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J. Biol. Chem., Vol. 279, Issue 42, 44046-44056, October 15, 2004
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From the Department of Biomedical Sciences, Anatomy Section, Colorado State University, Fort Collins, Colorado 80523-1617
Received for publication, May 12, 2004 , and in revised form, July 27, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1S and 
1a subunits of the DHPR. For all of the sites examined, attachment of the CFP-YFP tandem did not impair function of the DHPR as a Ca2+ channel or voltage sensor for excitation-contraction coupling. The free tandem displayed a 27.5% FRET efficiency, which decreased significantly after attachment to the DHPR subunits. At several sites examined for both 1S (N-terminal, proximal II-III loop of a two fragment construct) and 1a (C-terminal), the FRET efficiency was similar after expression in either dysgenic (1S-null) or dyspedic (RyR1-null) myotubes. However, compared with dysgenic myotubes, the FRET efficiency in dyspedic myotubes increased from 9.9 to 16.7% for CFP-YFP attached to the N-terminal of 1a, and from 9.5 to 16.8% for CFP-YFP at the C-terminal of 1S. Thus, the tandem reporter suggests that the C terminus of 1S and the N terminus of 1a may be in close proximity to the ryanodine receptor. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1S subunit and the auxiliary subunits 2-
, 1a, and 
(1-3). The ryanodine receptor is a calcium release channel composed of four identical subunits (4). One argument for conformational coupling between the DHPR and RyR1 is that depolarization of the plasma membrane elicits calcium release from the SR without the requirement for entry of extracellular Ca2+ (5). In addition to this orthograde signal from the DHPR to RyR1, there is also a retrograde signal whereby RyR1 increases the magnitude of the Ca2+ current via the DHPR (6). Morphological evidence strongly supports the idea that this bidirectional signaling involves physical links (direct or indirect) between DHPRs and RyRs in skeletal muscle. In particular, freeze-fracture images of the plasma membrane at junctions with the SR reveal clusters of intramembranous particles that appear to represent DHPRs arranged in groups of four (tetrads) such that each DHPR is apposed to a subunit of an RyR (7). Additionally, this arrangement of DHPRs requires the presence of RyR1 because the arrangement of clustered particles into tetrads does not occur in dyspedic (RyR1-lacking) myotubes (8).
In an attempt to determine whether, and at what sites, interactions occur between the DHPR and RyR1, a number of different approaches have been utilized. This work has yielded no consistent picture. As one example, application of peptides corresponding to 1S(II-III) loop residues 671-690 was found to activate isolated RyRs (9-12), but scrambling or deleting these residues did not interfere with the ability of 1S constructs to restore skeletal-type EC coupling in dysgenic myotubes (13-16). As another example, biochemical studies found that the 1S(II-III) and (III-IV) loops interact with residues 954-1112 of RyR1 but not with the corresponding RyR2 residues (17). However, analysis of RyR chimeras revealed that replacing RyR1 residues 1-1634 with those of RyR2 has little effect on bidirectional signaling (18). Other biochemical studies showed an interaction between RyR1 residues 3609 and 3643 and the proximal portion (1393-1527) of the 1S C-terminal (19). However, if this interaction actually occurs in vivo, it cannot be a sole determinant of skeletal-type EC coupling because replacing the II-III loop of 1C with that of 1S is sufficient to convert EC coupling from cardiac-type (Ca2+-entry dependent) to skeletal-type, independent of the identity (cardiac versus skeletal) of the C-terminal (20). Indeed, a subset of 1S residues (720-765) in the II-III loop have been shown to be both critical for bidirectional signaling (21) and to interact very weakly with RyR1 residues 1837-2168 in yeast two-hybrid studies (22). However, this interaction also seems unlikely to be the sole determinant of skeletal type coupling because (i) strong coupling is mediated in dyspedic myotubes by a chimera in which RyR1 residues 1645-2154 are replaced by RyR2 residues 1637-2118 (22), and (ii) weak skeletal-type coupling can be produced in dysgenic myotubes by an 1S lacking residues 720-765 as long as a segment of upstream sequence (671-690, peptide A) is also removed (15). In addition to the 1S(II-III) loop, a role for the 1a C-terminal has been suggested from analyses of cDNA constructs expressed in 1-null myotubes (23, 24). Moreover, preliminary biochemical studies have suggested that 1a binds to RyR1 in vitro (25).
