Dihydropyridine Receptor II-III Loop Research Articles

Cyclization of the Intrinsically Disordered α1S Dihydropyridine Receptor II-III Loop Enhances Secondary Structure and in Vitro Function

A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α(1S) subunit of the dihydropyridine receptor (DHPR). The II-III loop has been previously examined in vitro using a linear II-III loop with unrestrained N- and C-terminal ends. To better reproduce the loop structure in its native environment (tethered to the DHPR transmembrane domains), we have joined the N and C termini using intein-mediated technology. Circular dichroism and NMR spectroscopy revealed a structural shift in the cyclized loop toward a protein with increased α-helical and β-strand structure in a region of the loop implicated in its in vitro function and also in a critical region for EC coupling. The affinity of binding of the II-III loop binding to the SPRY2 domain of the skeletal ryanodine receptor (RyR1) increased 4-fold, and its ability to activate RyR1 channels in lipid bilayers was enhanced 3-fold by cyclization. These functional changes were predicted consequences of the structural enhancement. We suggest that tethering the N and C termini stabilized secondary structural elements in the DHPR II-III loop and may reflect structural and dynamic characteristics of the loop that are inherent in EC coupling.

MOLECULAR RECOGNITION OF THE DISORDERED DIHYDROPYRIDINE RECEPTOR II–III LOOP BY A CONSERVED SPRY DOMAIN OF THE TYPE 1 RYANODINE RECEPTOR

1. The dihydropyridine receptor (DHPR) II-III loop is an intrinsically unstructured region made up of alpha-helical and beta-turn secondary structure elements with the N and C termini in close spatial proximity. 2. The DHPR II-III loop interacts in vitro with a ryanodine receptor (RyR) 1 SPRY domain through alpha-helical segments located in the A and B regions. Mutations within the A and B regions in the DHPR II-III loop alter the binding affinity to the SPRY2 domain. 3. The A and C peptides derived from DHPR II-III loop show negative cooperativity in binding to the SPRY2 domain. 4. The SPRY2 domain of the RyR1 (1085-1208) forms a beta-sheet sandwich structure flanked by variable loop regions. An acidic loop region of SPRY2 (1107-1121) forms part of a negatively charged cleft that is implicated in the binding of the DHPR II-III loop. 5. The mutant E1108A located in the negatively charged loop of SPRY2 reduces the binding affinity to the DHPR II-III loop.

REGULATION OF RYANODINE RECEPTORS FROM SKELETAL AND CARDIAC MUSCLE DURING REST AND EXCITATION

1. In muscle, intracellular calcium concentration, hence skeletal muscle force and cardiac output, is regulated by uptake and release of calcium from the sarcoplasmic reticulum (SR). The ryanodine receptor (RyR) forms the calcium release channel in the SR. 2. Calcium release through RyRs is modulated by a wide variety of endogenous molecules, including small diffusible ligands such as ATP, Ca2+ and Mg2+. The regulation of RyR channels by ATP, Ca2+ and Mg2+ is a complex interplay of several regulatory mechanisms, which are still being unravelled. Consequently, it is not clearly known how RyRs are regulated in resting muscle and during contraction. 3. The present paper reviews factors controlling the activity of RyRs in skeletal and cardiac muscle with an emphasis on mechanistic insights derived from single channel recording methods. 4. In addition, the nature of dihydropyridine receptor (DHPR) control of RyRs in skeletal muscle derived from experiments with peptide fragments of the DHPR II-III loop is reviewed. 5. Finally, recent experiments on coupled RyRs in lipid bilayers and their potential for resolving the elusive mechanisms controlling calcium release during cardiac contraction are discussed.

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF INTERACTIONS BETWEEN THE DIHYDROPYRIDINE RECEPTOR II–III LOOP AND THE RYANODINE RECEPTOR

1. Excitation-contraction coupling in skeletal muscle is dependent on a physical interaction between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR). 2. A number of peptides derived from the II-III loop region of the DHPR have been shown to be functionally active in stimulating the release of calcium via RyR channels. Their function has been found to correlate with the presence of a basic helical region located at the N-terminus of the II-III loop. 3. The entire recombinant skeletal DHPR II-III loop is an efficient activator of RyR1 and RyR2. 4. The skeletal DHPR II-III loop is comprised of a series of a-helices, but its tertiary structure has been determined to be unstructured and flexible. 5. Fluorescence quenching experiments have been used to identify and measure the binding affinity of the II-III loop with fragments of the RyR.

