SCORPION PEPTIDES AS HIGH-AFFINITY PROBES OF RYANODINE RECEPTOR FUNCTION
Georgina B. Gurrola, Xinsheng Zhu, and Héctor H. Valdivia
Dept. of Physiology, University of Wisconsin Medical School, Madison, WI
INTRODUCTION
A variety of peptide toxins in scorpion venoms interact with ionic channels with high affinity and exquisite selectivity. Distinct toxins recognize specifically voltage-dependent channels,730 several subclasses of channels,730 and at least one subclass of channels.731 These scorpion peptides have become useful tools in the identification, purification and structural mapping of ionic channels.732
A subclass of scorpion peptides with high affinity for the release channel/ryanodine receptors (RyR) was reported for the first time in 1991.733 Toxins from the venom of the African scorpion Buthotus hottentota induced a rapid, specific and reversible activation of RyR 1, the RyR isoform that is mostly expressed in skeletal muscle. The finding was surprising because RyRs are intracellular channels that are supposedly inaccessible to the bulk of scorpion toxins, which are membrane-impermeable, ionized peptides targeted against external receptors. However, the ingenuity of nature is immense, and as we will see later, several mechanisms may account for penetration of these basic peptides into intracellular environments. Another peculiar finding was that these peptides activated, rather than blocked, RyRs by inducing the appearance of a long-lived subconducting state.733 While this effect resembled the mechanism of action of the classical ligand ryanodine,
the vast majority of scorpion toxins paralyze excitable tissues by antagonizing ion conduction.730
The first scorpion peptides purified to homogeneity that act on RyRs with nanomolar affinity were the imperatoxins, so called because they are derived from the scorpion Pandinus imperator.734 Imperatoxin I is a ~15 kDa heterodimeric protein with phospholipase activity that indirectly inhibits RyRs by releasing unsaturated fatty acids from membrane phospholipids.735 Imperatoxin A on the other hand, is a 3.7 kDa basic peptide that specifically increases binding to RyR1 (but not to RyR2, the cardiac isoform) by direct ligand-receptor interaction.736 More recently, two peptides from the venom of the scorpion Buthus martensis (BmK AS and BmK AS-1), each having a rather unusual number (4) of disulfide bridges and long chain (66 amino acid residues), were found to activate binding to RyR1.737 From Scorpio maurus palmatus venom was purified Maurocalcin (MCa), a 33-amino acid basic peptide that shares 82% homology with and also exerts many of functional effects.738 A novel subclass of scorpion peptides from Buthotus judaicus venom with activity against RyRs but with lower affinity than or MCa was recently purified and sequenced.739 Thus, a diverse group of scorpion peptides with distinct specificity and different affinity for RyRs is emerging.
Here, we will discuss the most prominent structural and functional characteristics of a representative example of this group of peptides.
STRUCTURAL FEATURES
is a 33-amino acid basic peptide with molecular weight = 3,759 Da.740 It is a thermostable and globular peptide due to 3 disulfide bridges that condense its backbone and stabilize its structure.740 This peptide does not display the cisteine pattern necessary to maintain the classical structure common to scorpion toxins that block or channels.741,742 Rather, displays a cisteine arrangement corresponding to the “inhibitor cisteine knot” found in and a subclass of spider and marine snail toxins, respectively, that block voltage- dependent channels.743,744 therefore, conforms structurally with more fidelity to spider and snail toxins that block channels than to scorpion toxins that block or channels.
Recently, the three-dimensional structure of 745 and MCa746 were solved by NMR. Owing to their high degree of homology and distinctive cisteine pattern, both peptides display the “inhibitor cisteine knot” fold, in which the disulfide bond between C16 and C32 penetrates through a 13- residue ring formed by the peptide backbone and the other two disulfide
bonds (Fig. 19-1). The structure of consists of two antiparallel formed by residues K20-K23 I) and K30-R33 II), connected by four reverse chains.745 The structure of MCa consists of three and four reverse chains.746 Both toxins display a surface rich in basic residues (K19-R24, R33) and an opposite surface rich in acidic residues (D2, D13-D15, E29). The main structural differences between and MCa are found near the ammo-terminal region, where residues 9-11 of MCa form an additional peripheral (Fig. 19-1).
