Structure-function relationships of peptides forming the calcin family of ryanodine receptor ligands

Abstract

Calcins are a novel family of scorpion peptides that bind with high affinity to ryanodine receptors (RyRs) and increase their activity by inducing subconductance states. Here, we provide a comprehensive analysis of the structure-function relationships of the eight calcins known to date, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs (RyR1). Primary sequence alignment and evolutionary analysis show high similarity among all calcins (≥78.8% identity). Other common characteristics include an inhibitor cysteine knot (ICK) motif stabilized by three pairs of disulfide bridges and a dipole moment (DM) formed by positively charged residues clustering on one side of the molecule and neutral and negatively charged residues segregating on the opposite side. [(3)H]Ryanodine binding assays, used as an index of the open probability of RyRs, reveal that all eight calcins activate RyR1 dose-dependently with Kd values spanning approximately three orders of magnitude and in the following rank order: opicalcin1 > opicalcin2 > vejocalcin > hemicalcin > imperacalcin > hadrucalcin > maurocalcin >> urocalcin. All calcins significantly augment the bell-shaped [Ca(2+)]-[(3)H]ryanodine binding curve with variable effects on the affinity constants for Ca(2+) activation and inactivation. In single channel recordings, calcins induce the appearance of a subconductance state in RyR1 that has a unique fractional value (∼20% to ∼60% of the full conductance state) but bears no relationship to binding affinity, DM, or capacity to stimulate Ca(2+) release. Except for urocalcin, all calcins at 100 nM concentration stimulate Ca(2+) release and deplete Ca(2+) load from skeletal sarcoplasmic reticulum. The natural variation within the calcin family of peptides offers a diversified set of high-affinity ligands with the capacity to modulate RyRs with high dynamic range and potency.

Figure 1.

Purification and characterization of vejocalcin. (A) 1.5 mg of soluble venom from V. mexicanus was separated by HPLC using a semi-preparative C18 reverse-phase column, eluted with a linear gradient from solution A to 60% solution B, run for 60 min. The fraction labeled with the asterisk was rechromatographed in an analytical C18 reverse-phase column and run from solution A to 40% solution B in 60 min. (B) The component with retention time of 28.09 min (marked with an asterisk) is vejocalcin. (C) The complete amino acid sequence of vejocalcin was obtained by a combination of direct Edman degradation and mass spectrometry, as described in Materials and methods.

Figure 2.

Calcin sequence alignment and evolutionary analysis. (A) The eight calcins known to date are aligned with opicalcin₁ as reference by Clustal Omega. The identity values to opicalcin₁ are shown on the right side. Positively charged residues lysine (K) and arginine (R), negatively charged residues aspartic acid (D) and glutamic acid (E), and the disulfide bond-forming cysteine (C) are highlighted by the colors blue, red, and gray, respectively. Columns with identical residue are marked by asterisks, whereas columns with only one difference are marked by colons on the bottom. (B) Three pairs of highly conserved disulfide bonds (Cys³-Cys¹⁷, Cys¹⁰-Cys²¹, and Cys¹⁶-Cys³²) and the residues forming part of β strands appear connected to form an ICK motif. (C) The evolutionary tree is built with the principle minimum evolution by MEGA 5.2. The genetic distance is measured by Poisson correction method with the formula d_AB = −ln(1 − f_AB), where f_AB is the fraction of different amino acids after the Poisson distribution between two sequences (dissimilarity).

Figure 3.

Net charge versus pH plot and hydrophobicity plot of calcins. (A) Net charge versus pH plot of calcins. The theoretical isoelectric points of calcins are highly basic with values ranging from 9.3 (vejocalcin) to 10.1 (urocalcin), whereas the net charges at pH 7.0 are positive for all calcins and vary from 5.8 (vejocalcin) to 8.7 (urocalcin). (B) Hydrophobicity plot of calcins. The ratio of hydrophilic residues (green) of calcins is between 52% (imperacalcin and hemicalcin) and 66% (hadrucalcin), indicating high solubility in water for all calcins.

Figure 4.