Role of some unconserved residues in the "C" region of the skeletal DHPR II-III loop

The actions of the recombinant skeletal dihydropyridine receptor II-III loop (SDCL), and the C region peptide (CS) on native skeletal muscle ryanodine receptor Ca2+ release channel (RyR1) have been examined. Three non conserved residues in the "C" region of the skeletal DHPR II-III loop were replaced by the equivalent cardiac residues in SDCLAFP-PTT (A739P, F741T and P742T) and single substitutions made in SDCLA-P, SDCLF-T and SDCLP-T. Wild type SDCL as well as SDCLF-T and SDCLP-T activated RyR1 in lipid bilayers with high affinity (10 nM to 1 microM). Wild type SDCL at higher concentrations inhibited RyR1. In contrast, SDCLAFP-PTT and SDCLA-P inhibited the channels at >or=10 nM. The inhibitory actions of these two skeletal loop mutants were distinctly different from the cardiac II-III loop (CDCL) which, like the wild-type SDCL, activated channels. In contrast to the full loop, the triple A739P, F741T and P742T mutation in peptide CS converted the peptides' function from skeletal-like to cardiac-like. The individual A739P mutation, but not F741T or P742T, reduced the functional efficacy of CS. None of the mutations significantly altered the NMR-based secondary structure of the C residues in SDCLAFP-PTT or CS. The CS peptide and its mutants, like the cardiac CC peptide, were all partially alpha helical at low temperatures. The results show that residue A739 is critical for the functional consequences of interactions between RyR1 and either the skeletal II-III loop or CS, but that none of A739, F741 or P742 are critical determinants of the structure of the C region.

Structural Requirements of the Dihydropyridine Receptor α1S II-III Loop for Skeletal-type Excitation-Contraction Coupling

Residues Leu720-Leu764 within the II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha1S subunit represent a critical domain for the orthograde excitation-contraction coupling as well as for retrograde DHPR L-current-enhancing coupling with the ryanodine receptor (RyR1). To better understand the molecular mechanism underlying this bidirectional DHPR-RyR1 signaling interaction, we analyzed the critical domain to the single amino acid level. To this end, constructs based on the highly dissimilar housefly DHPR II-III loop in an otherwise skeletal DHPR as an interaction-inert sequence background were expressed in dysgenic (alpha1S-null) myotubes for simultaneous recordings of depolarization-induced intracellular Ca2+ transients (orthograde coupling) and whole-cell Ca2+ currents (retrograde coupling). In the minimal skeletal II-III loop sequence (Asp734-Asp748 required for full bidirectional coupling, eight amino acids heterologous between skeletal and cardiac DHPR were exchanged for the corresponding cardiac residues. Four of these skeletal-specific residues (Ala739, Phe741, Pro742, and Asp744) turned out to be essential for orthograde and two of them (Ala739 and Phe741) for retrograde coupling, indicating that orthograde coupling does not necessarily correlate with retrograde signaling. Secondary structure predictions of the critical domain show that an alpha-helical (cardiac sequence-type) conformation of a cluster of negatively charged residues (Asp744-Glu751 of alpha1S) corresponds with significantly reduced Ca2+ transients. Conversely, a predicted random coil structure (skeletal sequence-type) seems to be prerequisite for the restoration of skeletal-type excitation-contraction coupling. Thus, not only the primary but also the secondary structure of the critical domain is an essential determinant of the tissue-specific mode of EC coupling.

Activating the ryanodine receptor with dihydropyridine receptor II-III loop segments: size and charge do matter.