Figure 19-1. Three-dimensional structures of MCa, and Peptide A. (A) Backbone alone, and (B) CPK representation, highlighting amino acids that participate in binding to RyR1. Taken, with permission, from Lee et al.745
Functional analogy between and a segment of the II- III loop of the DHPR
Using synthetic peptides corresponding to different segments of the II-III loop of the subunit of the skeletal dihydropyridine receptor (DHPR), Ikemoto’s group747 found that the amino-terminal region (peptide A, T671- L690) activates RyR1. Gurrola et al.748 noted that shares structural similarities with peptide A (Fig. 19-2). Both peptides display a cluster of basic amino acids (rectangle) followed by a hydroxylated residue (circle)
in and in peptide A). Based on
this observation, it was proposed that acts as a peptide mimetic of an endogenous activator of RyR1.748
Figure 19-2. Proposed homology between and Peptide A of the II-III loop of the DHPR. Taken, with permission, from Gurrola et al.748
To probe for active site, amino acid substitutions were performed using solid-phase peptide synthesis. Substitution of R23 (R23E) or T26 (T26A and T26E) in substantially decreased the capacity of this peptide to activate RyR1.748 Since substitution of K14 (K14E), another basic amino acid not encompassed in the aforementioned cluster, had no major effect, it was concluded that the structural domain cluster-hydroxylated residue was involved in the binding of to RyR1. Gurrola et al.748 also found that and peptide A likely activate RyR1 by a similar mechanism and appear to compete for the same binding site on RyR1.
A similar work involving MCa identified R24 as a critical amino acid in the toxin’s binding site.749 An R24A substitution rendered MCa ineffective to increase binding, to induce subconducting states on single RyR1, and to stimulate release in SR vesicles or intact myotubes.749 Other single substitutions (K8A, K19A, K20A, K22A, R23A and T26A) were capable of reducing affinity of the parent peptide but appeared less
critical components of MCa’s binding site. Supposedly, the alanine substituting each of the above residues hampers, but does not disrupt, MCa’s binding site.
Alanine scanning of also revealed that the amino acid residues responsible for activation of RyR1 are localized in the C-terminal region and correspond (in order of importance) to R24, R31, R33, K22 and R23.745 Furthermore, L7 appears to play an important role as point of hydrophobic interaction with RyR1. The 3-D structure of shows that these six residues along with others make up a critical domain of this peptide with a surface area of (Fig. 19-1). This structural domain would form a functional surface with a putative binding site that interacts with a cytosolic region of the RyR1.745
The ability of peptide A to activate RyR1 has been correlated with both its capacity to adopt an structure750-753 and with the orientation of its positive charges in a single surface of the molecule.752,753 Peptide A displays a basic surface with five basic residues (R681-K685) clustered in the C- terminal region of an whereas (and also MCa) aligns its basic residues (K22, R23, R24, R31 and R33) in a central region. In both and peptide A, this basic surface is totally exposed to solvent and displays a characteristic shape that may be directly involved in the activation of RyR1.
In the 3-D structure, the functional surface of is large
whereas the equivalent surface of peptide A is smaller (Fig. 19-1).
These differences could explain, at least in part, why and peptide A compete for the same binding site with different affinities.745
activates binding to RyR1 over a wide range of producing a generalized increment in the magnitude of the bell-
shaped of binding curve (Fig. 19-3). On the
other hand, displays different effects on RyR2 (cardiac isoform), activating binding moderately at low and inhibiting it, also moderately, at high These somewhat erratic effects of on RyR2 transform the bell-shaped binding curve into a sigmoidal relationship,736,754 suggesting that the and
sites of RyR2 are differently affected by
Figure 19-3. Effect of on the of binding curves to (A) skeletal RyR (RyR1) and (B) cardiac RyR (RyR2). Taken, with permission, from El- Hayek et al.736
Tripathy et al.754 showed that RyR2, although relatively insensitive to in binding assays, is affected by in a manner that is indistinguishable from RyR1. In single channel recordings, both RyRl and RyR2 bind and undergo conformational changes that induce the appearance of identical subconducting states (Fig. 19-4). therefore, is a rare example of a RyR2 ligand that produces divergent effects on
binding and single channel experiments.
Figure 19-4. Effect of on single channel behavior of skeletal and cardiac RyRs.
Taken, with permission, from Tripathy et al.754
and RyR isoforms
stimulates binding to RyR3 (brain, smooth and skeletal muscle isoform), but at higher concentrations than required for effects on RyR1.755 The effect of on RyR3 is observed over a wide range of to 10 mM), and single channel experiments show subconducting states similar to those observed in RyR1 and RyR2. These results indicate that is capable of inducing indistinguishable single channel effects on all three RyR isoforms, whereas its effect on binding is isoform-specific, with potencies ranking RyR1>RyR3>RyR2.755
The mechanism by which exerts different effects on RyR isoforms when tested on binding and identical effects on single channel experiments is unknown, but an appealing hypothesis may be advanced by dividing the effect of the toxin into two distinct events. The first would be an event common to all RyR isoforms which induces a conformational change that leads to the subconducting state observed in single channel recordings;
this event requires an open channel for binding and is favored by conditions that promote high ,754 The second event would be isoform- specific and affected by the RyR-ryanodine interaction, which is in turn dependent on the activity of the channel. As and ryanodine bind to different sites on RyRs,754 both sites may exert bi-modal cooperativity on each other. Thus, at low activity, binding is low and exerts positive cooperativity, leading to additional increment on binding parameters. At high activity, binding is high and may actually affect it adversely, by destabilizing the conformational state that favors binding of the alkaloid. This may explain why RyR1, which has intrinsically lower activity than RyR2, responds to at all
whereas binding to RyR2 is increased by only at low where its activity is low.