In evaluating the various results described above, one must take into account the limitations of each of the approaches. In the case of the biochemical and peptide experiments, it is possible that regions of the DHPR and RyR are artificially brought into contact with one another in a manner that cannot occur in vivo. In the case of the expression of cDNAs encoding DHPRs and RyRs, the observation that function is altered by changes introduced into a segment of primary sequence does not reveal whether that segment actually represents a site of intermolecular contact. Thus, we have undertaken a new approach in the attempt to obtain information about the molecular architecture of DHPRs and RyRs within plasma membrane-SR junctions of living muscle cells. This approach makes use of fluorescent resonant energy transfer (FRET) between donor and acceptor fluorophores (ECFP and EYFP in the present experiments) (26). FRET is an appropriate tool for analyzing the disposition of donors and acceptors that are separated by distances (
10 nm or less) that are relevant for interacting proteins in vivo (27). In addition to being very sensitive to donor-acceptor separation, FRET efficiency is also affected by the relative orientation of the donor and acceptor moieties (28). We have engineered cDNAs in which a CFP-YFP tandem has been incorporated at varying positions of 1S and 1a. This approach allows one to determine whether the FRET differs for the CFP-YFP tandem attached to these sites from the FRET of the free CFP-YFP tandem. At all sites examined, FRET efficiency was reduced when the tandem was attached to the DHPR subunits within junctional membranes. Comparison of FRET efficiency after expression in dysgenic and dyspedic myotubes allowed determination of whether RyR1 influenced the environment of the attachment sites. Interestingly, the presence of RyR1 did not affect the FRET signal of the CFP-YFP tandem attached to the C-terminal of 1a, the N-terminal of 1S, or the II-III loop of two-fragment 1S constructs. By contrast, the FRET efficiency of the tandem attached either to the N-terminal of 1a or the truncated C-terminal of 1S was significantly reduced by the presence of RyR1. Thus, the tandem reporter suggests that RyR1 may be in close proximity to these two sites of the DHPR. In the accompanying article (29), we describe another approach for probing the topology of junctions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1S—The coding sequence for rabbit skeletal muscle 1S (GenBankTM number M23919
[GenBank]
) was excised from its original vector (30) using a SalI site immediately before the ATG start codon and a SalI site that had been introduced via mutagenesis (QuikChange, Stratagene, La Jolla, CA) at nucleotide 5581 (immediately after amino acid 1860 of 1S, where 1873 is full-length). The SalI-SalI fragment containing the 1S coding sequence was inserted into the multiple cloning site of the SalI-digested pEYFP-C1 (or pEYFPN1) to create constructs encoding the N- and C-terminal labeled fusion proteins YFP-1S and 1S-YFP, respectively (these constructs contained a KpnI site 6 and 10 nucleotides, respectively, downstream from the end of the 1S coding sequence). Restriction digests and sequencing were used to verify these constructs.
Unlabeled 1S—To provide an unlabeled 1S construct with an expression level comparable with that of the fluorescently labeled subunits, 1S-YFP was digested with KpnI and MfeI (unique sites), and the small fragment containing the YFP sequence was discarded. The large fragment containing the 1S coding sequence was closed by ligating it to the 126-bp fragment of pEYFP-C1 that had been digested with the same enzymes.
CFP-YFP-1S—To label the N-terminal of 1S with the CFP-YFP tandem, the AgeI-XmaI fragment of pECFP-C1 (containing the CFP encoding sequence) was ligated to YFP-1S that had been opened with an AgeI cut proximal to the YFP coding sequence. In this construct, a 15-residue linker connected the tandem to the first 1S residue.
1Sshort-CFP-YFP—To label the C-terminal of 1S, a second KpnI site was introduced into 1S-YFP by PCR mutagenesis to change the wild-type 1S sequence 4999AATGTTGCC to 4999AAGGTACC. After digestion with KpnI to remove the sequence coding for the distal part of the 1S C-terminal, the plasmid was re-ligated to yield a cDNA encoding 1S with a truncated C-terminal ("1S-(1-1667)"). The YFP sequence was then replaced by the CFP-YFP tandem sequence by cutting both 1S-(1-1667)-YFP and the CFP-YFP plasmid with AgeI and MfeI and ligating the fragment containing 1S-(1-1667) to the one carrying the CFP-YFP sequence, giving 1S-(1-1667)-CFP-YFP (=1Sshort-CFP-YFP). In this protein, 1S was attached to the tandem via a 12-residue linker.
