junctions in Skeletal Muscle*
Valérie Leuranguer
,
Symeon Papadopoulos
, and
Kurt G. Beam1
From the
Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523-1617 and Physiologie, Medizinische Hochschule, 30625 Hannover, Germany
Received for publication, August 31, 2005
, and in revised form, November 15, 2005.
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ABSTRACT
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In skeletal muscle, dihydropyridine receptors (DHPRs) in the plasma membrane interact with the type 1 ryanodine receptor (RyR1) at junctions with the sarcoplasmic reticulum. This interaction organizes junctional DHPRs into groups of four termed tetrads. In addition to the principle 
1S subunit, the 
1a subunit of the DHPR is also important for the interaction with RyR1. To probe this interaction, we measured fluorescence resonance energy transfer (FRET) of 1a subunits labeled with cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP). Expressed in dysgenic (1S-null) myotubes, YFP-1a-CFP and CFP-1a-YFP were diffusely distributed in the cytoplasm and highly mobile as indicated by fluorescence recovery after photobleaching. Thus, 1a does not appear to bind to other cellular proteins in the absence of 1S. FRET efficiencies for these cytoplasmic 1a subunits were 
6-7%, consistent with the idea that <10 nm separates the N and C termini. After coexpression with unlabeled 1S (in dysgenic or 1-null myotubes), both constructs produced discrete fluorescent puncta, which correspond to assembled DHPRs in junctions and that did not recover after photobleaching. In 1-null myotubes, FRET efficiencies of doubly labeled 1a in puncta were similar to those of the same constructs diffusely distributed in the cytoplasm and appeared to arise intramolecularly, since no FRET was measured when mixtures of singly labeled 1a (CFP or YFP at the N or C terminus) were expressed in 1-null myotubes. Thus, DHPRs in tetrads may be arranged such that the N and C termini of adjacent 1a subunits are located >10 nm from one another.
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INTRODUCTION
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Excitation-contraction (EC)2 coupling in skeletal muscle involves conformational coupling between the dihydropyridine receptor (DHPR), a voltage-gated calcium channel present in the plasma membrane, and the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR). Specifically, the DHPR responds to membrane depolarization by inducing the opening of RyR1 and the release of Ca2+ from the SR. Although the DHPR can function as a calcium channel, skeletal type EC coupling does not require the entry of extracellular Ca2+, leading to the idea that DHPRs and RyR1 interact mechanically. Indeed, DHPRs in skeletal muscle are arranged in groups of four, termed tetrads, such that each DHPR within a tetrad is apposed to one of the four, identical subunits of RyR1 (1).
The DHPR consists of a principal 1S subunit, which contains the ion-conducting pathway and voltage sensing structures, together with auxiliary 1a, 2
-1, and 
1 subunits (for review, see Ref. 2). A significant body of evidence indicates that the cytoplasmic loop connecting 1S homology repeats II and III (the II-III loop) plays an important role in conformational coupling between the DHPR and RyR1. The auxiliary 1a subunit appears to be similarly important. One essential function of all calcium channel subunits (currently known to be encoded by four different genes) is that of facilitating trafficking to the plasma membrane, which occurs largely as a consequence of binding to the 1 I-II loop. Consequently, knock-out of the gene encoding the predominant form in skeletal muscle (1) causes a nearly complete absence of DHPRs in the plasma membrane (3) and a resultant loss of EC coupling (4). Significantly, truncating or altering the sequence of the 1a C-terminal preserves membrane expression but suppresses EC coupling (5, 6), indicating that this region is important for communication between the DHPR and RyR1.
All subunits can be subdivided into five regions: highly variable N- and C-terminal regions flanking a core segment with three regions. Crystal structures have been recently reported for this core segment (7-9), which confers the principal functional properties on full-length subunits. Within the core segment, the first region is highly conserved among subunits and is homologous to canonical Src homology 3 domains. The third region of the core segment is also highly conserved and is guanylate kinase-like. The Src homology 3- and guanylate kinase-like domains are linked by a more variable, flexible loop so that the core segment as a whole shares structural features with the membrane-associated guanylate kinases, a protein family that organizes signaling components near membranes (10).
