Mu8.1 defines a new class of conotoxins evolutionarily related to the con-ikot-ikots

Transcriptome analysis of the venom gland of C. mucronatus, a fish-hunting species from the Phasmoconus clade [20], revealed the presence of 2 previously unrecognized toxin transcripts. Their high degree of sequence similarity designated them to be allelic variants that we assigned the names Mu8.1 and Mu8.1ii [21]. The protein sequence of Mu8.1 displays the characteristic tripartite organization of conotoxins consisting of a predicted N-terminal signal sequence (21 residues), an intervening propeptide region (5 residues), and, lastly, the toxin-encoding region (89 residues) (Fig 1A). The Mu8.1 and Mu8.1ii sequences differed only in the substitution of 3 residues in the C-terminal region (Fig 1B).

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Fig 1. Mu8.1 belongs to a new conotoxin superfamily.

(A) Mu8.1 prepro-sequence annotated with colored bars indicating the predicted tripartite organization. Mauve: signal sequence; pink: propeptide region; green: mature Mu8.1. Cysteine residues are colored red. (B) Alignment of Mu8.1/ii and select sequences from the con-ikot-ikot (from C. striatus), Cluster 1 (from C. litteratus), and Cluster 2 (from C. ventricosus) classes. Top: multiple sequence alignment of the predicted signal and propeptide sequences (mauve and pink bars denote signal and propeptide organization for Mu8.1, and the shaded pink boxes indicate the propeptide sequences of con-ikot-ikot, Cluster 1 and Cluster 2). Bottom: alignment of the mature toxin sequences (green bar). The 3 sequence differences between Mu8.1 and Mu8.1ii are marked with black bars above the residues in the alignment. Precursor sequences provided in S1 Data. Grey: columns with at least 60% sequence identity; yellow: cysteine residues.

https://doi.org/10.1371/journal.pbio.3002217.g001

We initially performed a BLASTp search [22] using the Mu8.1 precursor as the query sequence to find Mu8.1-related sequences from other Conus species. Three of the identified Mu8.1-related toxins, Vx65, Vx78, and Vx79 from Conus vexillum, have previously been identified at the protein level in venom extracts by MS/MS [23]. Several of the identified sequences were annotated as con-ikot-ikot toxins. However, sequence alignment with the original con-ikot-ikot toxin [24] suggested that Mu8.1 may belong to a different class of conotoxins. To identify a more complete set of conotoxins belonging to the same class as Mu8.1, we mined the transcriptome data available in the National Center for Biotechnology Information (NCBI), DNA Databank of Japan (DDBJ), and China National Genebank (CNGB) sequence repositories. Searching 602,377 assembled transcripts from these venom gland datasets (S1 Table), we identified a total of 104 sequences from 38 species. These sequences included examples from the snail-hunting species Conus marmoreus, Conus textile, and Conus gloriamaris, as well as several from various worm-hunting species. The identified signal sequences are highly conserved and harbor a MDMKMTFSGFVLVVLVTTVVG consensus sequence (S1 Fig). Several of the sequences contain 2 additional N-terminal residues, MT. All sequences contain 10 conserved cysteine residues in the region of the mature toxin with identical loop sizes between them, with a few sequences containing 1 or 2 additional cysteines. As is common in conotoxins, there is a high variability between the mature sequences. Still, 10 residues (in addition to the cysteines) are strictly conserved among species (S1 Fig). Based on the structural similarity with proteins carrying a saposin domain (see further below), we refer to this new class of toxins as saposin-like conotoxins (SLCs).

The database annotation of several SLCs as con-ikot-ikots led us to investigate a potential evolutionary relationship between these 2 classes. To do so, we identified all published Conus sequences with sequence similarity to con-ikot-ikots (retrieved from GenBank) and grouped these using CLuster ANalysis of Sequences (CLANS) clustering analysis. This method uses all-against-all BLAST e-values to attract sequences with high similarity and repel sequences of little similarity, thereby forming clusters of highly similar sequences. The CLANS analysis (S2 Fig) showed 4 distinct but interconnected clusters corresponding to the identified toxin classes: the “classical” con-ikot-ikots, the SLCs, and 2 previously unrecognized conotoxin classes (Clusters 1 and 2). The numerous links interconnecting the 4 clusters suggest a common evolutionary origin. To further probe the putative evolutionary relationship between the sequences of the 4 classes, we compared the nucleotide sequences of their 5′ untranslated regions (UTRs) and the beginning of their open reading frames (ORFs) using randomly selected sequences from each class. This sequence alignment showed a high sequence identity of the 5′ UTR in the 4 toxin classes (S3 Fig), further suggesting a common evolutionary origin. The conclusion that the sequences of the 4 identified classes are related through an ancestral gene was corroborated by the finding that their gene structures are very similar, with all introns occurring in the same phase (phase 1) (S4 Fig). We concluded that the 4 identified classes—SLC, con-ikot-ikot, Cluster 1, and Cluster 2—together comprise a superfamily where the individual classes have related signal sequences, but distinct mature regions (Fig 1B).

Recombinantly expressed Mu8.1 purifies as a single, fully oxidized species from E. coli

To gain insight into the structure and function of the new SLC family of conotoxins, we expressed Mu8.1 using the csCyDisCo system [25]. This system, based on the original CyDisCo system [26,27], allows disulfide-bond formation in the cytosol of E. coli as a result of expression of the Erv1p oxidase along with 2 protein disulfide isomerases (human protein-disulfide isomerase (PDI) and a conotoxin-specific PDI) from an auxiliary plasmid [25,28]. Mu8.1 was expressed as a fusion protein with an N-terminal ubiquitin (Ub) tag containing 10 consecutive histidine residues inserted into a loop region followed by a tobacco etch virus (TEV)-protease recognition site (S5A Fig). The beneficial effect of the csCyDisCo system was confirmed by SDS-PAGE gel and western blot analysis, where the Ub-His₁₀-Mu8.1 fusion protein was found in the soluble fraction (to approximately 50%) only when coexpressed with Erv1p and the 2 PDIs (Fig 2A), as we have previously observed for other conotoxins [25,28]. TEV protease cleavage of Ub-His₁₀-Mu8.1 resulted in the liberation of the 89-residue mature conotoxin, which was purified (>95% purity; Fig 2B) in a final yield of approximately 1 mg per liter of culture.