Two-Fragment 1S-labeled with CFP-YFP—One plasmid ("1S(I-II)CFP-YFP") contained the coding sequence for residues 1-671 of rabbit 1S (including repeats I and II together with a small portion of the II-III loop), followed by an 11-residue linker and the CFP-YFP sequence. The second plasmid ("1S(III-IV)") encoded residues 686-1860 (including a large part of the cytoplasmic II-III loop in addition to repeats III and IV). To make 1S(I-II)-CFP-YFP, a second KpnI site was introduced into YFP-1S at a position corresponding to Ser672 of 1S. The mutated plasmid was then cut with KpnI to remove the sequence coding for 1S residues 672-1860 and re-ligated. The resulting YFP-1S-(1-671) was digested with BamHI and PshAI to yield a 1,142-bp fragment containing the sequence for 1S residues 1-671, which was ligated to the 6,389-bp fragment of the BamHI/PshAI-digested 1S-(1-1667)-CFP-YFP to obtain 1S(I-II)-CFP-YFP. The 1S(III-IV) expression vector was constructed by using PCR mutagenesis to introduce a HindIII just upstream of the ATG codon for Met686 into the coding sequence of expression vector for unlabeled 1S (described above). The plasmid was digested with HindIII, which cut both at this introduced site and a second already present upstream of the first ATG of 1S, and then re-ligated.
1S(I-II)-CFP-YFP-(III-IV)—This construct encodes 1S residues 1-671, followed by the CFP-YFP tandem, followed by 1S residues 686-1860 (1S-(1-671), 11 residue linker, CFP-YFP, 12 residue linker, 1S-(686-1860)). The plasmid was made by first opening the 1S(III-IV) construct (see above) with NheI and HindIII at the multiple cloning region preceding the 1S coding sequence and introducing the EYFP coding sequence to obtain YFP-1S(III-IV). The ECFP coding sequence was then added to this plasmid by opening YFP-1S(III-IV) proximal to the EYFP coding sequence with NheI and AgeI and inserting the NheI-XmaI fragment obtained upon digestion of ECFP-C1. The resulting CFP-YFP-1S(III-IV) was then cut with AgeI and MfeI, and the 5.2-kb fragment encompassing the CFP-YFP-1S(III-IV) sequence was finally subcloned into 1S(I-II)-CFP-YFP that had been treated with AgeI and MfeI to remove CFP-YFP.
CFP-YFP-labeled Rabbit 1a Subunit—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 or pEYFP-N1 to obtain the intermediate constructs YFP-1a and 1a-YFP, respectively. CFP-YFP-1a was made by cutting YFP-1a with AgeI and ligating it with the fragment containing the CFP sequence of AgeI/XmaI-digested pECFP-C1 (a 12-residue linker connected CFP-YFP to 1a). 1a-CFP-YFP (with a 13-residue linker connecting 1a to CFP-YFP) was created by first removing the YFP sequence from 1a-YFP via AgeI and DraIII cuts and then ligating the fragment containing the 1a sequence to a fragment containing the CFP-YFP sequence, obtained by digestion of the CFP-YFP tandem with the same enzymes. Correct orientation of the sequences was checked with appropriate restriction digests.
Unlabeled Rabbit 1a Subunit—To provide an unlabeled 1a construct with an expression level comparable with that of the fluorescently labeled subunits, 1a-YFP was digested with Acc651 and BsrGI (unique sites) and the small fragment containing the YFP sequence was discarded. The large fragment containing the 1a coding sequence was closed by ligation.
Cell Culture and cDNA Microinjection
Primary cultures of dysgenic (31) and dyspedic (8) and 1-null (23) myotubes were prepared from newborn mice as described previously (32). Briefly, myoblasts were plated into 35-mm culture dishes with collagen-coated glass coverslip bottoms (MatTek, Ashland, MA) 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 ng/µl for both the CFP-YFP tandem and the 1a constructs, and 50-80 ng/µl for the 1S constructs. These same concentrations were used for each of the individual plasmids when combinations of plasmids were injected. For the measurement of Ca2+ currents, the injected cDNA concentration was 20 ng/µl for all the 1a constructs and 100 ng/µl for all the 1S constructs except for the two fragment construct: 60 ng/µl for 1S(I-II)-CFP-YFP, and 100 ng/µl for 1S(III-IV). Myotubes expressing unlabeled 1S or 1a were usually identified by co-injection of 10 or 2 ng/µl, respectively, of a cDNA expression plasmid encoding EYFP.
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 via 100-V, 30-ms pulses applied via a patch pipette that contained physiological saline and was positioned near the myotube.