Based on sequence alignment of 1a with 2a (GenBankTM numbers: M25514
[GenBank]
and M80545
[GenBank]
), and on the crystal structure of the 2a core segment, leucine 74 and threonine 463 of 1a are likely separated by 42 Å. However there is little basis for predicting the structure of the 1a segments N-terminal (73 residues) and C-terminal (61 residues) to the core. Thus, one goal of the present experiments was to obtain a rough idea about the separation of the N and C termini of 1a and about whether or not there were large changes in the relative arrangements of the N and C termini as a consequence of the binding of 1a to 1S. A second goal was to obtain information about the arrangement of 1a subunits within tetrads. Toward these ends, we measured fluorescence resonance energy transfer (FRET) with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) used as the donor and acceptor, respectively (11). The 1a subunits were singly labeled on either the N or C terminus with either CFP or YFP or doubly labeled with CFP on one terminal and YFP on the other. These fluorescently labeled 1a subunits were expressed in dysgenic (1S-null) or 1-null myotubes. We found that the 1a subunit was incorporated into fluorescent puncta at the cell surface (indicative of junctional targeting) only when the 1S subunit was also present. Based on intramolecular FRET, it appears that the N and C termini of 1a are <10 nm apart. Additionally, the absence of intermolecular FRET between singly labeled subunits is consistent with the idea that the N and C termini of 1a subunits are directed away from the center of tetrads.
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MATERIALS AND METHODS
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Generation of the Constructs Expressed in Myotubes—Fig. 1 illustrates the 1a constructs that were used for this study. The coding sequence for rabbit skeletal muscle 1a (GenBankTM number M25514
[GenBank]
) was isolated by using PCR to introduce EcoRI and SalI sites immediately before the ATG start codon and immediately after the last codon, respectively. The EcoRI-SalI segment containing the 1a coding sequence was then ligated to EcoRI/SalI-digested pEYFP-C1, pEYFP-N1, pECFP-C1, or pECFP-N1 (Clontech, Palo Alto, CA) to obtain the constructs YFP-1a, 1a-YFP, CFP-1a, and 1a-CFP, respectively. To construct CFP-1a-YFP, CFP-1a and 1a-YFP were individually digested with MfeI, and the 5903-bp fragment obtained from CFP-1a and the 1128-bp fragment from 1a-YFP were ligated together. For the YFP-1a-CFP construct, YFP-1a and 1a-CFP were individually digested with MfeI, and the 5903-bp fragment from YFP-1a and the 1128-bp fragment from 1a-CFP were ligated together. Unlabeled 1a was obtained by digesting 1a-YFP with Acc651 and BsrGI, discarding the small fragment containing the YFP sequence, and re-ligating the large fragment containing the 1a coding sequence. Unlabeled 1S was obtained as described previously (12).
Cell Culture and cDNA Microinjection—Primary cultures of dysgenic (13) and 1-null (14) myotubes were prepared from newborn mice as described previously (15). Briefly, myoblasts were plated into 35-mm culture dishes (MatTek, Ashland, MA) with glass coverslip bottoms that had been coated with ECL (Upstate%20Biotechnology">Upstate Biotechnology, Lake Placid, NY) and grown for 6-7 days in a humidified 37 °C incubator with 5% CO2. The culture medium, Dulbecco's modified Eagle's medium supplemented with 20% fetal bovine serum, was then replaced by differentiation medium (Dulbecco's modified Eagle's medium supplemented with 2% horse serum), and after 2-4 days single nuclei of myotubes were microinjected with a small amount of plasmid DNA in water. The DNA concentration in the injection solution was 10-20 ng/µl for both 1a constructs and 40 ng/µl for 1S.
The ability of the 1S and 1a constructs to support EC coupling in dysgenic or 1-null myotubes, respectively, was assayed by the presence of spontaneous contractions and/or evoked contractions. To test for evoked contractions, the myotubes were placed in physiological saline (containing in mM: 140 NaCl, 1.5 KCl, 1 MgCl2, 2.5 CaCl2, 11 glucose, 10 HEPES, pH 7.4 with NaOH) and stimulated with 100-V, 15-30-ms pulses applied via a patch pipette that contained physiological saline and was positioned near the myotube. Images of these myotubes were acquired at a rate of 11 Hz. The contractions were quantified by measuring the movement of an identifiable portion of a myotube across the visual field.