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Fig 2. Recombinantly expressed Mu8.1 exists primarily as a noncovalent dimer in solution.

(A) Comparison of Ub-His₁₀-Mu8.1 expressed in the absence (−csCyDisCo) or presence (+csCyDisCo) of the csCyDisCo expression system in E. coli BL21(DE3) cells. Left panel: 15% SDS-PAGE gel stained with Coomassie Brilliant Blue showing the molecular weight marker (M), the total cell extract (T), resuspended pellet after lysis and centrifugation (P), and the soluble fraction (S) from cells expressing Ub–His₁₀–Mu8.1. Note that Ub-His₁₀-Mu8.1 and Erv1p migrate similarly. Protein levels are directly comparable between lanes. Right panel: western blot probed with an anti-His antibody (α-His) for detection of Ub-His₁₀-Mu8.1 in the same samples loaded on the SDS-PAGE gel. The line labeled with a red asterisk indicates the migration of what is likely reduced and partially oxidized species of Ub-His₁₀-Mu8.1. Original SDS-PAGE gel and western blot images provided in S1 Raw Images. (B) 15% Tris-Tricine SDS-PAGE gel analysis of purified Mu8.1 in the reduced (40 mM DTT; red) and oxidized (ox) state. Original SDS-PAGE gel and western blot images provided in S1 Raw Images. (C) Analytic gel filtration of Mu8.1 at 100 μM (orange) and 1 μM (blue) concentrations. Arrows denote the elution volumes of standard proteins relating to the blue trace. The dotted lines show the retention volumes of Mu8.1 in each sample. The peak intensity of the 2 samples was min-max normalized to allow comparison, and the orange plot was offset for clarity. (D) Normalized Kratky plot from small angle X-ray scattering analysis of 5 concentrations of purified Mu8.1 (1 mg/ml, orange; 3 mg/ml, yellow; 6 mg/mL, purple; 8 mg/mL, green; 13 mg/mL, light blue). The cross denotes the peak position of a globular protein. Source data for quantifications provided in S2 Data.

https://doi.org/10.1371/journal.pbio.3002217.g002

Disulfide-bond formation in Mu8.1 was verified by SDS-PAGE analysis and full oxidation by MALDI-TOF mass spectrometry. Upon reduction, a clear mobility shift was observed by SDS-PAGE (Fig 2B), and MALDI-TOF mass spectrometry of reversed-phase high pressure liquid chromatography (RP-HPLC)-purified Mu8.1 verified the presence of a single species with a mass of 10,181.7 Da (S5B Fig), a value that fits the theoretical average mass of fully oxidized Mu8.1 of 10,181.5 Da.

Mu8.1 is an α-helical, dimeric protein in solution

More detailed insight into the structural properties of Mu8.1 in solution was obtained using circular dichroism (CD) spectroscopy, analytical gel filtration, and small-angle X-ray scattering (SAXS) measurements.

The CD spectrum of Mu8.1 showed characteristic features of an α-helical structure with minima at 208 nm and 218 nm, and a global maximum at 195 nm (S5C Fig). To probe the quaternary structure of Mu8.1 in solution, we first performed analytical size exclusion chromatography on Mu8.1. Regardless of the concentration (1 μM or 100 μM), Mu8.1 eluted as a single, symmetric peak with an apparent molecular weight of 22 to 24 kDa, suggesting a dimer under these conditions (Fig 2C).

To corroborate this result, SAXS measurements were carried out at 5 protein concentrations ranging from 1 mg/mL to 13.2 mg/mL (100 μM to 1.3 mM). Here, at low q-values, we observed an increase in the intensity with increasing protein concentration, indicating an increase in size (S6 Fig and Table A in S2 Table). A dimensionless Kratky plot of the data revealed a shift in peak position with increasing concentration, suggesting a change from a spherical conformation concomitant with increasing concentration (Fig 2D). Singular value decomposition was employed to assess the number of species present, followed by calculation of their relative volume fractions (Table B in S2 Table). The results revealed the presence of 3 oligomeric species, with the dominant being a dimer (85% at 1 mg/mL) and increasing occurrence of tetrameric and hexameric species that correlated with increasing concentration. No monomer was observed.

Based on evidence from the analytic gel filtration and SAXS measurements, we concluded that Mu8.1 exists primarily as a dimer in solution.

The crystal structure of Mu8.1

To elucidate key structural features of the SLC superfamily, we determined crystal structures of Mu8.1 from 2 different crystal conditions referred to as Mu8.1_38 and 59 (see Materials and methods) at resolutions of 2.33 Å and 2.1 Å, respectively (Fig 3). The asymmetric unit of Mu8.1_59 accommodates 6 molecules that form 3 equivalent dimers (S7 Fig). The asymmetric unit of Mu8.1_38 includes 2 molecules that form a single dimer with an interface equivalent to the one present in the 3 dimers in Mu8.1_59. Each protomer of the Mu8.1 structure comprises 2 regions (Fig 3A and 3B). The first region comprises an N-terminal 3₁₀-helix (3₁₀N), followed by 2 α-helices (α1 and α2), and a 3₁₀-linker helix (3₁₀L). The second region comprises 2 α-helices (α3 and α4), and the C-terminal 3₁₀-helix (3₁₀C). The disulfide bonds are found primarily within each of these 2 regions. Within the first region, the Cys18-Cys34 and Cys22-Cys30 disulfide bonds connect α1 and α2. Further, the 3₁₀N-helix connects to α3 through the Cys10-Cys51 disulfide bond. In the second region, Cys61-Cys71 links α3 and α4, and Cys57-Cys89 tethers the C-terminus to α3 (Fig 3B).

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Fig 3. Crystal structure of Mu8.1.

