Primers for the frame-shift mutant, fs-α_1S, were originally designed to delete the 20-mer Thr671-Leu690 in the cytosolic loop between repeats II and III of α_1S and to generate a full-length α_1S carrying this internal deletion. A proofreading error during a PCR reaction resulted in an amplified DNA with the desired deletion but also containing an additional thymidine following the TTG codon for Leu670 (Fig. 1A). The one-base shift in reading frame introduced a serine at position 671 followed immediately by a stop codon (Fig. 1B). This frame-shift mutation was re-ligated into an otherwise full-length α_1S, subcloned into the mammalian expression vector pSG5, and transfected into dysgenic (α_1S null) myotubes. Fs-α_1S was abundantly expressed in primary dysgenic myotubes in culture (Fig. 1C) and produced the expected truncated α_1S protein (Fig 1D). Western blots using N-terminus T7-tagged fs-α_1S and T7-tagged full-length α_1S showed that the expressed full-length α_1S protein migrated with an apparent molecular weight of approximately 185 KDa under reducing conditions. This result is consistent with the mobility of the native purified skeletal muscle α_1S subunit 25. The fs-α_1S migrated with a molecular weight of approximately 90 KDa which is entirely consistent with the theoretical molecular weight of the expressed fragment which was 85.6 KDa. Furthermore, 5-fold overloading of the SDS-PAGE gel failed to detect any fragment of a size comparable to full-length α_1S (not shown).

EC coupling was investigated in voltage-clamped myotubes with simultaneous monitoring of intracellular Ca²⁺ using confocal fluorescence of fluo-4 26. Controls shown in Fig. 2A indicated that the overwhelming majority of non-transfected dysgenic muotubes (13 of 15 cells) did not produce detectable Ca²⁺ transients (<0.1 ΔF/Fo) or Ca²⁺ currents (<20 pA/cell) in response to depolarization under voltage-clamp. This is shown in the line-scan images of fluo-4 fluorescence in Fig. 2A and the corresponding traces of ICa²⁺ during a 50-ms depolarization to +30 mV and +90 mV delivered at the start of the line scan in the same cell. However, in two cells (2 of 15 cells) we observed Idys, the low-density endogenous Ca²⁺ current previously described in dysgenic myotubes 2728. The reason for the low abundance of this current in these cultured myotubes is unknown. Ca²⁺ currents and stimulated fluorescence for one of the cells expressing Idys is shown in Fig. 2B. We observed a peak ICa²⁺ density of approximately 0.8 pA/pF and a barely detectable fluorescence signal which in Fig. 2B is indicated by the arrow in the trace of integrated fluorescence at +30 mV. This small fluorescence signal disappeared entirely at +90 mV, suggesting it might be contributed directly by Idys or might be due to SR Ca²⁺ release induced by Idys. The voltage dependence of the fluorescence signal and ICa²⁺ are compared in Fig. 2C for the two cells expressing Idys and for the vast majority of cells which altogether did not express intracellular Ca²⁺ transients or ICa²⁺. The maximum fluorescence signal contributed by Idys, when Idys was present, was <0.2 ΔF/Fo units. Furthermore, the shape of the fluorescence vs. voltage relationship was bell-shaped and a mirror image of the ICa²⁺ vs. voltage curve. These controls indicated that non-transfected dysgenic myotubes are low-background cells that do not express voltage-activated Ca²⁺ signals of major consequence for the present studies.

Fig. 3 shows that fs-α_1S recovered a significant fraction of the voltage-activated Ca²⁺ transient compared to that express by full-length wt-α_1S. The magnitude of the fluorescence signal expressed by fs-α_1S was approximately 5-fold larger than the largest Ca²⁺ transient detected in non-transfected myotubes expressing Idys, >20-fold larger than the average Ca²⁺ transient detectable in non-transfected cells, and about 1/3 of the maximum SR Ca²⁺ release expressed by the control wt-α_1S construct. Thus, we are confident that the voltage-evoked Ca²⁺ transient in cells transfected by fs-α_1S cells was a direct consequence of the expressed protein. Also shown in Fig. 3 is ICa²⁺ activated by the 50-ms depolarization used to activate the Ca²⁺ transient. fs-α_1S did not express L-type Ca²⁺ current even though it was consistently able to activate the Ca²⁺ transient in 15 of 15 cells. Absence of ICa²⁺ was further verified using longer 500-ms depolarizing pulses (not shown). The skeletal nature of the EC coupling expressed by fs-α_1S is shown in Fig. 4A. The peak Ca²⁺ vs. voltage relationship expressed by fs-α_1S, like that of wt-α_1S, was sigmoidal in shape reaching a maximum at large positive potentials (>50 mV), a range in which ICa²⁺ is progressively small. The line scans of Fig. 4B further confirmed that a Ca²⁺ transient of similar shape and magnitude was observed in a fs-α_1S transfected myotube in the absence of external Ca²⁺. Hence the signaling mechanism, like that reported in normal myotubes and dysgenic myotubes expressing wt-α_1S, was Ca²⁺ entry independent 29. We also expressed fs-α_1S in cultured myotubes from two available gene knock-out (KO) mice, lacking the endogenous β1a isoform of the skeletal muscle DHPR 30 and lacking RyR1 31. As shown in Fig. 4B, we failed to detect EC coupling in these KO cells transfected with fs-α_1S. In summary, the EC coupling expressed by fs-α_1S is strictly skeletal-type and requires RyR1 and DHPR β1a.

