Excitation-Contraction Coupling Is Not Affected by Scrambled Sequence in Residues 681–690 of the Dihydropyridine Receptor II-III Loop*

A peptide corresponding to residues 681–690 of the II-III loop of the skeletal muscle dihydropyridine receptor α1 subunit (DHPR, α1S) has been reported to activate the skeletal muscle ryanodine receptor (RyR1)in vitro. Within this region of α1S, a cluster of basic residues, Arg681–Lys685, was previously reported to be indispensable for the activation of RyR1 in microsomal preparations and lipid bilayers. We have used an intact α1S subunit with scrambled sequence in this region of the II-III loop (α1S-scr) to test the importance of residues 681–690 and the basic motif for skeletal-type excitation-contraction (EC) coupling and retrograde signaling in vivo. When expressed in dysgenic myotubes (which lack endogenous α1S), α1S-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type α1S. The scrambled DHPR also rescued skeletal-type EC coupling, as indicated by electrically evoked contractions in the presence of 0.5 mmCd2+ and 0.1 mm La3+. Furthermore, the release of intracellular Ca2+, as assayed by the indicator dye, Fluo-3, had similar kinetics and voltage dependence for α1S and α1S-scr. These data suggest that residues 681–690 of the α1S II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.

in the sarcolemma, and ryanodine receptors (RyRs), calcium release channels in the sarcoplasmic reticulum membrane. The mechanism of EC coupling differs in skeletal and cardiac muscle. In cardiac muscle, calcium influx through the pore-forming subunit of the cardiac DHPR (␣ 1C ) activates RyRs (1). However, in skeletal muscle, EC coupling is independent of the entry of extracellular Ca 2ϩ (2) and may result instead from a mechanical coupling between the skeletal DHPR ␣ 1 subunit (␣ 1S ) and the skeletal muscle RyR isoform (RyR1). Expression of ␣ 1S /␣ 1C chimeras in dysgenic myotubes (which lack endogenous ␣ 1 subunits) has established that skeletal-type EC coupling depends upon skeletal sequence within the putative cytoplasmic region between repeats II and III (II-III loop, amino acids 666 -791 (3)). Chimeric DHPRs in which smaller segments of the skeletal DHPR were substituted into the cardiac DHPR subsequently identified residues 720 -765 within the II-III loop as critical for activation of skeletal-type EC coupling (4,5). Moreover, this same critical region is essential for "retrograde signaling," whereby RyR1 enhances the current density of ␣ 1S (6). On the other hand, observations in vitro indicate that a different region of the II-III loop, residues 671-690 ("peptide A"), is important for activation of RyR1, as indicated by ryanodine binding, single channel activity, and calcium release (7)(8)(9). Within peptide A, residues 681-690 have been identified as the "minimum essential region" of the DHPR II-III loop for activating ryanodine binding and Ca 2ϩ release (10), and it has been suggested that the integrity of a cluster of five basic residues (Arg 681 -Lys 685 ) is requisite for this region to serve as the physiological trigger for skeletal-type EC coupling (10,11). In an attempt to determine whether the specific sequence of residues 681-690 and the integrity of the cluster of positively charged residues are required for EC coupling in vivo, we have constructed a full-length DHPR with a scrambled sequence in residues 681-690 (␣ 1S -scr). Dysgenic myotubes expressing ␣ 1S or ␣ 1S -scr did not differ in calcium current density, voltage dependence of activation, electrically evoked contractions, or voltage dependence of intracellular calcium release. These results indicate that neither the specific sequence of these residues, nor the integrity of the cluster of positive charges, is required for skeletal-type EC coupling in muscle cells.