(A) Graphic representation of the Mu8.1 structure with disulfide bridges represented by brackets (numbered by Roman numerals) and α- and 3₁₀-helices represented by cylinders (numbered by Arabic numerals). Four amino acid residues at the N-terminus not resolved in the crystal structure are shown as dotted lines. (B) Cartoon representation of the Mu8.1 protomer containing three 3₁₀ helices interspersed with 4 α-helices labelled as α1-α4. The disulfide bridges are shown as yellow stick models and labelled with Roman numerals as in Panel A.

https://doi.org/10.1371/journal.pbio.3002217.g003

The 2 helical regions surround a hydrophobic core predominantly formed by aromatic residues (Phe15, Tyr31, Tyr58, Tyr59, Trp72; Fig 4A). Moreover, α1, α3, and α4 are held together by a network of contacts between Arg16, Tyr58, Glu68, and Trp72, where ionic interactions are present between the side chains of Arg16 and Glu68, whereas Tyr58 and Trp72 partake in T-shaped pi–pi interactions (Fig 4A). This network of residues is completely or highly conserved among the SLC superfamily toxins (S1 Fig). The only variations are seen in 4 sequences where Arg16 is substituted with a Lys and Tyr58 is substituted by a Phe.

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Fig 4. Crystal structure of dimeric Mu8.1.

(A) Cartoon representation of the Mu8.1 protomer, with the aromatic residues (F15, Y31, Y58, Y59, and W72) contributing to the hydrophobic core depicted as stick models and the corresponding van der Waals radii depicted as translucent spheres. Zoom: Select conserved amino acid residues—R16, Y58, E68, and W72 (shown as stick models)—whose interactions play a structural role (see main text for details). Ionic interactions are shown as yellow dotted lines. (B) “Side view” of the Mu8.1 dimer highlighting the amino acid residues (depicted as sticks) composing the dimer interface. Protomer A is shown in forest green, and protomer B in pale green. Hydrophobic residues are in dark blue, and polar residues in pink. Water molecules are represented by yellow spheres.

https://doi.org/10.1371/journal.pbio.3002217.g004

The dimer interface is characterized by both hydrophobicity as well as the presence of a water-filled cavity surrounded by the polar residues Thr36, Asn47, Thr52, and Thr56 (Fig 4B). The residues forming the largely dry (water-excluded) hydrophobic interface include Tyr31, Ala32, Phe49, Ile53, Tyr59, and Val83. While the residues involved in structural stabilization of the Mu8.1 monomer are highly conserved among SLC superfamily proteins, the amino acid residues comprising the dimer interface exhibit a higher degree of variability (S1 Fig). A potential functional role of 4 additional conserved residues without any apparent structural purpose—Lys55, Arg60, Lys66, and His70—remains speculative at present but could represent residues that interact with the molecular target of this toxin family.

Mu8.1 has structural similarity with con-ikot-ikot but does not modify GluA2 desensitization

Structural similarity often correlates with similar functional properties and, consequently, may provide important insight into protein function. We, therefore, searched the entire Protein Data Bank (PDB) for structures exhibiting similarity to Mu8.1. The search identified 48 hits showing a high degree of similarity to the Mu8.1 protomer structure, with most structures displaying a saposin-like fold (see below). Notably, con-ikot-ikot from Conus striatus also showed structural similarity with Mu8.1. The schematic overview of Mu8.1, con-ikot-ikot, and human saposin A (SapA) in Fig 5A reveals the similar architecture of these proteins.

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Fig 5. Monomeric Mu8.1 exhibits a saposin-like fold.

(A) Graphic representations of Mu8.1 (green), con-ikot-ikot (dirty violet), and SapA (blue). Con-ikot-ikot is a homodimer held together by 3 intermolecular disulfide bonds, with each subunit comprising 4 α-helices and 5 disulfide bonds [29]. SapA comprises 4 α-helices and 3 disulfide bonds. Disulfide bonds are depicted as brackets and labelled with Roman numerals. Amino acid residues not resolved in the crystal structure of Mu8.1 are shown as dotted lines. The yellow and light blue triangles denote structurally conserved disulfide bonds between Mu8.1 and con-ikot-ikot. (B) Superimposition of the structures of monomeric Mu8.1 (forest green) and con-ikot-ikot dimer (PDB:4U5H) (dirty violet) are shown in cartoon representation. Disulfide bonds are represented as yellow sticks. The superimposition was performed using the CLICK server as described in the Materials and methods section and visualized in Pymol. (C) Overlay of cartoon representations of monomeric Mu8.1 (forest green), monomeric con-ikot-ikot (dirty violet), and SapA (PDB: 2DOB) (blue). Con-ikot-ikot and Mu8.1 superposition was performed with the CLICK server as described in Materials and methods. SapA was manually superimposed with the 2 other molecules in Pymol using the sequence-independent program “super”. (D) Mu8.1 (green), con-ikot-ikot (dirty violet), and SapA (blue) shown separately in the same orientation as in Panel C.

https://doi.org/10.1371/journal.pbio.3002217.g005

Con-ikot-ikot targets the AMPA-type of ionotropic glutamate receptors and has been shown to inhibit desensitization of the receptor producing a sustained agonist receptor-mediated current [24,30]. The crystal structure of con-ikot-ikot, determined in complex with the GluA2-type AMPA receptor, has shown a homo-dimeric protein covalently linked by disulfide bonds formed between 3 of the 13 cysteine residues present in each monomer [29] (Fig 5A). Despite low sequence similarity between con-ikot-ikot and Mu8.1 (Fig 1B), the structures of the Mu8.1 protomer and con-ikot-ikot superimpose well with a root mean square deviation (RMSD) of 2.14 Å for all Cα atoms (Fig 5B). Nevertheless, despite the high degree of structural similarity between the 2 proteins, Mu8.1 failed to modulate the desensitization of the GluA2 AMPA receptor transiently transfected into HEK293 cells (S8 Fig).

Mu8.1 and con-ikot-ikot display a saposin-like fold

Saposin-like proteins (SAPLIPs) comprise a protein family with over 200 members that perform a variety of biological functions and are found in a phylogenetically diverse group of eukaryotes. Most of these involve lipid interaction and lead to local disordering of the lipid structure, membrane perturbation, or membrane permeabilization [31], although the fold is also associated with other functions [32]. SAPLIPs display a characteristic 4-helix bundle structure stabilized by a conserved pattern of 3 disulfide bonds (Fig 5A, 5C and 5D).