Excitation-contraction coupling in skeletal muscle is thought to depend on a physical interaction between II-III loop of the alpha1 subunit of the skeletal dihydropyridine receptor (DHPR) and ryanodine receptor (RyR). A peptide corresponding to II-III loop residues 671-690 of the skeletal DHPR (peptide A) is a high affinity activator of the RyR when it adopts an alpha-helical structure with critical basic residues aligned along one helical surface (1). Neither the structure of the full length II-III loop, or of sequences longer than 671-690 residues have been determined. Here we describe the structure and function of a 40 amino acid peptide corresponding to residues 671-710 (peptide AB) of the skeletal DHPR alpha1 subunit. This peptide contains the A region with a further 20 residues towards the C-terminus of the II-III loop. We predicted that peptide AB would strongly activate the RyR, because (a) it contains the active A sequence of basic residues and (b) it contains a greater proportion of the II-III loop. The structure of the AB peptide was determined and it was found to consist of two helical regions joined by an unstructured linker region. Surprisingly, although the structure of the A region was maintained, the 40 residue peptide was unable to release Ca2+ from skeletal SR. Strong activity was restored when four negatively charged residues in the C-terminal part of the peptide were replaced by neutral residues. The charge substitution caused minimal changes in the overall structural profile of the peptide and virtually no changes in the A portion of the peptide. The results suggest that the ability of the A region of the skeletal II-III loop to interact with the RyR could depend on the tertiary conformation of the II-III loop, which is thought to change during EC coupling.

A cardiac dihydropyridine receptor II-III loop peptide inhibits resting Ca(2+) sparks in ferret ventricular myocytes.

1. We studied the effect of a peptide (Ac-10C) on cardiac ryanodine receptor (RyR) opening. This decapeptide (KKERKLARTA) is a fragment of the cardiac dihydropyridine receptor (DHPR) from the cytosolic loop between the second and third transmembrane domains (II-III loop). Studies were carried out in ferret ventricular myocytes by simultaneously applying ruptured-patch voltage clamp and line-scan confocal microscopy with fluo-3 to measure intracellular [Ca(2+)] ([Ca(2+)](i)) and Ca(2+) sparks. 2. Inclusion of Ac-10C in the dialysing pipette solution inhibited resting Ca(2+) spark frequency (due to diastolic RyR openings) by > 50 %. This occurred without changing sarcoplasmic reticulum (SR) Ca(2+) content, which was measured via the caffeine-induced Ca(2+) transient amplitude and the caffeine-induced Na(+)-Ca(2+) exchange current (I(NCX)) integral. Ac-10C also reduced slightly the size of Ca(2+) sparks. 3. Ac-10C did not alter either resting [Ca(2+)](i) (assessed by indo-1 fluorescence) or DHPR gating (measured as L-type Ca(2+) current). 4. The SR Ca(2+) fractional release was depressed by Ac-10C at relatively low SR Ca(2+) content, but not at higher SR Ca(2+) content. 5. A control scrambled peptide (Ac-10CS) did not alter any of the measured parameters (notably Ca(2+) spark frequency or SR Ca(2+) fractional release). Thus, the Ac-10C effects may be sequence or charge distribution specific. 6. Our results suggest an inhibitory regulation of RyRs at rest via the cardiac DHPR II-III loop N-terminus region. The mechanism of the effect and whether this interaction is important in cardiac excitation-contraction coupling (E-C coupling) per se, requires further investigation.

Structural Determinants for Activation or Inhibition of Ryanodine Receptors by Basic Residues in the Dihydropyridine Receptor II-III Loop

The structures of peptide A, and six other 7–20 amino acid peptides corresponding to sequences in the A region (Thr 671- Leu 690) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca 2+ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at −40 mV), was seen with peptides containing the basic residues 681Arg Lys Arg Arg Lys 685, and was strongest when the residues were a part of an α-helix. Inhibition of channels (at +40 mV) was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor.