Measurement of Ionic Currents—Macroscopic Ca2+ currents were measured using the whole-cell patch clamp method. Patch pipettes of borosilicate glass had resistances of 1.8-2.2 M
when filled with an intracellular solution containing (mM): 110 CsCl, 3 MgCl2, 10 Cs2EGTA, 10 HEPES, 3 Mg-ATP, and 0.6 GTP, pH = 7.4 with CsOH. The external bath solution contained (mM): 10 CaCl2, 145 tetraethylammonium-Cl, 0.003 tetrodotoxin, 0.02 N-benzyl-p-toluene sulfonamide, and 10 HEPES, pH = 7.4 with tetraethylammonium-OH. To measure L-type current, myotubes were stepped from the holding potential (-80 mV) to -30 mV for 1 s (to inactivate endogenous T-type current), repolarized to -50 mV for 30-50 ms, depolarized to varying test potentials (Vtest) for 200 ms, and then returned to the holding potential. Cell capacitance was determined by integration of the capacity transient resulting from 30-mV hyperpolarizations from the holding potential and was used to normalize current amplitudes (pA/pF) obtained from different myotubes. Current-voltage curves were fitted using the Boltzmann expression: I = Gmax* (V - Vrev)/{1 + exp[- (V - V
)/kG]}, where I is the current for the test potential V, Vrev is the reversal potential, Gmax is the maximum Ca2+ channel conductance, V is the half-maximal activation potential and kG is the slope factor.
Measurement of FRET and Fluorescence Recovery after Photobleaching—Measurement of FRET was as described previously (12). Briefly, intact fluorescent myotubes were examined 48 h after cDNA microinjection using an LSM 510 META laser scanning confocal microscope (Zeiss, Thornwood, NY). In a typical experiment, an image of the cell region of interest was taken using standard spectroscopic settings: CFP and YFP were excited with separate sweeps of the 458 and 514 nm lines, respectively, of an argon laser (30 milliwatt maximum output, operated at 50% or 6.3 A) attenuated to 10 and 2%, respectively, and directed to the cell via a 458/514 nm dual dichroic mirror. The emitted fluorescence was split via a 515 nm dichroic mirror and for CFP was directed to a photomultiplier equipped with a 465-495 nm bandpass filter (Chroma, Rockingham, Vermont) and for YFP was directed to a photomultiplier equipped with a 530-nm-long pass filter. Confocal fluorescence intensity data (ICFPpre and IYFPpre) were recorded as the average of four line scans per pixel and digitized at 8 bits. Repeated scans (20-60) with unattenuated 514 nm illumination were used to photobleach YFP, which required 30-120 s at maximal scan rates. After completion of YFP bleaching, fluorescence intensity (ICFPpost and IYFPpost) was measured using the identical parameters as before bleaching. FRET efficiency (E) in percent was calculated as E = (ICFPpost - ICFPpre/ICFTpost) x 100%, where ICFPpre and ICFPpost are the background-corrected CFP fluorescence intensities before and after photobleaching YFP, respectively. Data are reported as mean ± S.D. Statistical significance was tested with an unpaired Student's t test.
To measure the recovery of yellow fluorescence after photobleaching of YFP, myotubes were first imaged for cyan and yellow fluorescence as already described above. Within this image, a smaller region-of-interest was designated for photobleaching of YFP, which was accomplished with repeated scans of non-attenuated 514 nm excitation. Complete bleaching required about 70 s (similar sized regions of interest were used for all these experiments). Images of cyan and yellow fluorescence were then obtained as above every 30 s, beginning immediately after, and continuing until 400 s after, completion of the bleaching.
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RESULTS
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1a Subunit Constructs Are Targeted to the Membrane in the Presence of 1S and Restore EC Coupling and L-type Ca2+ Current—To analyze the arrangement of 1a subunits at plasma membrane/SR junctions, both singly labeled (CFP-1a, YFP-1a, 1a-CFP, and 1a-YFP) and doubly labeled (CFP-1a-YFP and YFP-1a-CFP) constructs were examined. Confocal microscopy and functional measurements were used to verify that these fusions of one or two fluorescent proteins to the 1a subunit did not affect targeting or the ability of the DHPR to function as a voltage sensor for EC coupling. Fig. 2 illustrates confocal fluorescence sections of myotubes expressing CFP-1a-YFP. After expression in dysgenic myotubes (lacking 1S), CFP-1a-YFP was diffusely distributed within the cytoplasm of the cell (Fig. 2a). When the same construct was coexpressed with unlabeled 1S in dysgenic myotubes, it was arranged in fluorescent puncta, many of which were close to the surface of the cell (Fig. 2b). Similar, fluorescent puncta were also observed when CFP-1a-YFP was expressed in b1-null myotubes, which endogenously express 1S (Fig. 2c). Patterns of fluorescence like those illustrated for CFP-1a-YFP in Fig. 2 were also seen for YFP-1a-CFP (data not shown). The diffuse fluorescence of 1a in absence of 1S, and the punctate fluorescence in the presence of 1S, agrees with previous results with CFP-YFP-tagged 1a (12), as well as with results from GFP-tagged 1a (16). Furthermore, in the latter study, the puncta of GFP fluorescence colocalized with puncta of 1S and RyR1 as revealed by immunolabeling. Thus, the diffuse fluorescence appears to represent the 1a subunit free in the myoplasm of the cell, whereas the fluorescent puncta appear to correspond to DHPRs inserted into junctional regions of the plasma membrane.
