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J. Biol. Chem., Vol. 275, Issue 39, 29935-29937, September 29, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/39/29935    most recent C000464200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Proenza, C. Articles by Beam, K. G. Search for Related Content PubMed PubMed Citation Articles by Proenza, C. Articles by Beam, K. G. Social Bookmarking                      What's this?

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

Catherine Proenza^§, Christina M. Wilkens^¶, and Kurt G. Beam^¶

From the Departments of  Physiology and ^¶ Anatomy and Neurobiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523

Received for publication, July 14, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A peptide corresponding to residues 681-690 of the II-III loop of the skeletal muscle dihydropyridine receptor ₁ subunit (DHPR, _1S) has been reported to activate the skeletal muscle ryanodine receptor (RyR1) in vitro. Within this region of _1S, 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 _1S subunit with scrambled sequence in this region of the II-III loop (_1S-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 _1S), _1S-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type _1S. The scrambled DHPR also rescued skeletal-type EC coupling, as indicated by electrically evoked contractions in the presence of 0.5 mM Cd²⁺ 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 _1S and _1S-scr. These data suggest that residues 681-690 of the _1S II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Excitation-contraction (EC)¹ coupling in skeletal and cardiac muscle involves a functional interaction between dihydropyridine receptors (DHPRs), voltage-gated L-type calcium channels 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 Ca²⁺ (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 (7-9). 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 Ca²⁺ release (10), and it has been suggested that the integrity of a cluster of five basic residues (Arg⁶⁸¹-Lys⁶⁸⁵) 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 5'-AAGGCCAAGGCCGAGGAGAGGAAAATGAGGTCGAGGGGCAAGCTTCGC-3' and 5'-CTTCTCCTCCTCTCTCTTGTCAGGGCGAAGCTTGCCCCTCGACCTCAT-3'. The mutagenized product was amplified using the primer pair 5'-GGGTCCTTCTTCATCCTCAACCTGGTGCTGGGC-3' and 5'-GAGGATCTTTACCACGGAGATGGTGCTGGACT-3'. 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 µg/µl) were microinjected into single nuclei. 36-72 h after injection, myotubes expressing DHPRs were identified by accumulation of green fluorescence. 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 CdCl₂ and 0.1 mM LaCl₃ were added to the medium to block Ca²⁺ influx through DHPRs.

Macroscopic Ca²⁺ currents and intracellular Ca²⁺ 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 MgCl₂, 145 cesium glutamate, 10 HEPES, 2 CsCl, 0.1 EGTA, and 0.5 K₅-Fluo-3 (Molecular Probes, Eugene, OR). The composition of the bath solution was 10 CaCl₂, 145 tetraethylammonium chloride, 0.003 tetrodotoxin, and 10 HEPES (pH 7.4 with tetraethylammonium hydroxide). In some experiments, 0.5 mM CdCl₂ and 0.1 mM LaCl₃ were added to the extracellular solution. The voltage clamp command sequence was to step from a holding potential of 80 mV to 30 mV for 1 s, to 50 mV for 30 ms, to the test potential for 200 ms, and back to 80 mV. Test currents were digitally corrected for linear leakage and capacitive currents. Ca²⁺ currents were normalized by linear cell capacitance (pA/pF). All data are presented as mean ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 wild-type sequence has been disrupted in _1S-scr. Whole-cell calcium 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 Cd²⁺ and 0.1 mM La³⁺ (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 +40 mV were 5.06 ± 1.12 pA/pF (n = 14) and 5.11 ± 0.74 pA/pF (n = 10) for _1S and _1S-scr, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Scrambled sequence in the DHPR II-III loop does not alter calcium channel properties of the skeletal muscle dihydropyridine receptor. A, top, schematic illustration of the DHPR 1S subunit with the region of the II-III loop investigated indicated by a bold line. Bottom, sequence of residues 681-690 in the full-length 1S and 1S-scr constructs used in this study. B, representative whole-cell calcium currents recorded from dysgenic myotubes expressing 1S (left) or 1S-scr (right) in response to a voltage step to +40 mV. C, neither the average current density nor the voltage dependence of activation of 1S is altered by scrambled sequence in residues 681-690. Peak current densities for 14 cells expressing 1S (open circles) and 10 cells expressing 1S-scr (filled circles) are shown.

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 Ca²⁺ 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 Cd²⁺ and 0.1 mM La³⁺ to the bathing medium to block Ca²⁺ entry.

View larger version (70K): [in this window] [in a new window]   Fig. 2.   Scrambled sequence in the II-III loop does not prevent the ability of DHPRs to restore EC coupling to dysgenic myotubes. Dysgenic myotubes expressing either 1S or 1S-scr were bathed in normal medium (gray bars) or in medium containing 0.5 mM Cd2+ and 0.1 mM La3+ (hatched bars). The cells were stimulated electrically (10 ms, 90 V), and the percentage observed to contract is indicated.

As a further test of whether _1S-scr differs from _1S, we measured intracellular Ca²⁺ release in voltage-clamped cells by recording changes in fluorescence of the Ca²⁺ indicator dye, Fluo-3. In both normal medium and medium containing Cd²⁺ and La³⁺, 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).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Scrambled sequence in the II-III loop does not alter the ability of DHPRs to elicit voltage-activated intracellular calcium release. Representative Fluo-3 fluorescent transients recorded from myotubes injected with either 1S (A, C) or 1S-scr (B, D) and stimulated by a series of test pulses from 20 to +80 mV. F is given in arbitrary units and is the same for all traces. E, the voltage dependence of the intracellular Ca2+ release is not changed by scrambled residues 681-690. Normalized change in fluorescence (F) is plotted as a function of voltage for 1S (open circles, n = 6) and 1S-scr (filled circles, n = 13).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 Cd²⁺/La³⁺ and by voltage dependence of intracellular Ca²⁺ 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 skeletal 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 (7-9)). 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.

	ACKNOWLEDGEMENTS

We thank Katherine Parsons and Lindsay Grimes for expert technical assistance.

	FOOTNOTES

^* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: School of Kinesiology, Simon Fraser University, Burnaby, BC V5A 1S6, Canada.

To whom correspondence and reprint requests should be addressed: Dept. of Anatomy and Neurobiology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523. Tel.: 970-491-5277; Fax: 970-491-7907; E-mail: kbeam@lamar.colostate.edu.

Published, JBC Papers in Press, July 27, 2000, DOI 10.1074/jbc.C000464200

	ABBREVIATIONS

The abbreviations used are: EC, excitation-contraction; DHPR, dihydropyridine receptor; RyR, ryanodine receptor; GFP, green fluorescent protein; PCR, polymerase chain reaction; F, farad(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/39/29935    most recent C000464200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Proenza, C. Articles by Beam, K. G. Search for Related Content PubMed PubMed Citation Articles by Proenza, C. Articles by Beam, K. G. Social Bookmarking                      What's this?