Arg 615Cys Substitution in Pig Skeletal Ryanodine Receptors Increases Activation of Single Channels by a Segment of the Skeletal DHPR II-III Loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca 2+ release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr 671-Leu 690; 36 μM) enhanced the rate of Ca 2+ release from normal SR (SR N) and from SR of MH-susceptible muscle (SR MH) by 10 ± 3.2 nmole/mg/min and 76 ± 9.7 nmole/mg/min, respectively. Ca 2+ release from SR N or SR MH was not increased by control peptide NB (Gly 689-Lys 708). AS (scrambled A sequence; 36 μM) did not alter Ca 2+ release from SR N, but increased release from SR MH by 29 ± 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR MH) were up to about fourfold more strongly activated by peptide A (≥1 nM) than normal RyR channels (RyR N) at −40 mV. Neither NB or AS activated RyR N. RyR MH showed an ∼1.8-fold increase in mean current with 30 μM AS. Inhibition at +40 mV was stronger in RyR MH and seen with peptide A (≥0.6 μM) and AS (≥0.6 μM), but not NB. These results show that the Arg 615Cys substitution in RyR MH has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca 2+ release from SR in MH-susceptible muscle.

Effects of dihydropyridine receptor II-III loop peptides on Ca2+ release in skinned skeletal muscle fibers

In skeletal muscle fibers, the intracellular loop between domains II and III of the alpha(1)-subunit of the dihydropyridine receptor (DHPR) may directly activate the adjacent Ca(2+) release channel in the sarcoplasmic reticulum. We examined the effects of synthetic peptide segments of this loop on Ca(2+) release in mechanically skinned skeletal muscle fibers with functional excitation-contraction coupling. In rat fibers at physiological Mg(2+) concentration ([Mg(2+)]; 1 mM), a 20-residue skeletal muscle DHPR peptide [A(S(20)); Thr(671)-Leu(690); 30 microM], shown previously to induce Ca(2+) release in a triad preparation, caused only small spontaneous force responses in approximately 40% of fibers, although it potentiated responses to depolarization and caffeine in all fibers. The COOH-terminal half of A(S(20)) [A(S(10))] induced much larger spontaneous responses but also caused substantial inhibition of Ca(2+) release to both depolarization and caffeine. Both peptides induced or potentiated Ca(2+) release even when the voltage sensors were inactivated, indicating direct action on the Ca(2+) release channels. The corresponding 20-residue cardiac DHPR peptide [A(C(20)); Thr(793)-Ala(812)] was ineffective, but its COOH-terminal half [A(C(10))] had effects similar to A(S(20)). In the presence of lower [Mg(2+)] (0.2 mM), exposure to either A(S(20)) or A(C(10)) (30 microM) induced substantial Ca(2+) release. Peptide C(S) (100 microM), a loop segment reported to inhibit Ca(2+) release in triads, caused partial inhibition of depolarization-induced Ca(2+) release. In toad fibers, each of the A peptides had effects similar to or greater than those in rat fibers. These findings suggest that the A and C regions of the skeletal DHPR II-III loop may have important roles in vivo.

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 alpha(1) subunit (DHPR, alpha(1S)) has been reported to activate the skeletal muscle ryanodine receptor (RyR1) in vitro. Within this region of alpha(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 alpha(1S) subunit with scrambled sequence in this region of the II-III loop (alpha(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 alpha(1S)), alpha(1S)-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type alpha(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 alpha(1S) and alpha(1S)-scr. These data suggest that residues 681-690 of the alpha(1S) II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.

A Structural Requirement for Activation of Skeletal Ryanodine Receptors by Peptides of the Dihydropyridine Receptor II-III Loop

The solution structures of three related peptides (A1, A2, and A9) corresponding to the Thr(671)-Leu(690) region of the skeletal muscle dihydropyridine receptor II-III loop have been investigated using nuclear magnetic resonance spectroscopy. Peptide A1, the native sequence, is less effective in activating ryanodine receptor calcium release channels than A2 (Ser(687) to Ala substitution). Peptide A9, Arg(681)-Ser(687), does not activate ryanodine receptors. A1 and A2 are helical from their N terminus to Lys(685) but are generally unstructured from Lys(685) to the C terminus. The basic residues Arg(681)-Lys(685), essential for A1 activation of ryanodine receptors, are located at the C-terminal end of the alpha-helix. Peptide A9 was found to be unstructured. Differences between A1 and A2 were observed in the C-terminal end of the helix (residues 681-685), which was less ordered in A1, and in the C-terminal region of the peptide, which exhibited greater flexibility in A1. Predicted low energy models suggest that an electrostatic interaction between the hydroxyl oxygen of Ser(687) and the guanidino moiety of Arg(683) is lost with the Ser(687)Ala substitution. The results show that the more structured peptides are more effective in activating ryanodine receptors.