All of the singly labeled 1a constructs had the same type of expression pattern as shown in Fig. 2 for doubly labeled 1a. Moreover, when mixes of singly labeled 1a constructs (CFP-1a + 1a-YFP, CFP-1a + YFP-1a, or 1a-CFP + 1a-YFP) were expressed in 1-null cells, colocalized puncta of cyan and yellow fluorescence were observed (Fig. 3). Thus, the clusters of DHPRs, which produce the visible puncta, appeared to contain the CFP- and YFP-tagged 1a with approximately equal probability.
Previously, we demonstrated that the presence of a CFP-YFP tandem on either the N or C terminus of 1a did not affect function of the DHPR as either a voltage sensor for EC coupling or as an L-type Ca2+ channel (12). To determine whether the addition of fluorescent proteins to both the N and C termini affected function of 1a, we measured evoked contractions and L-type Ca2+ currents in 1-null myotubes expressing CFP-1a-YFP. Such myotubes produced robust contractions in response to focal extracellular stimulation (Fig. 4A and Table 1) and also produced large, whole-cell L-type Ca2+ currents (Fig. 4, B and C). Thus, the function of 1a did not appear to be altered by the attachment of fluorescent proteins.
Photobleaching of Labeled 1a Subunits—To further compare labeled 1a subunits in the absence and presence of 1S, subregions of cells were subjected to intense illumination at 514 nm to bleach YFP. Fig. 5 illustrates this kind of experiment for YFP-1a-CFP expressed in either a dysgenic myotube without 1S (A) or coexpressed in a dysgenic myotube together with unlabeled 1S (B). In the absence of 1S, both the yellow and cyan fluorescence had a diffuse appearance. The 1a subunits producing this diffuse pattern appeared to be freely mobile, since immediately after the bleaching episode (total duration of 70 s), there was a partial loss of yellow fluorescence outside the portion of the myotube exposed to bleaching illumination, and within the bleached region the intensity of yellow fluorescence gradually increased as the distance to the non-bleached portion of the myotube decreased (Fig. 5A, panel b). Moreover, by 400 s after the end of the bleaching episode, there was a restoration of uniform pattern of yellow fluorescence observed prior to bleaching (Fig. 5A, panel c). The time course of the yellow fluorescence intensity in Fig. 5C clearly shows recovery within the bleached region relative to the non-bleached region, while the cyan fluorescence remains relatively stable. Note the small increase in cyan fluorescence immediately after the bleaching of YFP, indicative of the FRET from CFP to YFP prior to bleaching. These results appear to indicate that there is negligible binding of the labeled 1a subunits to cellular structures. By contrast, the majority of 1a subunits in the presence of 1S appeared to be immobile because of anchoring within junctional membranes. Thus, there was a sharp demarcation in yellow fluorescence between bleached and non-bleached regions of the myotube and no loss of yellow fluorescence in the non-bleached regions (Fig. 5B, panel b). Furthermore, even 400 s after the completion of bleaching (Fig. 5B, panel c), there was essentially no recovery of yellow fluorescence within puncta inside the bleached region, although a small amount of diffuse fluorescence did recover within the bleached region. The corresponding time course of YFP fluorescence intensity within (YELLOW 2) as well as outside (YELLOW 1) the bleached area is shown in Fig. 5D. The small continuous decrease of YELLOW 1 can be attributed to bleaching due to repetitive scanning whereas the slow recovery of YELLOW 2 may reflect diffusional invasion of 1a subunits not associated with 1S subunits. Results similar to those illustrated in Fig. 5A were obtained from a total of 6 dysgenic myotubes without 1S and similar to those illustrated in Fig. 5B from a total of 3 dysgenic myotubes plus unlabeled 1S and a total of 2 1-null myotubes.