Characterization of Ca2+ Currents
Macroscopic Ca2+ currents were measured with the whole cell patch clamp method (33). Patch pipettes of borosilicate glass had resistances of 2-3.5 M
when filled with an intracellular solution containing (mM): 140 CsCl, 5 MgCl2, 10 Cs2EGTA, and 10 HEPES, pH 7.4, with CsOH. The external bath solution contained (mM): 10 CaCl2, 145 tetraethylammonium-C1, 0.003 tetrodotoxin, and 10 HEPES, pH 7.4, with tetraethylammonium-OH. To measure the L-type current, myotubes were stepped from the holding potential (-80 mV) to -20 mV for 1 s (to inactivate endogenous T-type current), repolarized to -50 mV for 50-100 ms, depolarized to varying test potentials (Vtest) for 200 ms, repolarized to -50 mV for 100 ms, and then returned to the holding potential. Test currents were corrected for linear components of leak and capacitive current by digitally scaling and subtracting the average of 11 control currents elicited by a hyperpolarizing step to -110 mV delivered from the holding potential prior to each test pulse. Cell capacitance was determined by integration of a transient from -80 to -70 mV using Clampex 8.0 (Axon Instrument, Foster City, CA) and was used to normalize current amplitudes (pA/pF). Current-voltage (I-V) curves were fitted using the following Boltzmann expression,
 | (Eq. 1) |
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.
Measurements of FRET
Intact fluorescent myotubes were examined 24-48 h after cDNA microinjection using the confocal laser scanning microscope LSM 510 META (Zeiss, Thornwood, NY). With physiological saline, spontaneous contractions occurred in a large fraction of dysgenic myotubes expressing the 1S constructs and in 1-null myotubes expressing 1a constructs. Because such contractions would interfere with analysis of FRET, the contractions were eliminated by bathing the cells in a Ca2+/Mg2+-free saline (containing in mM: 155 NaCl, 5 KCl, 11 glucose, 10 HEPES, pH 7.4, with NaOH). Based on measurements of steady-state inactivation of sodium currents (100-ms prepulses), this solution appeared to shift the effective transmembrane potential by at least +25 mV (not shown).
An area of 500 to 2500 µm2 was selected from the field of view (x63 oil objective, 1.4 NA), which included the part of the myotube to be analyzed and also an adjacent, non-cellular region for measurement of background fluorescence. 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 6.3A) directed to the cell via a 458/514 nm dual dichroic mirror. The emitted fluorescence was split via a 515-nm long-pass, and for CFP was directed to a photomultiplier equipped with a 465-495-nm bandpass filter (Chroma, Rockingham, VT) and for YFP was directed to a photomultiplier equipped with a 530-nm long-pass filter. With this arrangement, there was no cross-talk between the CFP and YFP fluorescence because CFP is not excited at 514 nm and because YFP does not emit in the 465-495 nm range. Confocal fluorescence intensity data (ICFPpre and IYFPpre) were recorded as the average of four line scans per pixel and digitized at 8-bits, with photomultiplier gain adjusted such that maximum pixel intensities were no more than 70% saturated (particularly for CFP, which increased in intensity after photobleaching of YFP). Repeated scans (20-60) with 514 nm at maximum laser intensity (20-100-fold greater than for image capture) 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,
 | (Eq. 2) |
where ICFPpre and ICFPpost are the background corrected CFP fluorescence intensities before and after photobleaching YFP, respectively.
In most cases, the constructs tagged with the CFP-YFP tandem were localized in many, spatially separated foci localized near the cell surface. For such constructs, it was important to calculate the FRET efficiency only for pixels contained in these foci. For this purpose, we used the software package "3D for LSM" (Zeiss), which allows the construction of a binary mask with the value of unity for all pixel intensities within a user-specified range and a value of zero elsewhere. The software then allows calculations based on only those pixels of the original images corresponding to the positions having the value of unity in the mask. We used this software to produce a digital mask from the YFP image prior to photobleaching (Fig. 6). With the mask, a "CFP-difference image," which corresponded to the numerator of Equation 2, was calculated by subtracting pixel-by-pixel the background-corrected intensities prior to bleaching (ICFPpre) from those after bleaching (ICFPpost). Finally, the FRET efficiency was calculated by dividing the sum of all the pixel intensities for the CFP difference image by the sum of all the pixel intensities of the background-corrected post-bleach CFP image (ICFPpost). At times, slight movements of the cells during the measurements, along with a small variability of the scan-head, made it necessary to slightly shift the ICFPpost image to obtain perfect overlap with ICFPpre for analysis. Statistical comparisons were made on the basis of the unpaired t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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100 s that was evidently sufficient to allow diffusional mixing of tandems in the z direction) and (ii) FRET efficiencies similar to those in myotubes were found for the CFP-YFP tandem expressed in much flatter cells (HEK293 cells, data not shown).
