Maurocalcine and Domain A of the II-III Loop of the Dihydropyridine Receptor Cav 1.1 Subunit Share Common Binding Sites on the Skeletal Ryanodine Receptor

Abstract

Maurocalcine is a scorpion venom toxin of 33 residues that bears a striking resemblance to the domain A of the dihydropyridine voltage-dependent calcium channel type 1.1 (Ca v 1.1) subunit. This domain belongs to the II-III loop of Ca v 1.1, which is implicated in excitation-contraction coupling. Besides the structural homology, maurocalcine also modulates RyR1 channel activity in a manner akin to a synthetic peptide of domain A. Because of these similarities, we hypothesized that maurocalcine and domain A may bind onto an identical region(s) of RyR1. Using a set of RyR1 fragments, we demonstrate that peptide A and maurocalcine bind onto two discrete RyR1 regions: fragments 3 and 7 encompassing residues 1021-1631 and 3201-3661, respectively. The binding onto fragment 7 is of greater importance and was thus further investigated. We found that the amino acid region 3351-3507 of RyR1 (fragment 7.2) is sufficient for these interactions. In skeletal muscle cells, the activation of the dihydropyridine receptor (DHPR) 1 induces, through the ryanodine receptor (RyR1), a massive release of the calcium stored in the sarcoplasmic reticulum. The bi-directional communication between RyR1 and DHPR (i.e. orthograde and retrograde signals) has been shown to be enabled by the physical interactions of the two Ca 2ϩ channels (1-4). The DHPR ␣ 1 subunit forms the pore region, carries the voltage sensitivity of the channel, and is also directly involved in the functional coupling of the DHPR with RyR1 (2, 5). Indeed, the cytoplasmic loop linking domains II and III of the ␣ 1 subunit has been proposed to be responsible for the mechanical coupling between the DHPR and the RyR1 (6, 7). Within this loop, two regions (domains A and C) have been shown to regulate ryanodine binding and channel gating of RyR1 in vitro (8).RyR1 activity is regulated in vitro by a number of chemical compounds such as ATP, Ca 2ϩ , Mg 2ϩ , and proteins such as calmodulin and FK506-binding protein (FKBP12) (9, 10). Nevertheless, due to (i) the extremely large size of RyR1, (ii) the small number of high affinity effectors, and (iii) the absence of atomic resolution structure, the mapping of the functional sites of RyR1 is still far from completion. As part of the search for molecules able to strongly modify RyR1 properties, toxins isolated from scorpion venoms have been described as the most active effectors of RyR1. Among them, imperatoxin A and maurocalcine (MCa) have been extensively characterized both in terms of their effects on RyR1 and their three-dimensional structure (11-16). These two toxins present some amino acid sequence homology with the domain A of DHPR ␣ 1 subunit, and structural studies strongly suggest that their ␤-sheet structure could mimic that of the domain A (15, 16). In a previous work, we demonstrated that nanomolar concentrations of MCa induces a dramatic conformational change of RyR1, witnessed by the increase in [ 3 H]ryanodine binding (7-fold) and the induction of long lasting channel openings (13,14). The observed homologies in amino...

Small Efficient Cell-penetrating Peptides Derived from Scorpion Toxin Maurocalcine

“…The interaction of MCa with RyR1 has been witnessed by increased [ 3 H]ryanodine binding onto purified RyR1 (3,5). The binding site for MCa on RyR1 has also been mapped and shown to correspond to domain(s) that have a predicted localization within the cytoplasm (6). Second, similarly to imperatoxin A (7) for which this was first noted, MCa has an interesting sequence homology with the II-III loop of the L-type calcium channel Ca v 1.1 subunit over a domain that is slightly larger than the second ␤-strand of MCa (see Fig.…”

“…1A) (5). This loop is predominantly involved in excitation-contraction coupling through direct molecular interactions with RyR1 (6,8). This homology has been a source of inspiration to an understanding of how toxins may interfere with the process of excitation-contraction coupling (4,8,9).…”

“…The toxin belongs to a family of peptide that folds according to an inhibitor cystine knot motif and thus contains three disulfide bridges with a Cys 1 -Cys 4 , Cys 2 -Cys 5 , and Cys 3 -Cys 6 connecting pattern (2). The solution structure, as defined by 1 H NMR, illustrates that MCa contains three ␤-strands (strand 1 from amino acid residues 9 -11, strand 2 from 20 -23, and strand 3 from 30 -33).…”

Administration de molécules actives dans les cellules

“…Ces observations avaient laissé entrevoir la possibilité que la maurocalcine devait traverser la bicouche lipidique des cellules afin d'exercer sa fonction d'activateur de canal calcique. Cette hypothèse était largement renforcée par le fait que le site d'interaction de la maurocalcine sur RyR1 est localisé dans une zone du cytoplasme [7]. La démonstration que la maurocalcine était effectivement un CPP est venue des observations suivantes : (1) l'application extracellulaire du peptide à des myotubes induit une libération calcique quasi instantanée [8] ; et (2) la greffe d'une protéine streptavidine fluorescente sur la maurocalcine biotinylée permet sa translocation intracellulaire [5].…”

d-Maurocalcine, a Pharmacologically Inert Efficient Cell-penetrating Peptide Analogue

“…First, it was demonstrated that an extracellular application of L-MCa triggers an almost immediate Ca 2ϩ release from internal stores in myotubes, suggesting rapid access of L-MCa to its binding site (11). Second, it was discovered that the binding site of this toxin is located on the cytoplasmic side of RyR (12). Kinetic and RyR topological considerations therefore suggest that cell entry of L-MCa more likely relies on membrane translocation to achieve rapid cytoplasmic accumulation than on endocytosis.…”