Hemicalcin, a new toxin from the Iranian scorpion Hemiscorpius lepturus which is active on ryanodine-sensitive Ca2+ channels

Abstract

In the present work, we purified and characterized a novel toxin named hemicalcin from the venom of the Iranian chactoid scorpion Hemiscorpius lepturus where it represents 0.6% of the total protein content. It is a 33-mer basic peptide reticulated by three disulfide bridges, and that shares between 85 and 91% sequence identity with four other toxins, all known or supposed to be active on ryanodine-sensitive calcium channels. Hemicalcin differs from these other toxins by seven amino acids at positions 9 (leucine/arginine), 12 (alanine/glutamic acid), 13 (aspartic acid/asparagine), 14 (lysine/asparagine), 18 (serine/glycine), 26 (threonine/alanine) and 28 (proline/isoleucine/alanine). In spite of these differences, hemicalcin remains active on ryanodine-sensitive Ca2+ channels, since it increases [3H]ryanodine binding on RyR1 (ryanodine receptor type 1) and triggers Ca2+ release from sarcoplasmic vesicles. Bilayer lipid membrane experiments, in which the RyR1 channel is reconstituted and its gating properties are analysed, indicate that hemicalcin promotes an increase in the opening probability at intermediate concentration and induces a long-lasting subconductance level of 38% of the original amplitude at higher concentrations. Mice intracerebroventricular inoculation of 300 ng of hemicalcin induces neurotoxic symptoms in vivo, followed by death. Overall, these data identify a new biologically active toxin that belongs to a family of peptides active on the ryanodine-sensitive channel.

Figure 1. Purification of HCa from the venom of H. lepturus

(A) The extracted venom was fractionated by G-50 gel-filtration chromatography columns (2×K26/50) equilibrated with 20 mM ammonium acetate, pH 4.7. (B) HCa was purified from the neurotoxic fraction (II') by reverse-phase HPLC on a C₈ column using a gradient of buffer B (0.1% TFA in acetonitrile) as described in the Experimental section. HCa was collected at 18.93 min. OD_{280 nm}, A₂₈₀.

Figure 2. Sequence alignment of HCa with related toxins active on the RyR

(A) Sequence alignment of HCa with four analogous toxins, MCa, IpTx A, opicalcine 1 and opicalcine 2. MCa and IpTx A are two peptides known to be active on the RyR. Opicalcine 1 and 2 have not been tested for pharmacological activity. All five toxins have the same number of positively charged amino acid residues (the N-terminal glycine residue, six or seven lysine residues and four or five arginine residues). (B) A sequence alignment of HCa with peptide A of the II–III loop of Ca_vα_1.1 subunit from the DHPR is also provided. The strongest homology stretches from Lys¹⁹ to Thr²⁶ of HCa.

Figure 3. HCa affects [³H]ryanodine binding on heavy SR vesicles

Dose-dependent effect of HCa on [³H]ryanodine binding on to heavy SR vesicles (○). [³H]Ryanodine binding was measured at pCa 5 in the presence of 5 nM [³H]ryanodine for 3 h at 37 °C. Non-specific binding remained constant at all HCa concentrations and was less than 200 fmol/mg. Data were fitted with a logistic function y=y₀+{a/[1+(x/EC₅₀)^b]} where y₀=316±46 in the absence of HCa, a=2799±118 fmol/mg is the maximum [³H]ryanodine binding, b=−1.1±0.1 (the slope coefficient), and EC₅₀=71±6 nM (the concentration of HCa for half stimulation of [³H]ryanodine binding). Overall, HCa stimulates [³H]ryanodine binding 11.8-fold at saturation level. For comparison, the data are also shown for MCa (●; y₀=364±129, a=3502±127 fmol/mg, b=−1.0±0.1, and EC₅₀=25±2 nM; stimulation factor of 16.7 at saturation).

Figure 4. Ca²⁺ release from heavy SR vesicles induced by HCa

(A) Absorbance measured in the absence of SR vesicles and in response to sequential Ca²⁺ additions in the medium or to 40 nM HCa addition. The data indicate the lack of Ca²⁺ in the purified material. (B) Heavy SR vesicles were actively loaded with Ca²⁺ by four sequential additions of 20 μM CaCl₂ in the monitoring chamber. The absorbance was monitored to show that the added Ca²⁺ was taken up by the SR vesicles. The trace relaxed close to its original baseline with CaCl₂ additions constituting approx. 70–80% of the SR loading capacity. These Ca²⁺ additions were used to calibrate the Ca²⁺ release. Addition of 125 nM HCa produces a long-lasting Ca²⁺ release from SR vesicles. Ca²⁺ (20 μM) was added along with HCa to control the extravesicular concentration of Ca²⁺. Also, external Ca²⁺ appears to act as a cofactor to the HCa effect. Residual Ca²⁺ from SR vesicles is released by the addition of 1 μM ionophore A23187. Further addition of 0.5 mM EGTA buffers the released and the basal Ca²⁺ from the system. (C) Loading of heavy SR vesicles with Ca²⁺ by six sequential additions of 20 μM CaCl₂ in the monitoring absorbance chamber. The fifth application did not produce a similar calcium release to the one observed by the co-application of 20 μM Ca²⁺ and 125 nM HCa in (B). (D) Loading of heavy SR vesicles with Ca²⁺ by four sequential additions of 20 μM CaCl₂ and a fifth one at 40 μM in the monitoring absorbance chamber. The fifth application produces a slower loading of the vesicles, but no sustained response.

Figure 5. HCa alters gating kinetics and stabilizes subconductances of RyR1 single-channel activity in BLMs

RyR1 single channel was incorporated by inducing fusion of skeletal muscle junctional SR vesicles into BLMs. The channel activities were recorded and analysed as described in the Experimental section. RyR1 single channel in the absence of HCa was used to serve as control and was recorded for 3 min (A). Sequential additions of HCa to achieve a final concentration of 175 nM (B) and 350 nM (C) were made into the cis chamber, and the channel activity was recorded for 5 min under each condition. The broken lines indicate the maximum current amplitude of the native RyR1 channel (37.2 pA) when the channel is fully open (o). The arrow marked ‘c’ shows the zero current level when the channel is in the fully closed state, and the arrow marked ‘s’ shows the HCa-stabilized subconductance state of the channel. The data are representative of a total of five independent bilayer experiments with RyR1 channels from one junctional SR protein preparation and one purified RyR1 preparation.

Figure 6. Homology model of HCa

Backbone ribbon representation of the model of HCa and comparison with the structure of MCa (PDB code 1C6W [11]), which, besides IpTx A, is one of the two structures used as template for molecular modelling. Disulfide bridges are in stick representation. Basic amino acid residues present on the same face of MCa and HCa (Lys¹⁹, Lys²⁰, Lys²², Arg²³, Arg²⁴ and Lys³⁰) are shown. The side-chain bonds of Lys¹¹, Lys¹⁴, Asp¹³ and Ala¹² in HCa and Lys¹¹, Lys¹⁴, Asn¹³ and Glu¹² in MCa are also shown.