The EC coupling recovered by fs-α_1S could be due either to the activity of the N-terminal half of α_1S alone or to protein-protein complementation between the N-terminal half and a fragment expressing the C-terminal half of α_1S. The C-terminal half of α_1S could have been translated by the fs-α_1S expression vector if the ATG codon (Met701), which is downstream from the TGA termination codon and is in-frame with the wild-type message (Fig 1B) served as open reading frame for translation of the second half of the wt message. Although this would be unusual, the fact that the codon for Met701 is only 25 bases downstream from the termination codon could have substantially increased the possibility of a re-start of the translation of the second half of the message at Met701. This phenomenon has been described in eukaryotic cells and in viral-infected mammalian cells and is known as translation by leaky ribosomal scanning 3233. To test this explanation, the presumptive restart condon, Met701, was mutated to Ile701 in the fs-α_1S template. If fs-α_1S recovered EC coupling by virtue of expressing a single protein fragment, then fs-α_1SM701I should also recover EC coupling since the mutation was introduced downstream from the stop codon. Fig. 5 shows that this was not the case. Fs-α_1SM701I did not recover Ca²⁺ transients in 9 of 9 tested cells, consistent with leaky ribosomal scanning. As a positive control, we coexpressed fs-α_1SM701I and the C-terminus half of α_1S, namely α_1SΔ1–700, cloned into a separate pSG5 vector. The results in Fig. 5 indicated that α_1SΔ1–700 alone was inactive. However, when myotubes were cotransfected with fs-α_1SM701I and α_1SΔ1–700, each in a separate pSG5 vector, there was a robust recovery of Ca²⁺ transients in 5 of 5 cells. Fig. 6A shows fluorescence vs. voltage relationships for the fs-α_1SM701I mutant and for this mutant coexpressed with α_1SΔ1–700. The combined expression of the two complementary fragments of α_1S resulted in a robust recovery of EC coupling with sigmoidal Ca²⁺ release vs. voltage characteristics. A summary of the maximum fluorescence during the Ca²⁺ transient in response to a depolarization to +90 mV is shown in Fig. 6B. The magnitude of the Ca²⁺ transient expressed by fs-α_1SM701I + α_1SΔ1–700 was indistinguishable from that of wt-α_1S (t-test significance p = 0.671, see figure legend). To confirm expression of the C-terminus half of α_1S in cells transfected with fs-α_1S, we used the II-III loop polyclonal antibody SKI 34 directed against epitope Ala739-Ile752 which is downstream from Met701. Fig. 6C shows that the II-III loop antibody recognized the C-terminus half when cells were transfected with fs-α_1S but not when myotubes were transfected with fs-α_1SM701I. The C-terminal protein migrated with a molecular weight of approximately 126 KDa which is consistent with the theoretical molecular weight of 132 KDa. Finally, Fig. 6D shows that fs-α_1SM701I was abundantly expressed in myotubes in the absence or presence of the C-terminal fragment. This indicated that the absence of EC coupling observed in myotubes expressing fs-α_1SM701I was not due the production of a labile protein. In summary, the recovery of EC coupling by coexpression of two functionally inactive proteins (Fig. 5) taken together with the immunoblots (Fig. 6C) favor the explanation that 1) fs-α_1S recovers DHPR function by virtue of expressing two complementary fragments of α_1S and 2) the expression of the C-terminal half of α_1S by fs-α_1S is likely to occur by leaky ribosomal scanning.