EXPERIMENTAL PROCEDURES
An expression plasmid encoding the pore-forming subunit of the skeletal muscle DHPR (␣ 1S (12)) with scrambled residues 681-690 (␣ 1S -scr) was constructed by overlapping PCR mutagenesis (13) using ␣ 1S as template. This construct is schematically illustrated in Fig. 1A. The internal forward and reverse primers encoding the scrambled sequence were 5Ј-AAGGCCAAGGCCGAGGAGAGGAAAATGAGGTCGA-GGGGCAAGCTTCGC-3Ј and 5Ј-CTTCTCCTCCTCTCTCTTGTCAGG-GCGAAGCTTGCCCCTCGACCTCAT-3Ј. The mutagenized product was amplified using the primer pair 5Ј-GGGTCCTTCTTCATCCTCAACCT-GGTGCTGGGC-3Ј and 5Ј-GAGGATCTTTACCACGGAGATGGTGCTG-GACT-3Ј. This final PCR product was digested with XhoI and EcoRI and was ligated into an expression plasmid encoding green fluorescent protein (GFP) fused to the N terminus of ␣ 1S (GFP-␣ 1S (14)). For this, GFP-␣ 1S was digested with EcoRI (at nucleotide 1007 in the ␣ 1S coding sequence, generated by a partial digest) and XhoI (at nucleotide 2653 in the ␣ 1S coding sequence). The altered region of ␣ 1S -scr was confirmed by automated DNA sequencing.
Primary cultures of myotubes were prepared from newborn dysgenic mice as described previously (15). Approximately 1 week after plating, plasmids carrying cDNA for wild-type or mutant DHPRs (0.1-0.2 g/l) were microinjected into single nuclei. 36 -72 h after injection, myotubes expressing DHPRs were identified by accumulation of green fluores-* This work was supported by National Institutes of Health Grant NS24444 (to K. G. B.) with a minority supplement (for C. P.). 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.
cence. Expressing cells bathed in tissue culture medium (Dulbecco's modified Eagle's medium, Sigma) were examined for ability to contract in response to electrical stimulation (80 -90 V, 10 -30 ms). In some cases, 0.5 mM CdCl 2 and 0.1 mM LaCl 3 were added to the medium to block Ca 2ϩ influx through DHPRs.
Macroscopic Ca 2ϩ currents and intracellular Ca 2ϩ transients were measured simultaneously (16) using borosilicate glass patch pipettes with resistances of 1.5-3.0 M⍀ when filled with an internal solution containing (in mM) 1 MgCl 2 , 145 cesium glutamate, 10 HEPES, 2 CsCl, 0.1 EGTA, and 0.5 K 5 -Fluo-3 (Molecular Probes, Eugene, OR). The composition of the bath solution was 10 CaCl 2 , 145 tetraethylammonium chloride, 0.003 tetrodotoxin, and 10 HEPES (pH 7.4 with tetraethylammonium hydroxide). In some experiments, 0.5 mM CdCl 2 and 0.1 mM LaCl 3 were added to the extracellular solution. The voltage clamp command sequence was to step from a holding potential of Ϫ80 mV to Ϫ30 mV for 1 s, to Ϫ50 mV for 30 ms, to the test potential for 200 ms, and back to Ϫ80 mV. Test currents were digitally corrected for linear leakage and capacitive currents. Ca 2ϩ currents were normalized by linear cell capacitance (pA/pF). All data are presented as mean Ϯ S.E.

