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

Abstract

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, Arg 681 -Lys 685 , 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 excitationcontraction (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 Cd 2؉ and 0.1 mM La 3؉ . Furthermore, the release of intracellular Ca 2؉ , 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. 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 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 (7-9). Within peptide A, residues 681-690 have been identifi...

Maurocalcine and Peptide A Stabilize Distinct Subconductance States of Ryanodine Receptor Type 1, Revealing a Proportional Gating Mechanism

“…These studies have identified a different region (amino acid residues 720 -765) to be the critical site for DHPR and RyR interaction in skeletal muscle (9). Also, skeletal type EC coupling was unaffected when an ␣ 1s -DHPR with a scrambled sequence corresponding to pA (residues 681-690) was expressed by dysgenic myotubes (16). Since both active and inhibitory effects of peptide A on RyR channels have been reported, the mechanism of the interaction of peptide A with RyR1 must be complex (12,15).…”

“…These actions of pA were also found to extend to RyR2, suggesting a modulatory influence downstream of the skeletal ␣ 1s -DHPR/RyR1 interaction (12). However, expression of ␣ 1s -DHPR chimeras in dysgenic myotubes has shown that the pA region of the II-III loop was not essential for engaging skeletal type EC coupling (16), whereas a 46-amino acid fragment (Leu 720 -Gln 765 ) was essential for bidirectional signaling (9,(17)(18)(19). Although the action of pA and related peptides may not be directly involved in mediating bidirectional signaling in skeletal EC coupling, they are very useful in defining basic properties of RyR gating.…”

Functional implications of modifying RyR‐activating peptides for membrane permeability

“…A strict alignment (Block et al, 1988) allows physical coupling between the II-III loop of the DHPR and the RyR to initiate skeletal-type excitation-contraction (EC) coupling (which is independent of external Ca 2 þ ). Although the A region of the II-III loop is not required for EC coupling (Proenza et al, 2000;Wilkens et al, 2001), it has been implicated in the overall physical interaction between the DHPR and RyR (Ahern et al, 2001).…”

Ca2+-dependent Dual Functions of Peptide C

“…However, most of our previous studies were carried out at [Ca 2ϩ ]s higher than 0.2 M; many experiments were carried out at or higher than 1.0 M. Because the in vivo studies of E-C coupling described in the Introduction (3,4,16) Concentration Ranges in an RyR1-specific Manner- Fig. 1, A and B shows the effects of 50 M peptide A (a maximally activating concentration) on the ryanodine binding activity of the RyR of skeletal muscle and cardiac muscle microsome preparations, respectively, at different Ca 2ϩ concentrations in the assay solution.…”

“…However, according to the recent report of Proenza et al (16), scrambling of the amino acid sequence in the peptide A-10 region, which produced a severe loss of the activating function of peptide A-10 in our in vitro experiments (11), produced no detectable changes in E-C coupling in the dysgenic myotubes. Similarly, chimeric construct, in which the Leu 720 -Leu 764 region is the skeletal muscle-type sequence, but the rest is identical to the housefly II-III loop with the sequence quite dissimilar to the skeletal muscle sequence, produced essentially the same E-C coupling activity as the wild-type skeletal muscle system (17).…”