Except for the magnitude, the SR Ca²⁺ release signal expressed by fs-α_1S was entirely typical of skeletal myotubes with sigmoidal voltage-dependence, proceeding in the absence of external Ca²⁺ and requiring RyR1. A comparison of the maximum fluorescence (ΔF/Fo max) at +90 mV (Fig. 6B) shows that the signal generated by fs-α_1S was significantly smaller than that generated by the control construct (wt-α_1S vs. fs-α_1S t-test significance p = 0.014) and smaller than that generated by the two coexpressed fragments (fs-α_1S vs. fs-α_1SM701I + α_1SΔ1–700 t-test significance p = 0.013). These observation suggests that the magnitude of the Ca²⁺ release appears to be limited by the low yield of expression of the C-terminal fragment achieved by leaky ribosomal scanning of the second half of the fs-α_1S message. To test this explanation further, we coexpressed fs-α_1S and the C-terminal half of α_1S each in a separate pSG5 vector. We found that fs-α_1S and α₁SΔ1–700 together expressed Ca²⁺ transients with a ΔF/Fo max similar to wt-α_1S control (not shown). Thus we are certain that the EC coupling expressed by fs-α_1S is mechanistically similar to control skeletal-type EC coupling but limited in magnitude by a comparatively lower density of functional DHPRs that are assembled in cells expressing fs-α_1S. It is conceivable that the functional integrity of the fragmented α_1S protein is maintained in part by the β subunit of the DHPR which spans both halves of the α1 pore subunit by binding to the I-II loop and the C-terminus 3536. Consistent with this explanation, we failed to detect EC coupling recovery when fs-α_1S was expressed in β1-null myotubes.

Earlier studies in the voltage-gated Na⁺ channel had shown that pore function was not compromised when the II-III linker or III-IV linker was cut and the two fragments were coexpressed each in a separate vector 37. We would thus conclude that in the case of the Ca²⁺ channel, an intact II-III loop is essential for this function since neither fs-α_1S nor the combined expression of the two truncated fragments (not shown) was able to rescue Ca²⁺ current. This result is entirely consistent with the identification of the II-III loop as critical for enhancement of L-type Ca²⁺ current expression by the RyR1 38. However EC coupling, per se, can clearly proceed with a cut in the II-III loop. This was shown here by the behavior of the fs-α_1S construct and elsewhere by expressing the N-terminal half (α_1S1–670) and the C-terminal half (α_1S701–1873), each with entirely wt sequence and each in a separate expression vector 20. The fact that Thr671-Leu690 region known as Peptide A 15 is missing in fs-α_1S suggests this 20-mer domain is not critical for the conformation change transmitted from the DHPR to the RyR1. To test this further, we generated an in-frame deletion of this region, as originally intended, that showed normal function 20. Another domain critical for EC coupling is Csk35 downstream from Peptide A (Leu720-Gln765). This region was identified using chimeras of α_1S and α_1C16. Since Csk53 is present in α_1SΔ1–700 and was detected by the II-III loop antibody which is directed against the center portion of Csk53 34, the participation of this domain in the EC cannot be ruled-out.

In eukaryotic cells, translation starts at the AUG codon nearest to the 5' end of the mRNA, and this initiator site is found by sequential ribosomal scanning in the 5' to 3' direction 32. However, translation initiation at internal AUGs due to leaky ribosomal scanning has been documented, especially for mRNAs consisting of a short leader ORF upstream from the main ORF. In some cases, reassembly of a new ribosomal initiation complex after the terminator codon of the leader ORF serves to re-initiate translation at the initiator AUG codon for the main ORF, and thus two proteins are generated 39. In other cases, leaky scanning by the ribosomal initiation complex bypasses the 5'-located leader ORF entirely, and only the "internal" ORF is translated 32. In the case of fs-α_1S, we are not entirely certain which mechanism best applies. A bypass of the ORF at the 5' end of the α_1S mRNA in favor of a presumed ORF at Met701 is unlikely because a C-terminal fragment of the size expressed by α_1SΔ1–700 has not been detected in skeletal muscle with the SKI antibody 34 or other antibodies 25. Thus Met701 is not an internal initiation site under normal circumstances. Entry of a new ribosomal complex at Met701 seems a more likely explanation. However, re-initiation after a stop has only been described for cases in which the leader ORF is no longer than 30 codons because initiation factors fall off shortly after recognition of the initiator AUG 32. In the case of fs-α_1S, what can be considered the "leader" ORF encodes for a protein of 671 residues. Hence we are not certain if re-initiation of translation after a stop signal, as currently described in the literature, would apply here. At the same time, it is important to point out that in the present studies, fs-α_1S expression is under the control of a viral promoter and that in this hybrid viral-mammalian expression system, the rules pertaining to leaky ribosomal scanning may be different. The mechanism of translation of the fs-α_1S clearly deserves closer scrutiny in the future.