RESULTS
To test the importance of residues 681-690 for DHPR channel function, for retrograde signaling, and for EC coupling, we constructed a mammalian expression plasmid encoding the full-length pore-forming subunit of the skeletal DHPR (␣ 1S ) with a scrambled sequence in this region (␣ 1S -scr, Fig. 1A). Note that the cluster of charged residues present in the wildtype sequence has been disrupted in ␣ 1S -scr. Whole-cell cal-cium currents recorded from dysgenic myotubes expressing ␣ 1S -scr closely resembled those recorded from myotubes expressing wild-type ␣ 1S (Fig. 1B) and, like the wild-type currents, were abolished by application of 0.5 mM Cd 2ϩ and 0.1 mM La 3ϩ (data not shown). Fig. 1C compares average peak I-V relationships for the two constructs, showing that they were similar in both voltage dependence and magnitude. Peak current densities at ϩ40 mV were Ϫ5.06 Ϯ 1.12 pA/pF (n ϭ 14) and Ϫ5.11 Ϯ 0.74 pA/pF (n ϭ 10) for ␣ 1S and ␣ 1S -scr, respectively.
To assay the ability of ␣ 1S -scr to mediate skeletal-type EC coupling, dysgenic myotubes expressing either ␣ 1S or ␣ 1S -scr were tested both for contraction in response to extracellular stimulation and for depolarization-induced Ca 2ϩ release. As illustrated in Fig. 2, wild-type ␣ 1S restored evoked contractions in 70% of fluorescent cells tested and ␣ 1S -scr restored contractions in 79% of cells tested. Myotubes expressing either ␣ 1S (67%) or ␣ 1S -scr (53%) retained the ability to contract even after the addition of 0.5 mM Cd 2ϩ and 0.1 mM La 3ϩ to the bathing medium to block Ca 2ϩ entry.
As a further test of whether ␣ 1S -scr differs from ␣ 1S , we measured intracellular Ca 2ϩ release in voltage-clamped cells by recording changes in fluorescence of the Ca 2ϩ indicator dye, Fluo-3. In both normal medium and medium containing Cd 2ϩ and La 3ϩ , calcium transients generated by ␣ 1S -scr were similar, in time course and size, to those produced by wild-type ␣ 1S (Fig. 3, A-D). The voltage dependence of calcium release was also similar and for both constructs showed a sigmoidal response that saturated at strong depolarizations (Fig. 3E).

DISCUSSION
In this paper we have shown by expression in muscle cells that wild-type ␣ 1S and ␣ 1S -scr (␣ 1S with scrambled sequence in residues 681-690 of the II-III loop) do not differ in density or voltage dependence of calcium currents or in skeletal-type EC coupling, as indicated by evoked contractions in Cd 2ϩ /La 3ϩ and by voltage dependence of intracellular Ca 2ϩ release. Thus, the function of ␣ 1S as a calcium channel and its ability to participate in EC coupling appear to be unaffected by either the specific sequence of residues 681-690 or the integrity of a cluster of basic residues in this region.
The ability of ␣ 1S -scr to mediate skeletal-type EC coupling is consistent with earlier work on ␣ 1S /␣ 1C chimeras, which showed that strong skeletal-type EC coupling occurred when a critical domain of the II-III loop, residues 720 -765, was skele- tal in origin (4,5). The ability of these chimeras to produce skeletal coupling was independent of whether there was skeletal or cardiac sequence for residues 681-690. The interchangeability of cardiac and skeletal sequence in this region could simply be a consequence of sequence conservation or could mean that the sequence of these residues is unimportant for skeletal-type EC coupling. The present results strongly support the latter conclusion.
In vitro studies have shown that RyR1 is activated by a peptide composed of residues 681-690 (see Fig. 1A; Ref. 10) or by a slightly larger peptide (peptide A; residues 671-690 (7)(8)(9)). This activation is dependent on the integrity of a group of five basic residues within this region (10,11). We have now shown that skeletal-type EC coupling still occurs in muscle cells expressing ␣ 1S bearing a scrambled sequence in the peptide A region, even though the same scrambled sequence abolished the ability of residues 681-690 to activate RyR1 (10). Thus it seems unlikely that in vitro activation of RyR1 by peptides implies in vivo activation of RyR1 by the corresponding region of the II-III loop. The activation by loop peptides may occur as a consequence of action at sites inaccessible to the intact II-III loop or may result from free solution conformations of the peptides that do not occur natively.
In conclusion, it is clear from our results that residues 681-690 are not required for EC coupling in vivo. However, our results do not exclude the possibility that residues 681-690 play some role in EC coupling, since moderate decreases in calcium release or efficacy of EC coupling would be difficult to detect with our methods.