RyR binding site(s) for and stoichiometry of binding
The similarity in structure and function of and MCa suggest that both peptides may share the same binding site in RyR1 and that this site may be overlapped with the activation site for peptide A of the DHPR,748,750 although this scheme may not be that simple. Recently, it’s been proposed that displays three independent functional effects on RyR1 as well as on RyR1 Accordingly, binds to three different sites on RyRs, one of high affinity, another of low affinity, and yet another of intermediate affinity that is responsible for inducing the subconducting states.718
Single channel experiments754 and studies using frog skeletal fibers756 concluded that one molecule of interacts with one RyR tetramer during the induction of long releasing events. This 1:1
stoichiometry contrasts with results produced in binding748 and electron microscopy112 experiments, which suggest that up to 4 molecules may bind to 1 RyR tetramer. The results may be reconciled if it is postulated that binding of a single molecule of to any of its four potential binding sites is sufficient to activate the channel. In this scheme, both the long release events observed in permeabilized skeletal fibers756 and the subconductance state observed in single channel recordings754-756 represent long-dwelling events of a single onto a single RyR channel.
The three-dimensional structure of RyR1 determined by cryo-electron microscopy revealed that the channel has a mushroom-like form with a squarely-shaped, bulky cytoplasmic domain containing four peripheral clamps.57,106,107 The transmembrane (TM) region is composed of four subdomains (one for each subunit) that form the stem of the mushroom-like channel. Comparison of RyR1 in open and closed states show that there is a great range of conformational changes in the stem of the channel accompanied by opening of a central pore106,112 The TM domain in the open state is rotated ~4° with respect to closed state in a movement similar to the movement of an iris and potentially involving the four TM subdomains.106 A single molecule binds to a single RyR1 subunit in a crevice between domain 3 and domain 7/8112 (which is probably the site binding MCa as well). Domain 3 and domain 7/8 are connected to the central conducting vestibule through short “bridges”. Based on this structural information, occupation of the toxin sites may transmit a great range of conformational changes through the region of the “bridges” that introduces a constriction of the conduction pathway. In this manner, binding of the toxin to any of the four RyR subunits may modify the conformational changes of the four subunits. Accordingly, or MCa (and peptide A, if bound to the same site) may limit the counter-clockwise rotation predicted by cryo-electron microscopy to less than the ~4° required for a normal, fully-conducting opening, thus producing a partial rotation that leads to subconducting states (Fig. 19-5).757
Accessibility of binding sites in intact cells
It has been long recognized that ionized peptide toxins are incapable of penetrating the plasmatic membrane unless aided by an active mechanism of transport. It is therefore logical that peptide toxins lack intracellular targets, although some peptide toxins are known to mimic external receptors and to unfold a series of intracellular events. and MCa, however, are extremely basic peptides (net charge of +7 at physiological pH), a property that could confer them the capacity to permeate membranes.758,759 Specifically, highly basic peptides translocate to cytosolic compartments
after local perturbation of the lipid bilayer,760 apparently by interaction of positively-charged basic amino acids with the negatively-charged polar heads of the phospholipids of the external membrane. Indeed, MCa has been recently shown to induce release of from intracellular stores of intact myotubes.749 As the release of was evoked in the absence of external it was inferred that MCa penetrated the external membrane and reached intracellular stores to open release channels.
Figure 19-5. Proposed mechanism of action of MCa and Peptide A on RyRs. Taken, with permission, from Chen et al.757
Alternative hypotheses abound that could explain how these toxins reach their intended targets. RyRs have recently been found in unusual places, such as mitochondria761 and possibly external membranes. It is possible that these oddly-localized RyRs are more frequent in insects and reptiles, scorpions’ natural prey, than in mammals. Also, scorpion venoms contain a rich assortment of phospholipases735 that may aid in the permeation of ionized molecules.
CONCLUDING REMARKS
and MCa are representative of a group of high-affinity scorpion peptides that induce conformational changes in the RyR protein that ultimately evoke the release of The specificity, high affinity, and reversibility of the peptide-RyR interaction make MCa and the emerging group of new toxins useful tools for the study of the structural determinants of RyR gating and conduction. At the cellular level, this subclass of toxins may aid in the dissection of the chain of events that lead to the opening of RyRs during excitation-contraction coupling.