Primary cultures were prepared from hind limbs of day 18 embryos (E18) as described previously 23. cDNAs of interest and a separate expression vector encoding the T-cell membrane antigen CD8 were subcloned into the mammalian expression vector pSG5 (Stratagene, CA) and were mixed and cotransfected with the polyamine LT-1 (Panvera, WI). Whole-cell recordings and immunostaining were done 3–5 days after transfection. Cotransfected cells were recognized by incubation with CD8 antibody beads (Dynal, Norway). The coincidence of expression of CD8 and a cDNA of interest was >85%.

All cDNA constructs were sequenced twice or more using BigDye technology (Perkin Elmer, CA) at a campus facility. For epitope tagging and expression in mammalian cells, the unmodified full-length rabbit α_1S cDNA encoding residues 1–1873 (Genebank #M23919 nucleotide coordinates nt 226 to nt 5847) was fused in frame to the first 11 amino acids of the phage T7 gene 10 protein in pSG5 using AgeI and NotI cloning sites. All constructs were made using the T7 tagged α_1S as template in PCR-based strategies, some previously described 2021. All primers were HPLC-purified (Operon, CA) and a phosphate was tagged to the 5'-end of the sense primer. Genebank #M23919 nucleotide coordinates are used below to describe primers.

Whole-cell recordings were performed as described previously 22 using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). All experiments were performed at room temperature. Patch pipettes had a resistance of 1–2 MΩ . The external solution was (in mM) 130 TEA-Methanesulfonate, 10 CaCl₂, 1 MgCl₂, 10 HEPES-TEA(OH), pH 7.4. The pipette solution was (in mM) 140 Cs-aspartate, 5 MgCl₂, 0.1 EGTA (for Ca²⁺ transients) or 5 EGTA (for Ca²⁺ current), 10 MOPS-CsOH, pH 7.2. The voltage dependence of peak intracellular Ca²⁺ (ΔF/Fo) was fitted according to a Boltzmann distribution (Eqn. 1) A = A_max/(1+exp(-(V-V_1/2)/k)). A_max is ΔF/Fo_max; V_1/2 is the potential at which A = A_max/2; and k is the slope factor. ΔF/Fo=(F-Fo)/Fo where F is the fluorescence during a Ca²⁺ transient and Fo is the resting fluorescence of the cell immediately before the stimulation.

Line-scans were performed as described 26 in cells loaded with 4 mM fluo-3 AM (fluo-3 acetoxymethyl ester, Molecular Probes, OR) for ~30 minutes at room temperature. Cells were viewed with an inverted Olympus microscope with a 20X objective (N.A. 0.4) and a Fluoview confocal attachment (Olympus, NY). Excitation light was provided by a 5 mW Argon laser attenuated to 20% with neutral density filters. For immunofluorescence, confocal images had a dimension of 1024 by 1024 pixels (0.35 microns/pixel) and were obtained with a 40X oil-immersion objective (N.A. 1.3).

Cells were fixed and processed for immunofluorescence as described 420. The N-terminal fragment expressed by fs-α_1S or wt-α_1S was identified with a mouse monoclonal antibody against a T7 epitope fused to the N-terminus of α_1S. The anti-T7 antibody (Novagen, WI) was used at a dilution of 1:1000. Secondary antibodies were a fluorescein-conjugated goat anti mouse IgG (Boehringer Mannheim, IN) used at a dilution of 1:1000 and a fluorescein-conjugated donkey anti-rabbit IgG (Chemicon, CA) used at a dilution of 1:1000.

The C-terminal fragment was identified with SKI, a rabbit polyclonal antibody against the II-III loop of α_1S (Ala739-Ile752) previously characterized 34. Cells were scrapped from tissue cultures dishes with cold PBS plus protease inhibitors and spun in a cold table-top centrifuge. Cells were homogenized in a glass-teflon homogenizer in a minimal volume of PBS and diluted 1:1 (vol:vol) with SDS-gel loading buffer composed 100 mM Tris-Cl (pH 6.8), 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue and 20% glycerol. Samples were incubated at 100°C for 20 minutes. Approximately 10 mg of total protein was applied to a 5–15% SDS polyacrylamide gel and electrophoresed for 2 hours at 40 mA. Proteins were transferred to PVDF membranes and analyzed with either anti-T7 or SKI antibodies and the appropriate secondary antibodies. The subunits were visualized using SuperSignal ECL reagent (Pierce, Rockford, IL). The images were captured on a Chemi-Imager (Alpha Innotech, San Leandro, CA) set to a level just below saturation.