SapA was one of the top hits in our search, although the SapA and Mu8.1 sequences only share 21.6% sequence identity. The SapA and Mu8.1 have similar structural topologies and superimpose with an RMSD of 1.81 Å for all Cα atoms, with the 2 first α-helices superimposing especially well (Fig 5C). A significant difference is constituted by the disulfide pattern in the 2 proteins (Fig 5A and 5D). The functional implications of this observation suggest that Mu8.1 is unlikely to be involved in lipid binding (see Discussion). Overall, this analysis revealed a previously undiscovered structural resemblance between con-ikot-ikot and SAPLIPs that extends to Mu8.1.

Mu8.1 modulates depolarization-induced Ca²⁺ influx in sensory neurons

To assess the bioactivity of Mu8.1, we next tested the effects of Mu8.1 on Ca²⁺ influx in somatosensory DRG neurons that relay sensory input to the CNS. These cells were isolated from transgenic reporter mice in which the regulatory elements of the calcitonin gene-related peptide (CGRP) drive the expression of green fluorescent protein (GFP), whereby GFP labeling identifies peptidergic nociceptive neurons. Overall, 7 major classes of sensory neurons responsible for processing the sensations of cold, heat, mechanical cues, and pain were assigned based on cell size, GFP labeling, and isolectin B4 staining [33], as well as their responses to mustard oil (AITC), menthol, capsaicin, and conotoxin RIIIJ, according to the functional classification of somatosensory neurons proposed by Giacobassi and colleagues [11].

Representative examples from neurons belonging to all somatosensory subclasses assayed are shown in Fig 6A. DRG neurons incubated in observation solution (4 mM KCl) were sequentially depolarized by pulses (15 seconds) of high potassium (25 mM KCl) extracellular solution, which caused stereotypical rises in intracellular Ca²⁺ concentration evidenced by the increase in Fura-2 signal. Incubation with 10 μM Mu8.1 produced a decrease in subsequent Ca²⁺ peaks elicited by the high K⁺ stimulus in 20.3 ± 3.0% of the neurons analyzed (n = 2,365; Fig 6B pie chart), predominantly in peptidergic nociceptors (68% of all affected cells, green bar Fig 6B). In addition to the peptidergic nociceptors, 10 μM Mu8.1 reduced the calcium signals of DRG neurons identified as large-diameter mechanosensors (12.6% of all affected cells) and C-low threshold mechanoreceptors (C-LTMRs; 7% of all affected cells) as shown in Fig 6B (magenta and lilac bars, respectively).

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Fig 6. Mu8.1 inhibits calcium entry into mouse sensory neurons.

(A) Intracellular calcium changes in response to sequential pharmacological treatments from 7 classes of DRG neurons (labelled on the right). Each trace represents the calcium signal (ΔF/F) of the neuron pictured on the left (GFP-CGRP+: peptidergic nociceptors; Alexa Fluor 647-Isolectin B4+: nonpeptidergic nociceptors, and bright-field). KCl depolarization pulses (25 mM) are indicated by light grey shading. In the presence of Mu8.1 (10 μM, mauve box) KCl-induced calcium peaks are reduced in peptidergic nociceptors (traces 1–4), large-diameter mechanoreceptors (traces 5–6), and C-LTMRs (traces 7–8). Class-defining pharmacology: RIIIJ (1 μM, blue box); AITC (100 μM; mustard flower), menthol (400 μM; peppermint leaf), and capsaicin (300 nM; chili pepper). A pulse of KCl (40 mM) was used to elicit maximum calcium signal at the end of the experiment. Horizontal lines flanking breaks within a trace signify graphical adjustment of trace amplitude to avoid overlap of neighboring traces. (B) Pie chart representing the population of neurons analyzed (n = 2,365), highlighting the 20.3 ± 3% of Mu8.1 (10 μM)-sensitive sensory neurons. The bar graph provides the percentage of each class of Mu8.1-sensitive DRG neurons. (C) Quantification of the relative change f(x) in KCl-induced intracellular calcium signal observed in the presence of Mu8.1 (10 μM) (see Materials and methods for normalization details). Two-tailed t test * p ≤ 0.05; **** p < 0.0005. Source data for individual traces and quantifications found in S3 Data. AITC, allyl isothiocyanate; CGRP, calcitonin gene-related peptide; C-LTMR, C-low threshold mechanoreceptor; DRG, dorsal root ganglia; GFP, green fluorescent protein.

https://doi.org/10.1371/journal.pbio.3002217.g006

Mu8.1 inhibitory effects on cytosolic Ca²⁺ concentration was estimated after min-max normalization (see Materials and methods) and evaluated by two-tailed t tests (Fig 6C). Mu8.1 (10 μM) significantly curtailed the total influx of Ca²⁺ elicited by KCl application in peptidergic nociceptors (f(x) = −0.14 ± 0.01, n = 596; p <0.001), C-LTMRs (f(x) = −0.06 ± 0.01, n = 190; p <0.001), and large-diameter mechanosensors (f(x) = −0.03 ± 0.01, n = 48; p = 0.0279).

Mu8.1 inhibits recombinant and native Cav2.3-mediated currents

Given the ability of Mu8.1 to inhibit cellular Ca²⁺ influxes in DRG neurons, we assessed the modulatory effects of Mu8.1 over a comprehensive panel of recombinant voltage-gated calcium, sodium, and potassium channels (Cav, Nav, and Kv, respectively) by automated patch clamp (APC) electrophysiology as well as a large array of GPCRs by high-throughput screening assays. Among all the recombinant channels tested (S3 Table), Mu8.1 exhibited the highest potency against Cav2.3 channels. Fig 7 shows representative current traces of human Cav2.3-mediated whole-cell currents exposed to increasing concentrations of Mu8.1 recorded from HEK293 cells (Fig 7A) and the resulting concentration–response curve rendering an IC₅₀ of 5.8 μM and a Hill coefficient close to unity (0.97) (Fig 7B). Mu8.1 reversibly inhibited Cav2.3 peak currents (S9 Fig) without significantly affecting the voltage dependence of activation for the channel (S10 Fig).