A new fold in the scorpion toxin family, associated with an activity on a ryanodine-sensitive calcium channel.

We determined the structure in solution by (1)H two-dimensional NMR of Maurocalcine from the venom of Scorpio maurus. This toxin has been demonstrated to be a potent effector of ryanodyne-sensitive calcium channel from skeletal muscles. This is the first description of a scorpion toxin which folds following the Inhibitor Cystine Knot fold (ICK) already described for numerous toxic and inhibitory peptides, as well as for various protease inhibitors. Its three dimensional structure consists of a compact disulfide-bonded core from which emerge loops and the N-terminus. A double-stranded antiparallel beta-sheet comprises residues 20-23 and 30-33. A third extended strand (residues 9-11) is perpendicular to the beta-sheet. Maurocalcine structure mimics the activating segment of the dihydropyridine receptor II-III loop and is therefore potentially useful for dihydropyridine receptor/ryanodine receptor interaction studies. Proteins 2000;40:436-442.

Activation and Inhibition of Skeletal RyR Channels by a Part of the Skeletal DHPR II-III Loop: Effects of DHPR Ser687 and FKBP12

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2>A1>A1S>NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by ∼30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at −40mV, with 10μM A1 or 50nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100nM cis Ca2+. Inhibition by A1 was greatest at +40mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100μM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.

Conversion of an inactive cardiac dihydropyridine receptor II-III loop segment into forms that activate skeletal ryanodine receptors

A 25 amino acid segment (Glu 666–Pro 691) of the II-III loop of the α 1 subunit of the skeletal dihydropyridine receptor, but not the corresponding cardiac segment (Asp 788–Pro 814), activates skeletal ryanodine receptors. To identify the structural domains responsible for activation of skeletal ryanodine receptors, we systematically replaced amino acids of the cardiac II-III loop with their skeletal counterparts. A cluster of five basic residues of the skeletal II-III loop ( 681RKRRK 685) was indispensable for activation of skeletal ryanodine receptors. In the cardiac segment, a negatively charged residue (Glu 804) appears to diminish the electrostatic potential created by this basic cluster. In addition, Glu 800 in the group of negatively charged residues 798EEEEE 802 of the cardiac II-III loop may serve to prevent the binding of the activation domain.

Involvement of the Glu724–Pro760 Region of the Dihydropyridine Receptor II-III Loop in Skeletal Muscle-type Excitation-Contraction Coupling

Our previous study (El-Hayek, R., Antoniu, B., Wang, J. P., Hamilton, S. L., and Ikemoto, N. (1995) J. Biol. Chem. 270, 22116-22118) suggested the hypothesis that skeletal muscle-type excitation-contraction coupling is regulated by two domains (activating and blocking) of the II-III loop of the dihydropyridine receptor alpha1 subunit. We investigated this hypothesis by examining conformational changes in the ryanodine receptor induced by synthetic peptides and by transverse tubular system (T-tubule) depolarization. Peptide A, corresponding to the Thr671-Leu690 region, rapidly changed the ryanodine receptor conformation from a blocked state (low fluorescence of the conformational probe, methyl coumarin acetamide, attached specifically to the ryanodine receptor) to an activated state (high methyl coumarin acetamide fluorescence) as T-tubule depolarization did. Peptide C, corresponding to the Glu724-Pro760 region, blocked both conformational changes induced by peptide A and T-tubule depolarization. Its ability to block peptide A-induced and depolarization-induced activation was considerably impaired by replacing the portion of peptide C corresponding to the Phe725-Pro742 region of the loop with cardiac muscle-type sequence. These results are consistent with the model that depolarization-induced activation of excitation-contraction coupling and blocking/repriming are mediated by the peptide A region and the peptide C region (containing the critical Phe725-Pro742 sequence) of the II-III loop, respectively.