Measurement of Intra- and Intermolecular FRET—For doubly labeled 1a, FRET efficiencies were compared for subunits diffusely distributed in the cytoplasm with the efficiencies for subunits associated with 1S subunits in plasma membrane/SR junctions (Fig. 6). Diffuse cytoplasmic distribution was studied by expression in dysgenic myotubes without 1S (see Fig. 2a). For CFP-1a-YFP and YFP-1a-CFP in the cytoplasm, the FRET efficiencies were 6.9 ± 1.8 and 5.9 ± 1.9, respectively. For the doubly labeled subunits targeted to junctional membranes, the FRET efficiency varied depending on the conditions of expression. When coexpressed in dysgenic myotubes with unlabeled 1S (see Fig. 2b) the FRET efficiencies were reduced compared with those of the same constructs in the cytoplasm, being 3.4 ± 1.1 and 3.3 ± 1.6% for CFP-1a-YFP and YFP-1a-CFP, respectively. Two issues complicate the interpretation of this result. First, the measured FRET efficiency for doubly labeled 1a subunits in junctions could contain contributions from both intra- and intermolecular FRET. Second, dysgenic myotubes endogenously express 1a subunits, which could have reduced intermolecular FRET. To investigate the latter, the doubly labeled constructs were expressed in 1-null myotubes (see Fig. 2c). Under these conditions, the FRET efficiencies were 7 ± 2.8 and 5.6 ± 2.6% for CFP-1a-YFP and YFP-1a-CFP, respectively. Thus, FRET efficiencies for doubly labeled 1a in junctions were higher in 1-null myotubes (Fig. 6, iii) than in dysgenic myotubes (Fig. 6, ii). To confirm that this difference was a consequence of the presence of unlabeled 1a, FRET efficiencies were measured for the doubly labeled constructs coexpressed with unlabeled 1a in 1-null myotubes, which yielded FRET efficiencies of 3.1 ± 0.9% for CFP-1a-YFP and 3.0 ± 1.9% for YFP-1a-CFP (Fig. 6, iv). Thus, the presence of unlabeled 1a under two conditions (dysgenic myotubes + unlabeled 1S: Fig. 6, ii; and 1-null myotubes + unlabeled 1a: Fig. 6, iv) yielded FRET efficiencies about half those of the doubly labeled, junctionally targeted constructs in the absence of unlabeled 1a (Fig. 6, iii).
One way to explain the results in Fig. 6 is to suppose that (i) intramolecular FRET is lower for doubly labeled 1a in junctions compared with that in cytoplasm and (ii) the measured FRET efficiency for doubly labeled 1a in junctions contains contributions from both intra- and intermolecular FRET. Accordingly, the presence of unlabeled 1a would reduce the contribution of the inter-molecular component. If this explanation were correct, one would expect to be able to measure intermolecular FRET with singly labeled constructs. This was tested by coexpression in 1-null myotubes of 1:1 mixtures of CFP-1a + YFP-1a, 1a-CFP + 1a-YFP, or CFP-1a + 1a-YFP. For none of these mixtures was detectable FRET observed (Table 2), which argues against a contribution of intermolecular FRET for the doubly labeled 1a constructs in 1-null myotubes.
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DISCUSSION
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The four major findings of the present work are as follows. First, the addition of fluorescent protein to the N terminus, C terminus, or both termini of 1a does not interfere with its targeting or function. In the presence of 1S (in 1-null myotubes or coexpressed with 1S in dysgenic myotubes), the 1a constructs are incorporated into DHPRs that are present in punctate regions of the plasma membrane that form junctions with the SR. The DHPRs containing these labeled 1a subunits retain the ability to mediate excitation-contraction coupling. Second, in the absence of 1S, there were no foci of 1a indicative of binding to other cellular structures and these 1a subunits appeared to be freely diffusible in the cytoplasm. Third, in the cytoplasm: the 6-7% intramolecular FRET efficiency of the doubly labeled constructs (CFP-1a-YFP or YFP-1a-CFP) expressed in dysgenic myotubes indicates that the N and C termini of cytoplasmic 1a subunits are likely separated by <10 nm. Fourth, within junctions: there was no measurable intermolecular FRET for 1:1 mixtures of CFP-1a + YFP-1a, 1a-CFP + 1a-YFP, or CFP-1a + 1a-YFP expressed in -null myotubes. This result is consistent with the idea that then N and C termini of adjacent 1a subunits are >10 nm apart, which would occur if they were oriented away from the center of tetrads.