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Fig 7. Mu8.1 inhibits recombinant human Cav2.3-mediated currents in a concentration-dependent manner.

(A) Representative whole-cell Cav2.3-mediated currents in control (green) and in the presence of 1 μM (blue), 3 μM (purple), and 10 μM (mauve) Mu8.1. Depolarization-activated currents were elicited by a 50-ms test pulse to −10 mV (Vh −80 mV; 0.1 Hz). (B) Concentration–response relationship for the inhibition of Cav2.3 peak currents (IC₅₀ = 5.8 μM; nH: 0.97; n = 5). Source data showing the recorded currents and quantifications provided in S4 Data. (C) Mu8.1 and SNX-482 target overlapping populations of peptidergic nociceptors and C-LTMRs. Each trace represents the calcium signal (ΔF/F) of the neuron pictured on the left (GFP-CGRP+: peptidergic nociceptors; Alexa Fluor 647-Isolectin B4+: nonpeptidergic nociceptors, and bright-field). Cells were treated with 3 μM Mu8.1 (purple box), 10 μM Mu8.1 (mauve box), and 100 nM SNX-482 (pink box). All other labels as in Fig 6A. Horizontal lines flanking breaks within a trace signify graphical adjustment of trace amplitude to avoid overlap of neighboring traces. (D) Relative quantification of peptidergic nociceptors (pep nocicep), C-LTMRs, and nonpeptidergic nociceptors (non-pep nocicep) sensitive to 3 μM and 10 μM Mu8.1 (purple and mauve, respectively) and 100 nM SNX-482 (pink). Percentages were calculated from the total number of cells recorded per class. Bars represent mean ± SD from 2 independent DRG isolations (190 and 810 neurons per group). Source data of individual traces and quantifications found in S4 Data. CGRP, calcitonin gene-related peptide; C-LTMR, C-low threshold mechanoreceptor; DRG, dorsal root ganglia; GFP, green fluorescent protein.

https://doi.org/10.1371/journal.pbio.3002217.g007

At concentrations above 30 μM, Mu8.1 displayed weak inhibitory effects over Cav2.1, Cav2.2, Cav3.1–3, and Kv1.1 channels with near equipotency (IC₅₀s calculated from inhibition at a single concentration of Mu8.1 are summarized in S3 Table), whereas no obvious modulatory effects over heterologously expressed Cav1.2, Nav1.4, Nav1.7, hERG, Kv1.2, Kv1.3, or Kv4.3 channels were noted (S3 Table). The same was observed for a large panel of recombinant GPCRs and other receptors assessed through β-arrestin recruitment and radioligand binding assays (S11 Fig). Overall, our comprehensive functional screening identified the R-type Cav2.3 channel as the highest affinity mammalian target of Mu8.1 (although inhibition occurs with modest potency). This finding aligns with the observed effects of Mu8.1 on DRG subpopulations, including peptidergic nociceptors and C-LTMRs, which are known to express abundant levels of Cav2.3 channels [34–36].

Using constellation pharmacology, we demonstrated that Mu8.1 inhibition of DRG neuron calcium signals were largely reversible shortly after removal of the peptide as well as concentration dependent. Reversibility of Mu8.1 actions can be surmised from the grey shading after Mu8.1 application in Figs 6A and 7C, while 3 μM and 10 μM Mu8.1 inhibited 32 ± 10% and 48 ± 13% of subsequent calcium peaks in peptidergic nociceptors, respectively (Fig 7C and 7D). Although overall reversibility was observed, 7.8% of peptidergic nociceptors did not recover after several full-bath media exchanges (S12 Fig).

SNX-482 is a spider venom peptide commonly used to study Cav2.3-mediated currents in native tissues [37], and, in contrast to Mu8.1, it is also a potent inhibitor of the Kv4 family of voltage-gated potassium channels [38]. We applied SNX-482 (100 nM) to DRG neurons after Mu8.1 inhibition was reversed, verifying that these 2 venom-derived peptides target overlapping populations of somatosensory neurons, predominantly peptidergic nociceptors, and C-LTMRs (Fig 7C and 7D). Interestingly, SNX-482 also decreased Ca²⁺ signals from approximately 28% of nonpeptidergic nociceptors. These results strongly indicate that the decrease in Ca²⁺ influx observed upon exposure to Mu8.1 is mechanistically supported by the inhibition of Cav2.3 channels. In DRG neurons, SNX-482 not only reduced depolarization-induced Ca²⁺ signals via inhibition of Cav2.3 channels but also displayed direct and indirect Ca²⁺ signal amplification effects (S13 Fig) akin to the inhibition of voltage-gated K⁺ conductances likely mediated by Kv4 potassium channel isoforms that are abundantly expressed in these neurons [39].

Venom gland transcriptome analysis

RNA extraction and transcriptome sequencing and assembly were performed as described previously [57,58]. Assembled transcripts were annotated using a BLASTx search [22] (E-value setting of 1 × 10⁻³) against a combined database derived from UniProt, Conoserver [59], and an in-house cone snail venom transcript library. The 2 toxins, SLC_Mu8.1 and SLC_Mu8.1ii, abbreviated as Mu8.1 and Mu8.1ii, were named according to [21]. Here, Mu describes the 2-letter species abbreviation (Mu for C. mucronatus), 8 describes the cysteine scaffold, and the number 1 represents the first toxin to be described from this gene family. The suffix ii is given to a sequence variant that likely represents an allelic variant.

Transcriptome mining of the NCBI, DDBJ, and CNGB databases

To find sequences that share similarity with Mu8.1, we mined the transcriptomes of 37 cone snail venom gland transcriptomes available in the NCBI, DDBJ, and CNGB repositories using the precursor sequence of Mu8.1 as query (accession numbers provided in S1 Table). Transcriptome assemblies were done as described previously [57,58]. Signal and propeptide sequences of the identified homologous sequences were predicted using ProP v. 1.0 [60]. Mature toxin sequences were predicted to begin after the last basic amino acid residue preceding the first cysteine in the sequence. Therefore, further trimming of sequences to meet this criterion was executed manually as needed. Multiple sequence alignments were carried out using the MAFFT version 7 multiple alignment online interface [61] and visualized in Jalview version 1.0 [62].