All 1S Constructs Are Correctly Targeted to Junctions and Restore EC Coupling—Because the goal of the present work was to use the CFP-YFP tandem to probe the environment of specific regions of 1S and 1a in the context of a functional EC coupling apparatus, it was important that the sites of incorporation meet a number of requirements. Specifically, it was important that some of these sites be close to regions that might be involved in signaling interactions with RyR1, whereas others be in regions not expected to be involved (and thus to serve as controls). Additionally, it was important that incorporation of the tandem not destroy the ability of the tagged protein to function as a voltage sensor for EC coupling and to target to junctions between the plasma membrane and SR. With these requirements in mind, one site chosen was the N-terminal of 1S because labeling at this site with a single green fluorescent protein had been shown previously not to interfere with targeting and function (30).
Fig. 3, a and b, illustrates a dysgenic (null for endogenous 1S) myotube that was injected with cDNA encoding CFP-YFP-1S and then examined with confocal fluorescence microscopy at a site relatively distant from the injected nucleus. In an optical section close to the surface of the myotube, both the yellow (Fig. 3a) and cyan fluorescence (not shown) were present in discrete, irregularly shaped foci that were about 1-2 µm in size. The preferential association of these fluorescent foci with the surface was also evident in an optical section taken roughly midway between the lower and upper surfaces of the myotube (Fig. 3b), although some fluorescent foci together with diffuse fluorescence were present in the interior of the cell. The prevalence of discrete foci within the interior varied somewhat between myotubes, perhaps as a function of maturation. Additionally, the diffuse interior fluorescence could be relatively intense at regions close to the injected nucleus. Overall, the size and distribution of the fluorescent foci was similar to that of the small clusters of DHPRs and RyRs as revealed by immunostaining of wild-type myotubes (34). Thus the fluorescent foci seem likely to represent groupings of DHPRs at presumptive contacts between the surface membrane and SR and/or between nascent transverse tubules and the SR. However, some of the fluorescent foci may also have represented DHPRs being delivered to the plasma membrane. Fig. 3c shows that a dyspedic myotube expressing CFP-YFP-1S displayed fluorescent foci similar to those of dysgenic myotubes expressing the same construct (e.g. Fig. 3, a and b). The presence of such fluorescent foci in a dyspedic myotube are consistent with previous results that demonstrate both that junctions form between the plasma membrane and SR, and that DHPRs target to those junctions, in muscle cells lacking RyR1 (35).
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1S, the CFP-YFP tandem was also placed within the II-III loop and at the C-terminal. In the latter case, the tandem was placed in a construct (1Sshort-CFP-YFP) that terminated at 1S residue 1667 (the native coding sequence terminates at residue 1873) to avoid the potential proteolytic cleavage that is believed to occur within 1S residues 1685-1699 (36). Truncation of the C-terminal after residue 1662 does not alter the function of unlabeled 1S as a calcium channel or voltage sensor for EC coupling (37). In the case of the II-III loop, two different constructs were examined. One of these, 1S(I-II)-CFP-YFP + 1S(III-IV), which separated the 1S sequence into two separate fragments, was based on previous work (15, 16) showing that similar two-fragment constructs (without the tandem) can complement one another to form DHPRs that function as both Ca2+ channels and voltage sensors for EC coupling. The second construct (1S(I-II)-CFP-YFP-(III-IV)) corresponded to a fusion of 1S(I-II)-CFP-YFP and 1S(III-IV) into a single protein. For both the single and double fragment constructs, 1S residues 672-685, which correspond roughly to the "peptide A" region (9), were deleted entirely and the CFP-YFP tandem was placed appreciably upstream of residues 720-765 that have been found to be critical for bidirectional signaling (21).