Three-dimensional modeling of calcins. (A) Imperacalcin, resolved by ¹H-NMR (Lee et al., 2004), was taken as the template molecule. (B–H) Other calcins, including opicalcin₁ (B), opicalcin₂ (C), maurocalcin (D), hemicalcin (E), vejocalcin (F), urocalcin (G), and hadrucalcin (H), were simulated by Swiss-PdbViewer 4.1.0 and viewed by Discovery Studio 3.5. Solvent-accessible PSAs were calculated using PyMOL, whereas DMs were analyzed by the online Protein Dipole Moments Server. For each calcin, the solid ribbon with line atom model may be found in Fig. S1, and the charged CPK model with frontal side (middle) and dorsal side (right) are displayed here. Positively charged residues (Lys and Arg), negatively charged residues (Asp and Glu), and neutral residues are colored by blue, red, and gray, respectively.

Figure 5.

[³H]Ryanodine binding stimulation by calcins. (A) Dose-dependent activation of RyR1 by calcins, with boundaries set at 1 pM to 20 µM (n = 3–5). Heavy SR from rabbit skeletal muscle was incubated with 5 nM [³H]ryanodine in the absence (control) and the presence of the indicated concentrations of calcins. Binding conditions were specified in Materials and methods. The K_d was determined with the formula B = (B_max)/[1 + (K_d/[calcin])^nH), where B is specific binding of [³H]ryanodine, B_max is the maximum binding stimulated by calcin, and nH is the Hill coefficient. (B) Effect of calcins (100 nM each; n = 3–5) on the Ca²⁺-dependent [³H]ryanodine binding curve. Ca²⁺-dependent activation and inactivation were fitted with the formula B = ((B_max·[Ca²⁺])/(K_a + [Ca²⁺]))·(K_i/(K_i + [Ca²⁺])), where B is the specific binding of [³H]ryanodine, B_max is the maximum binding stimulated by calcin, K_a is the activation constant, and K_i is the inactivation constant. The specific [³H]ryanodine binding was standardized with the value of the control at pCa5 as 100%. (C–E) Radar charts illustrating the effect of calcins on K_a, K_i, and maximal amplitude (A_max; percentage of stimulation with respect to control). The black line corresponds to control (no calcin) and is included in all charts for reference. Caffeine (C, dashed line) moves K_a but has little effect on A_max and no effect on K_i. This effect contrasts with that of calcins (C–E, color lines as indicated), which affect K_a, K_i, and A_max.

Figure 6.

Fractional subconductance value induced by calcins on single RyR1 channels. RyR1 channel from rabbit skeletal muscle SR vesicles was reconstituted in lipid bilayers and recorded and analyzed as described in Materials and methods. (A) Single RyR1 channel in the absence of calcin is presented as control. (B–I) The eight calcins were added separately into the cis (cytosolic) chamber at the indicated concentration. c represents the zero current level when the channel is in the fully closed state, whereas s and o display the subconductance state and full opening of the channel, respectively. Current histograms from 10,000–30,000-ms segments of channel activity are shown at the right side of the traces for each calcin. The number of experiments varied for each calcin. Imperacalcin, maurocalcin, and hadrucalcin: n = 12, 8, and 5 different channels. All other calcins were tested at least two or three times.

Figure 7.

Calcin-induced Ca²⁺ release from skeletal heavy SR vesicles. (A) Typical trace of Ca²⁺ release by calcins. A concentration of 20 nM imperacalcin elicited abrupt Ca²⁺ release from SR vesicles, and further additions had incremental effects only. The fraction of calcin-induced Ca²⁺ release was calculated by adding the Ca²⁺ ionophore A23187 (5 µM). (B) Except urocalcin, all calcins elicited abrupt Ca²⁺ release within 100 nM (n = 3–4). (C) At 100 nM, all calcins except urocalcin elicited Ca²⁺ release to ∼60% of total Ca²⁺ load from heavy SR. Mean ± SEM is shown. **, P < 0.01; calcins versus A23187, t test.

(Scheme 1)

(Scheme 2)

Figure 8.

DM, fractional conductance, and Ca²⁺ release appear unrelated to the K_d of calcins. (A) Plot of DM versus K_d of [³H]ryanodine binding. The DMs have a poor correlation with the binding affinity (r² = 0.15). (B) Subconductance versus K_d of [³H]ryanodine binding. The position of subconductance induced by calcin is unrelated to the K_d of [³H]ryanodine binding (r² = 0.07). (C) Ca²⁺ release versus [³H]ryanodine binding. No correlation is seen between calcin concentration that induces Ca²⁺ release and K_d of [³H]ryanodine binding (r² = 0.04).