Plasmid generation

The plasmid for bacterial expression of Mu8.1 was generated by uracil excision cloning, as described previously [63]. Polymerase chain reaction was carried out using Phusion U Hot Start polymerase (Thermo Fisher Scientific) according to the manufacturer’s instructions. Based on the transcriptome data for Mu8.1, the sequence of the mature toxin was predicted as GENSDNLTHCRLFEFRLCLLECMSLTLDHCYARCTTVITQIHGSDTNRFDCTIFKTCYYRCYVLGKTEDHCWKGTATSVTGDVGDLEFC. A codon-optimized DNA sequence for bacterial expression was generated using the CodonOpt tool. Using this codon-optimized DNA sequence as a template, the following 2 primers were designed:

Mu8.1_sense: ACACGGAUCGGACACCAATCGTTTTGATTGCACAATCTTCAAGACCTGCTACTACCGGTGCTACGTTCTTGGTAAAACAGAAGACCATTGCTGGAAAGGGACGGCAACGTCAGTGACAGGTGATGTCGGAGATTTGGAATTTTGCTAAGAATTCGAGCTCCGTCGACAG;Mu8.1_antisense:

ATCCGTGUATCTGGGTAATAACTGTGGTACATCTCGCATAGCAGTGGTCTAATGTAAGCGACATACACTCCAACAAACACAGCCGGAACTCGAATAATCTACAATGGGTAAGGTTGTCTGAGTTTTCGCCCTGAAAATACAGATTCTCAC.

These primers were subsequently used to clone Mu8.1 into the pET39_Ub19 expression vector [64]. The resulting plasmid encoding Ub-His₁₀-tagged Mu8.1 (Ub-His₁₀-Mu8.1) is referred to as pLE601. The fusion protein produced from pLE601 also contains a TEV protease recognition site following the Ub–His₁₀-tag. Primers were purchased from Integrated DNA Technologies, and the sequence encoding Ub-His₁₀-Mu8.1 was confirmed by Eurofins.

Protein expression

Chemically competent E. coli BL21 (DE3) cells were transformed with pLE601 with or (as a control) without the csCyDisCo plasmid (pLE577) [25]. Cells were plated on lysogeny broth (LB) agar supplemented with kanamycin (50 μg/mL) with (when cotransforming with pLE577) or without chloramphenicol (30 μg/mL). A single colony was picked to inoculate the LB medium containing the same type and concentration of antibiotic as used on the LB agar plates. The overnight culture was incubated for approximately 16 hours at 37°C at 200 rpm in an orbital shaker.

For initial small-scale expression tests (50 mL), LB medium containing appropriate antibiotics and supplemented with 0.05% glucose was inoculated with 2% overnight culture and grown at 37°C with shaking at 200 rpm until the desired OD₆₀₀ of 0.6 to 0.8 was reached. Expression was induced by adding isopropyl ß-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and the cultures grown for 18 hours at 25°C with shaking at 200 rpm to allow protein expression. Large-scale expression (1 L culture volume) was performed in autoinduction media prepared as described previously [65]. Briefly, terrific broth medium containing kanamycin (100 μg/mL) and/or chloramphenicol (30 μg/mL) was supplemented with sterilized stocks of the following: 0.05% glucose, 0.2% lactose, 50 mM KH₂PO₄/Na₂HPO₄, 50 mM NH₄Cl, 50 mM Na₂SO₄, 0.1 mM FeCl₃, 2 mM MgSO₄, 0.1 mM CaCl₂, and 1 × metal mix (203 g/L MgCl₂ 6·H₂O, 2.1 g/L CaCl₂ 2·H₂O, 2.7 g/L FeSO₄ 7·H₂O, 20 mg/L AlCl₃ 6·H₂O, 10 mg/L CoSO₄ 7·H₂O, 2 mg/L KCr(SO₄)₂ 12·H₂O, 2 mg/L CuCl₂ 2·H₂O, 1 mg/L H₃BO₄, 20 mg/L KI, 20 mg/L MnSO₄ H₂O, 1 mg/L NiSO₄ 6·H₂O, 4 mg/L Na₂ MoO₄ 2·H₂O, 4 mg/L ZnSO₄ 7·H₂O, 21 g/L citric acid monohydrate). Cultures were grown at 37°C at 200 rpm until OD₆₀₀ reached 0.8, at which point cells were moved to 25°C for expression performed with shaking at 200 rpm for 18 hours.

Harvest and clarification of bacterial cultures

Induced cultures were harvested by centrifugation at 5,000g for 20 minutes. The cell pellets were resuspended in 5 mL lysis buffer (50 mM Tris (pH 8), 300 mM NaCl, 20 mM imidazole) per gram pellet. Cell resuspensions were supplemented with approximately 12 units Benzonase Nuclease (Merck Millipore)/L culture to minimize viscosity due to the presence of nucleic acids post-lysis. Cell lysis was performed using a UP200S ultrasonic processor (Hielscher) keeping the cells on ice throughout. Cells were lysed with 8 × 30-second pulses at 90% power with 30-second rests between each pulse. Cell debris was pelleted by centrifugation at 30,000g for 45 minutes. The cleared lysates were filtered through 0.45 μm syringe filters and transferred to fresh tubes, whereas the pellets were resuspended in an equal volume lysis buffer containing 8 M urea for SDS-PAGE analysis.

Protein purification

Ub-His₁₀-Mu8.1 was affinity purified from the clarified lysate on an ÄKTA START system equipped with a 5-mL prepacked HisTrap HP (Cytiva) column equilibrated in lysis buffer. The lysate was applied to the column and washed with approximately 20 column volumes (CVs) of lysis buffer before elution of Ub-His₁₀-Mu8.1 with a gradient of 0% to 100% elution buffer (50 mM Tris (pH 8), 300 mM NaCl, 400 mM imidazole) applied over 20 CVs. Pooled fractions were dialyzed twice against 2 L anion exchange (AEX) buffer (50 mM NaH₂PO₄/Na₂HPO₄ (pH 6.8), 20 mM NaCl). AEX chromatography was performed on an ÄKTA Pure system equipped with a 10/100 Tricorn column (Cytiva) packed with Source 15Q ion exchange resin (Amersham Biosciences, GE Healthcare) equilibrated in AEX buffer. Ub-His₁₀-Mu8.1 was eluted using a gradient from 15% to 50% AEX elution buffer (50 mM NaH₂PO₄/Na₂HPO₄ (pH 6.8), 1 M NaCl) developed over 6 CVs.