Expression in both dysgenic and dyspedic myotubes of 1Sshort-CFP-YFP, of 1S(I-II)-CFP-YFP + 1S(III-IV), or of 1S(I-II)-CFP-YFP-(III-IV) gave rise to the formation of small fluorescent patches that were similar to those illustrated in Fig. 3 for CFP-YFP-1S. As mentioned above, the size and distribution of these fluorescent patches is similar to those previously reported for DHPRs in junctions between the plasma membrane and SR, as revealed by immunostaining (34). Moreover, all the tagged 1S constructs were able to restore EC coupling as indicated by contractions in response to focal, electrical stimulation (Table I). Although not quantified, spontaneous contractions were also frequently observed in dysgenic myotubes expressing these constructs. Thus, the presence of the CFP-YFP tandem did not appear to grossly interfere with either the targeting or function of 1S. However, it should be noted that in myotubes expressing 1S(I-II)-CFP-YFP + 1S(III-IV), a variable amount of bright fluorescence was observed in the vicinity of the injected nucleus (those regions were omitted in FRET measurements). This fluorescence likely represented 1S(I-II)-CFP-YFP backed up in the biosynthetic pathway, although measurements of charge movement indicate that similar two-repeat 1S constructs are able to reach the plasma membrane (16, 38). However, the delivery of 1S(I-II) to the surface may be less efficient because such constructs do not target to junctional membranes without accompaniment by 1S(III-IV) (16).
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1a Interacts with 1S—The CFP-YFP tandem was attached to either the N- or C-terminal of 1a to produce CFP-YFP-1a and 1a-CFP-YFP, respectively. Under all conditions examined, the two constructs produced a similar pattern of fluorescence; typical examples are illustrated for 1a-CFP-YFP in Fig. 4. After expression in dysgenic myotubes lacking 1S, the CFP-YFP-labeled 1a produced a diffuse intracellular fluorescence similar to that of the free CFP-YFP tandem (Fig. 2), except that the tandem-labeled constructs were excluded from the nuclei (Fig. 4, left). By contrast, multiple fluorescent foci were present when the tandem-tagged constructs were expressed in cells that contained an 1S subunit; i.e. co-expressed with 1S in dysgenic myotubes, or expressed in either RyR1-null (dyspedic) or 1-null myotubes, as shown in Fig. 4. The diffuse fluorescence of CFP-YFP-tagged 1a in cells lacking 1S and punctate fluorescence in cells having 1S is consistent with previous work using green fluorescent protein-tagged 1a and immunolabeling with antibodies directed toward 1S and the ryanodine receptor (39). Thus the diffuse fluorescence probably reflects tagged 1a subunits that are free within the myoplasm, whereas the foci represent DHPRs that contain both 1S and the tagged 1a and have been inserted into junctional membranes. In support of this idea, when the YFP was completely photobleached within a subregion (a stripe of 10-20-µm width), the diffuse yellow fluorescence recovered quickly (<2 min) in dysgenic myotubes expressing the tandem-tagged 1a subunits, whereas there was no recovery of fluorescence in foci (over the same time period) in cells that expressed both a labeled construct and 1S. In addition to displaying the expected subcellular distribution, the tandem-tagged subunits were functional as assayed by restoration of EC coupling in 1-null myotubes (Table I).
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1S and 1a Constructs Form Functional Calcium Channels—To determine whether attachment of the CFP-YFP tandem affected calcium channel function, the patch clamp technique was used to measure whole cell currents in dysgenic myotubes expressing the tagged 1S constructs and in 1-null myotubes expressing the tagged 1a constructs. Fig. 5 illustrates peak current-voltage relationships for the various constructs. As shown in Fig. 5A, the amplitude and voltage dependence of calcium currents produced by either CFP-YFP-1S or 1Sshort-CFP-YFP were similar to those of unlabeled 1S. Interestingly, peak currents were slightly larger for both the one- and two-fragment 1S constructs containing CFP-YFP in the II-III loop (Fig. 5B), although the voltage dependence was similar to that of the untagged 1S. Both CFP-YFP-1a and 1a-CFP-YFP produced currents with magnitude and voltage dependence like those of unlabeled 1a (Fig. 5C). The currents for all the 1a constructs were larger, and had a steeper, left-shifted voltage dependence, compared with those of the 1S constructs. The kinetics of the currents produced by the CFP-YFP-tagged 1S and 1a subunits did not differ obviously from those of the untagged subunits (not shown).