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FIGURE 5.
Photobleaching of yellow fluorescence associated with YFP-1a-CFP. Images of cyan and yellow fluorescence of YFP-1a-CFP expressed in dysgenic myotubes without (A) or with (B) unlabeled 1S are shown. The images were acquired immediately before (panel a), immediately after (panel b), and 400 s after (panel c) the bleaching. The dotted red rectangle indicates the region of yellow fluorescence bleaching (see "Materials and Methods" for details). Note that the yellow fluorescence shows strong recovery in A, panel c, but little recovery in B, panel c. Also note that the fluorescence in A has a quite uniform appearance because the illustrated myotube lacked nuclei in this region. Bars, 5 µm. For the cells illustrated in A and B, the time courses are shown in C and D, respectively, for fluorescence intensity within the indicated subregions. In C, the time course of the average intensity in subregion 1 divided by the average intensity in subregion 2 is shown for both yellow ( ) and cyan ( ), normalized to the value obtained prior to bleaching. This ratio of region 1 to region 2 was used to correct for the loss of fluorescence outside of the directly bleached area. In D, the intensity of the yellow puncta labeled 1 ( ) and 2 ( ) is shown as a function of time, normalized by the prebleach values.
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After expression in dysgenic myotubes lacking 1S, doubly labeled 1a was diffusely distributed (Figs. 2a and 5A). Such a diffuse distribution would seem to indicate that in the absence of 1S, the 1a subunits do not bind to immobile cellular structures. Consistent with this idea, yellow fluorescence at the ends of dysgenic myotubes showed substantial recovery within several minutes following the selective bleaching of YFP within YFP-1a-CFP or CFP-1a-YFP. The observation here that doubly labeled 1a is diffusely distributed in the absence of 1S is consistent with earlier results showing a diffuse distribution after expression in dysgenic myotubes of either 1a subunits tagged with a CFP-YFP tandem (12) or with GFP (16). However, small, weak puncta on a diffuse background were observed following immunostaining for endogenous 1a in dysgenic myotubes, from which it was concluded that the isolated 1a subunits could bind to some component of plasma membrane/SR junctions (14). One possible explanation for the discrepancy between the results with immunostaining and expressed fluorescent protein-labeled 1a is that immunostaining puncta in dysgenic myotubes reflect nonspecific sites of anti-1a antibody binding. Alternatively, the presence of fluorescent protein might interfere with the ability of isolated 1a to bind to other cellular proteins, although if this occurred it would be difficult to explain why fluorescent protein labeling had no observable effect on 1a function (12, 16).
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FIGURE 7.
Schematic model of 1a subunit structure and localization. The 1a subunits are indicated by dark gray ovals, with CFP and YFP indicated by the small, cyan and yellow ovals. In the absence of 1S (A), the 1a subunits are not bound to other cellular proteins and are thus diffusely distributed in the cytoplasm at an average distance too large for intermolecular FRET to occur from one 1a subunit to another. Within a single 1a subunit, the N- and C-terminal fluorescent proteins are separated by <10 nm and thus support intramolecular FRET. In the presence of 1S (B), the 1a subunits are present within the multiprotein complex that constitutes the DHPR (light gray oval), which assemble into a group of four (a tetrad) in association with RyR1 (outlined by the black square). The view shown is looking from the extracellular space in toward the SR and is based on the model of Wolf et al. (17) with respect to the general size and shape of the entire DHPR complex, the arrangement of the DHPRs within a tetrad, and approximate position, size, and shape of the 1a subunit with the DHPR. According to this model, the 1a subunits within tetrads are too far separated to support intermolecular FRET. The dotted circle has a diameter of 10 nm, the practical maximum separation consistent with measurable FRET.
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Within junctions, the FRET efficiency (E) for the doubly labeled 1a was 6-7% in 1-null myotubes (where no unlabeled 1a was present) and decreased to about 3% when unlabeled 1a was present (expression in dysgenic myotubes or expression together with unlabeled 1a in 1-null myotubes). The interpretation of this result requires one to consider the possibility that measured FRET efficiency within junctions ("jun") contained contributions from both intra- and intermolecular FRET (Ejun = ajun + e), where a and e are the intra- and intermolecular FRET efficiencies, respectively). For doubly labeled 1a in the cytoplasm ("cyt"), only intramolecular FRET would occur (Ecyt = acyt). One could then imagine a number of possibilities for why the presence of unlabeled 1a would cause a decrease in E.