The Ub-His₁₀-Mu8.1 fusion protein was cleaved using His₆-tagged TEV protease, expressed, and purified as described previously [25]. A molar ratio Ub-His₁₀-Mu8.1:His₆-TEV protease of 1:20 was used. To avoid reducing the disulfides in Mu8.1, His₆-TEV protease—preactivated with 2 mM dithiothreitol (DTT) for 30 minutes at room temperature—was diluted to approximately 0.002 mM DTT by 3 rounds of dilution/concentration in an Amicon Ultra 15 mL 3K Centrifugal Filter (Merck Millipore). TEV protease cleavage was performed overnight at room temperature.

To remove uncleaved Ub-His₁₀-Mu8.1, the Ub-His₁₀ tag, and His₆-TEV, the cleavage mixture was applied to a gravity flow column packed with 8 mL TALON cobalt resin (Takara) equilibrated in AEX buffer. The flow-through and the first wash fraction were collected. The presence of cleaved Mu8.1 in each fraction was investigated by analysis on 15% tricine SDS-PAGE gels, and the protein-containing fractions pooled. The cleaved Mu8.1 was subjected to size exclusion chromatography on a Superdex 75 Increase 10/300 GL column (Cytiva) equilibrated in 200 mM NH₄HCO₃ buffer (pH 7.8). Fractions containing purified Mu8.1 were pooled and lyophilized.

Analytical gel filtration

To analyze the oligomeric state of Mu8.1, the protein was analyzed at 2 concentrations, 100 μm and 1 μm, on a Superdex 75 Increase 10/300 GL column (Cytiva) equilibrated in 10 mM NaPi (pH 7.8), 150 mM NaCl at a flowrate of 0.5 mL/min. An excess of the protein was loaded onto a 100 μL loop to ensure analysis of the same volume in each run.

SDS-PAGE analysis

Samples from bacterial expression and subsequent purification steps were separated on 15% glycine SDS-PAGE or 15% tricine SDS-PAGE gels [66]. Where indicated, reduced samples were treated with 40 mM DTT. Protein bands were visualized with Coomassie Brilliant Blue, and images were recorded with a BioRad Chemidoc Imaging System. For western blotting, proteins separated by SDS-PAGE were transferred to a PVDF membrane (Immobilon-P, Merck Millipore) in a Mini Trans-Blot (Bio-Rad) transfer system. A mouse monoclonal His-tetra (Qiagen) antibody (1:1,000 dilution) was used in combination with a horseradish peroxidase–conjugated α-mouse secondary antibody (Pierce) (1:100,000 dilution). Chemiluminescence detection was performed using ECL Select Peroxide and Luminol solutions (GE Healthcare) according to the manufacturer’s directions.

Concentration determination

Concentrations were determined by measuring absorbance at 280 nm and using the theoretical extinction coefficient provided by the Expasy ProtParam tool available through the Expasy bioinformatics resource web portal [67]. Concentrations used for bioassays, constellation pharmacology, and electrophysiology assumed a monomeric state of Mu8.1.

Determination of molecular mass in solution by small angle X-ray scattering (SAXS)

Prior to SAXS analysis, Mu8.1 was run through a Superdex 75 Increase 10/300 GL column to ensure a monodisperse sample. The protein was subsequently dialyzed into a solution containing 150 mM NaCl and 10 mM NaPi (pH 7.8), and the dialysis buffer was reserved for measurement of background scattering. Immediately preceding SAXS measurements any precipitates were removed from the sample by centrifugation at 20,000g for 15 minutes at 4°C. Six dilutions were prepared ranging from 0.5 mg/mL to 12.4 mg/mL (49 μM to 1.2 mM) in dilution buffer. SAXS data were collected by the beamline staff at CPHSAXS using a Xenocs BioXolver L equipped with a liquid gallium X-ray source (λ = 1.34 Å). A sample-to-detector distance of 632.5 mm was used, corresponding to a Q-range of 0.013 to 0.5 Å⁻¹. Samples and buffer were measured at room temperature, and automatic loading was performed robotically from a 96-well plate. Data were collected as a minimum of 10 frames with 120-second exposure per frame. The longer exposure time was to account for the lower concentration and the expected presence of multimeric species. Data reduction and primary analysis were performed using RAW [68], singular value decomposition (SVD), and oligomer analysis performed with OLIGOMER (ATSAS program package) [69], and scattering curves plotted in Matlab R2020b.

X-ray crystallography

Freeze-dried Mu8.1 was dissolved in Milli-Q water to a concentration of 5 mg/mL. Crystallization screening experiments were performed with the Structure screen II (Molecular Dimensions, MD1-02) and the Index screen (Hampton Research, HR2-144) by the hanging drop vapor diffusion method. The crystal drops were mixed using 1 μL of protein and 1 μL precipitant solution as hanging drops on siliconized glass cover slides and equilibrated against 500 μL of precipitant solution in a 24-well plate setup. Wells were sealed with immersion oil (Sigma-Aldrich) and incubated at 21°C. Initial crystals of Mu8.1 appeared in several conditions in a few days to weeks. Diffracting crystals were obtained from the Structure screen II condition 38 (0.1 M NaOAc (pH 4.6), 0.1 M CdCl hemi(pentahydrate), 30% v/v PEG400), abbreviated as Mu8.1_38, and from the Index screen condition 59 (0.1 M HEPES (pH 7.5), 0.02 M magnesium chloride hexahydrate, 22% w/v polyacrylic acid sodium salt 5,100), abbreviated as Mu8.1_59.