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1a constructs, because there could be a substantial fraction of these constructs free within the cytoplasm. Because the fluorescent foci have an irregular shape and lack clearly defined boundaries, the approach taken was to use the pre-bleach yellow image (IYFPpre) to create a binary mask having the value of unity for all yellow pixels above an adjustable threshold intensity and a value of zero below the threshold (Fig. 6). The threshold was adjusted so that all low intensity pixels outside the fluorescent puncta vanished, leaving a binary mask that was congruent with the majority of the obvious foci of yellow fluorescence. The cyan intensity values (ICFPpre and ICFPpost) were then measured only for those pixels having the value of unity within the binary mask (Fig. 6). Additional details are provided under "Materials and Methods." FRET efficiencies for all the various constructs examined are summarized in Table I.
FRET Efficiency Is Altered by Junctional Insertion of 1a—Fig. 7 compares the FRET efficiencies of the free CFP-YFP tandem with the FRET efficiencies of the tandem attached to 1a. Compared with the free tandem, the tandem attached to either the N- or C-terminal of cytoplasmic 1a had a significantly (p < 0.001) reduced FRET efficiency (see Table I for values). These FRET efficiencies appear to truly reflect energy transfer between the linked CFP and YFP moieties because no measurable FRET was observed for dysgenic myotubes injected with isomolar mixtures (10 ng/µl each) of cDNAs for CFP-1a (or 1a-CFP) and YFP-1a (not shown). After co-expression with unlabeled 1S in dysgenic myotubes, the FRET efficiency was much lower both for tandem at the N-terminal and C-terminal, compared with cytoplasmic 1a (Fig. 7). These reduced FRET efficiencies suggest that membrane association and/or the presence of other proteins within the junctions alter the environment of the N- and C-terminals of 1a.
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1a but Not That of the C-terminal—To determine whether the presence of RyR1 contributed to the reduced FRET efficiencies of the CFP-YFP tandem attached to 1a in junctions, the tandem-tagged constructs were expressed in dyspedic myotubes. As shown in Fig. 4, tandem-tagged 1a subunits target to junctions in dyspedic myotubes, despite the lack of RyR1. For 1a-CFP-YFP, the FRET efficiency in dyspedic myotubes did not differ from that in dysgenic myotubes in which 1S was co-expressed (Fig. 7). However, for CFP-YFP-1a, the FRET efficiency in dyspedic myotubes was significantly greater (by 69%) compared with the same construct co-expressed with 1S in dysgenic myotubes. Thus, the presence of RyR1 causes a large reduction in FRET efficiency for the tandem at the C-terminal of 1a but has no effect on the efficiency of the tandem at the N-terminal, suggesting that RyR1 may be in closer proximity to the N-terminal than to the C-terminal.
RyR1 Alters the Environment of the C-terminal of 1S but Has Little Effect on the Proximal Portion of the II-III Loop—Fig. 8 summarizes the FRET efficiencies for the CFP-YFP tandem attached to sites of 1S. In all instances, the efficiency was lower for tandem attached to 1S than for the free tandem. We have no way of determining how much of this decrease was attributable to attachment per se and how much was attributable to the targeting of the constructs to junctions, because all of the tandem-tagged 1S constructs targeted to junctions.
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1S), there was no difference in efficiency between dysgenic and dyspedic myotubes. This result suggests that there may not be close proximity between RyR1 and the N-terminal of 1S. The similar FRET efficiency of CFP-YFP-1S in dysgenic and dyspedic myotubes also suggests that the FRET signal is dominated by energy transfer within tandems rather than between tandems attached to adjacent 1S subunits. Specifically, intermolecular FRET might be expected to decrease in dyspedic myotubes where, unlike dysgenic myotubes, the tagged 1S subunits would not organize into tetrads. As already just mentioned, no such decrease was observed.