One possibility is that the only effect of unlabeled 1a was to reduce the contribution of intermolecular FRET in junctions (because the unlabeled 1a reduced the number of adjacent doubly labeled 1a subunits within tetrads). If this were the case, one would have to conclude that e 
4% and that ajun 
3%, which is substantially less than acyt (6-7%). This change in intramolecular FRET could be a consequence of a conformational change in 1a that occurred upon binding to 1S (for example, an increase in separation between the N and C termini). However, x-ray crystallography demonstrates that the structure of the subunit core is little affected by binding to the AID of the 1 I-II loop (9). Moreover, this interpretation suffers from the weakness that intermolecular FRET was not detected for combinations of singly labeled 1a subunits (Table 2).
A second possibility is that e = 0 and that the conformation of the doubly labeled 1a subunit depends upon whether or not its neighboring 1a subunit had attached fluorescent proteins. Specifically, one could imagine that the presence of two fluorescent proteins on all four 1a subunits within a tetrad produces "molecular crowding" that results in a decreased distance between the N and C termini of each of the individual 1a subunits. The presence of one or more unlabeled 1a subunits could then allow a relaxation of the doubly labeled 1a subunits to the native conformation for 1a subunits bound to 1S. An argument against molecular crowding is that previous work has shown that addition of two fluorescent proteins per 1a subunit (tagging with CFP-YFP tandems) has no discernible affect on DHPR function as channel or voltage sensor for EC coupling (12).
A third possibility is that e = 0, the observed FRET signal from puncta is entirely intramolecular, and the effect of unlabeled 1a can be attributed to errors in measurement of cyan intensity. In particular, the contrast between the cyan fluorescence signal and cellular autofluorescence can sometimes be low. The presence of unlabeled 1a within the puncta would decrease the cyan fluorescent signal and cause a further loss of contrast. Because the autofluorescence is unaffected by bleaching of YFP, its presence would cause an underestimate of FRET efficiency, which would become worse as the contrast between cyan fluorescence and autofluoresence decreased as a result of unlabeled 1a.
Whatever the explanation for the effect of unlabeled 1a on the FRET signal produced by doubly labeled 1a, it remains the case that we were unable to measure directly any intermolecular FRET from N to N terminus, from C to C terminus or from N to C terminus of adjacent 1a subunits in tetrads. Taken together with the presence of N- to C-terminal intramolecular FRET for cytoplasmic and junctional 1a, our results are consistent with the model presented in Fig. 7. Individual, 1a subunits are sufficiently compact (<10 nm) that FRET occurs between fluorescent proteins on the N and C termini. In the absence of 1S, the 1a subunits do not appear to bind to other cellular structures and are randomly distributed in the cytoplasm at an average distance too large to allow intermolecular FRET (Fig. 7A). In the presence of 1S, the 1a subunits are assembled into DHPRs, which are organized into groups of four (tetrads), where each tetrad is associated with a single RyR1 (Fig. 7B). Within tetrads, intramolecular FRET occurs within individual doubly labeled 1a subunits, but adjacent subunits are separated by too great a distance to allow FRET. The specific model illustrated in Fig. 7B is based on the arrangement proposed by Wolf et al. (17), but our data are also consistent with the tetradic arrangement of DHPRs suggested by Paolini et al. (18). Independent of any particular arrangement of DHPRs into tetrads, our data are most easily explained by the hypothesis that the N and C termini of 1a lie outside a 10-nm diameter circle at the center of each tetrad. An important goal of future work will be to refine our knowledge of the spatial orientation of defined DHPR regions with respect to tetrads and with respect to defined regions of RyR1.
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FOOTNOTES
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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
1 To whom correspondence should be addressed: Dept. of Biomedical Sciences, 1617 Campus Delivery, Colorado State University, Fort Collins, CO 80523-1617. Tel.: 970-491-5277; Fax: 970-491-7907; E-mail: kbeam{at}lamar.colostate.edu.
2 The abbreviations used are: EC, excitation-contraction; DHPR, dihydropyridine receptors; RyR, ryanodine receptor; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; GFP, green fluorescent protein; SR, sarcoplasmic reticulum.
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