As for the N-terminal, tandem in the II-III loop of two fragment constructs (1S(I-II)-CFP-YFP + 1S(III-IV)) produced similar FRET in dysgenic and dyspedic myotubes (Fig. 8), consistent with the idea that RyR1 does not closely approach the part of the loop (residue 671) to which the tandem was attached. However, this portion of the loop may be indirectly affected by association of RyR1 with 1S because the FRET of tandem at the same position within the loop of a single fragment construct (1S(I-II)-CFP-YFP-(III-IV)) actually displayed a slightly decreased efficiency in dyspedic myotubes compared with dysgenic myotubes. Whatever the interpretation, the tandem clearly exists in a different environment within the intact II-III loop than within the loop of two-fragment constructs. In the case of tandem on the (shortened) C-terminal of 1S, FRET efficiency was significantly greater in dyspedic myotubes compared with dysgenic myotubes. Thus, RyR1 may be in close proximity to the C-terminal.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1a in the cytoplasm (i.e. expressed in the absence of 1S), the FRET efficiency of the tandem attached to either the N- or C-terminal (CFP-YFP-1a, 1a-CFP-YFP) was lower than that of the unattached CFP-YFP tandem. An additional decrease in FRET efficiency occurred when either CFP-YFP-1a or 1a-CFP-YFP was inserted into junctions (by co-expression with 1S). Thus, the environment of N- and C-terminals of 1a is affected by proximity to the membrane and/or by the presence of junctional proteins. For the N-terminal, a portion of this decreased FRET efficiency was attributable to RyR1. In all cases for 1S (N-terminal, C-terminal, and within the II-III loop), the FRET efficiency of the attached CFP-YFP tandem was lower than that of the free CFP-YFP tandem. However, it was not possible to determine how much of this decreased efficiency was simply a consequence of attaching CFP-YFP to these sites independent of junctional targeting (because all the constructs targeted to junctions). The FRET efficiency for the 1S N-terminal was unaffected by the presence of RyR1, whereas that of the C-terminal was substantially higher when RyR1 was absent. For the 1S(II-III) loop, the influence of RyR1 depended on the construct examined. When 1S was expressed as two fragments divided at the II-III loop (1S residues 1-671-CFP-YFP co-expressed with 1S residues 686-1860), the efficiency was unaffected by RyR1. However, the presence of RyR1 increased the FRET efficiency when the tandem was inserted into the II-III loop of a single fragment construct, 1S-(1-671)-CFP-YFP-(686-1860).
Fig. 9 summarizes the effects of RyR1 on FRET efficiency at the sites examined for 1S and 1a. For those regions that display RyR1-dependent FRET efficiencies, a number of different mechanisms could be postulated. The possibility portrayed in Fig. 9 is that the decreases in FRET efficiency occurred because RyR1 interacts with nearby sites of the 1S and 1a subunits of DHPR and thus closely approaches the attached CFP-YFP tandem. If the postulated interaction between RyR1 and 1a actually occurs, it must require 1S. In particular, 1a expressed without 1S showed only a diffuse distribution (Fig. 4), whereas significant binding to RyR1 should have resulted in a punctate distribution. With respect to mechanism, a close approach between RyR1 and the tandem could produce a decrease in FRET efficiency by increasing the separation between the two fluorophores, by restricting their dipoles to a less favorable orientation with respect to one another, or by shifting the spectral properties of one or both fluorophores (e.g. by a change in local pH). There is also the possibility that association with RyR1 at one site could affect the environment of a far-removed second site by, for example, inducing a conformational change at the second site. This might explain why the presence of RyR1 increased the FRET efficiency of the CFP-YFP tandem within the II-III loop of the one-piece 1S construct. In this construct, the tandem was introduced 50 residues upstream from the critical domain (720-765; Ref. 40), which may interact with RyR1 (22). Thus, the conformational change affecting the CFP-YFP tandem might be propagated via the loop segment connecting the tandem to this critical domain. Such an explanation is attractive because the CFP-YFP tandem in the loop of the two-fragment 1S construct showed no dependence of FRET efficiency on the presence of RyR1. In any case, the effect of RyR1 on FRET efficiency of the tandem in the II-III loop of the one-fragment 1S construct seems unlikely to be of great functional significance, because there were no obvious differences between the one- and two-fragment 1S constructs with regard to behaviors as calcium channels and voltage sensors for EC coupling.
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Independent of the exact physical interpretation of the CFP-YFP FRET signals, our results demonstrate that functional triad junctions can accommodate the presence of substantial additional mass (56 kDa) placed 11-15 residues away from the sites illustrated in Fig. 1. Of course, it is possible that the structure of the DHPR, RyR, or other junctional proteins becomes altered to accommodate these fluorescent proteins (each of which occupies a cylindrical space 4.2 nm in height and 2.4 nm diameter; Ref. 41), but it seems unlikely that such structural alterations could occur in domains of functional importance. In this regard, it is very interesting that the single particle reconstruction of RyR1 shows pits and holes that are large enough (about 5 nm) to accommodate the CFP-YFP tandem. For RyR2, single particle analysis of frozen-hydrated material has revealed the three-dimensional localization of green fluorescent protein added at different positions within the linear sequence (42, 43). Similar information would be of great value for the fluorescent proteins introduced into the 1a and 1S subunits of the DHPR. This information would place an important limit on how DHPRs can be oriented with respect to the RyR1, which in turn would provide clues about regions of potential interaction between the two proteins. Another important goal for the future will be to