Crystals were harvested using mounted CryoLoops (Hampton Research) and flash-cooled in liquid nitrogen. Cryoprotection was performed by quickly dipping the crystal in approximately 17% ethylene glycol (EG) prepared by mixing 1 μL 50% EG with 2 μL of the reservoir condition specific to each crystal condition. Both native crystals and iodide-soaked crystals were prepared from all 3 conditions. Single iodide crystals (Sigma-Aldrich) were added to the abovementioned cryo condition for each of the crystal conditions. Crystals from each condition were transferred to the iodide-containing cryo conditions and left to soak 5 to 10 seconds before harvesting them.

Data collection

Flash-cooled crystals were shipped to the beamline for remote data collection. Data were collected at 100K on a PILATUS detector at BioMax (MAX-IV, Lund, Sweden). A full sweep of 360° data was collected with an oscillation degree of 0.1°, with 0.050-second exposure at 12,700 eV and 7,000 eV. Complete data set was processed from 360° (3,600 images) with the X-ray beam reduced to 5% intensity.

Data processing

Native data were collected for Mu8.1_59 at 12,700 eV, and only Mu8.1_38 showed an anomalous signal from the data collected at 7,000 eV. All data were processed with xia2 using the 3dii pipeline [70,71] (Table 1).

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Table 1. Data collection and refinement statistics.

https://doi.org/10.1371/journal.pbio.3002217.t001

The phases for Mu8.1_38 were experimentally determined using autosol in the PHENIX package [72] with 13 iodide sites identified and an initial figure of merit (FOM) of 0.4. Density resembling helical structures were visible in the electron density map. The following AutoBuild wizard within the PHENIX package [72] was able to build a preliminary structure with the main helices in place. This structure was used as initial search model for molecular replacement and performed with the program Phaser [73] against the highest-resolution dataset Mu8.1_59.

All structures were manually refined using phenix.refine [72], and final model building was performed in Coot [74]. Data collection and refinement statistics are summarized in Table 1. Molecular graphics were presented with the PyMOL Molecular Graphics System, Version 2.2r7pre, Schrödinger, LLC. Electrostatic potentials were modelled using the Adaptive Poisson-Boltzmann Solver (APBS) plugin in PyMOL2.3 [75].

Structure search and topological comparisons

Structures similar to Mu8.1 were identified using PDBeFold at the European Bioinformatics Institute (https://www.ebi.ac.uk/msd-srv/ssm/) [76,77]. Monomeric Mu8.1 was used as query molecule to identify similar structures with a minimum acceptable match set to 60% or higher from the entire PDB database. Structural overlays were generated with the CLICK structural alignment tool (http://cospi.iiserpune.ac.in/click/) selecting “CA” as representative atoms and visualized using the PyMOL Molecular Graphics System [78] or executed manually in PyMol.

Constellation pharmacology

Primary cell cultures were dissociated from CGRP-GFP mice, STOCK Tg(Calca-EGFP)FG104Gsat/Mmucd, ages 34 to 38 days old as described previously [11]. In brief, lumbar DRG from vertebrae L1 to L6 were dissected, trimmed, and treated with 0.25% trypsin for 20 minutes. Following trypsinization, the DRGs were mechanically triturated using fire polished pipettes and plated in poly-l-lysine–coated plates. All plated cells were kept overnight at 37°C in a minimal essential medium supplemented with 10% fetal bovine serum, 1X penicillin/streptomycin, 10 mM HEPES, and 0.4% (w/v) glucose. One hour before the experiment, the dissociated cells were loaded with 4 μM Fura-2-AM dye (Sigma-Aldrich) and kept at 37°C. During each experiment, all dissociated cells were exposed to different pharmacological agents utilizing an automatic perfusion system and were imaged at 340/380 nm at 2 frames per second. In brief, cells were incubated with the pharmacological agents for 15 seconds followed by 6 consecutive washes and a 5-minute incubation period with extracellular solution for controls or Mu8.1. Five different pharmacological agents were used for cell classification: mustard oil (AITC) at 100 μM, menthol at 400 μM, capsaicin at 300 nM, K⁺ at 25 and 40 mM, and conotoxin κM-RIIIJ at 1 μM as described previously [11]. SNX-482 (Alomone Labs) was used at 100 nM. At the end of each experiment, all cells were incubated for 7 minutes with a 2.5-μg/mL Alexa Fluor 647 Azolectin B4 (IB4). All data acquisition was performed using the Nikon NIS-Elements platform. CellProfiler [79] was used for region of interest (ROI) selection, and a custom-built script in python and R was used for further data analysis and visualization. The package used to visualize, analyze, and deploy models for constellation pharmacology experiments is available at https://github.com/leeleavitt/procPharm. All procedures were approved by the University of Utah institutional animal care and use committee (IUCAC) Protocol number: 17–05017.

Statistical analysis of cellular calcium imaging

We utilized a min-max normalization (f(x)) to assess the effects of Mu8.1 on the peak height of the calcium signal induced by high concentrations of potassium using the formula: where K⁺ test is the peak height after the incubation with Mu8.1, and K⁺ control is the peak height before the incubation with Mu8.1 [11]. If f(x) = 0, this suggests that the conotoxin did not affect the Ca²⁺ concentration in the cytosol induced by the high concentration of potassium. An f(x) > 0 indicates that the conotoxin increased the cytosolic calcium concentration after a high potassium concentration, resulting in amplification. Finally, if f(x) < 0, the conotoxin decreased calcium concentration in the cytosol resulting in a calcium block. After calculating the f(x) for all sensory neuron cell types, we performed a two-tailed t test to assess if the f(x) values calculated for every cell type were significantly different from 0.

Electrophysiology

APC recordings were performed in a Patchliner Octo (Nanion Technologies GmbH, Munich, Germany) equipped with 2 EPC-10 quadro patch clamp amplifiers (HEKA Electronics). PatchControlHT (Nanion) was used for cell capture, seal formation, and establishment of the whole-cell configuration, while voltage was controlled, and currents sampled with PatchMaster (HEKA Electronik). Recordings were performed under the whole-cell configuration using single-hole planar NPC-16 chips (resistance of approximately 2.5 MΩ) at room temperature. Stably transfected cell lines (D.J. Adams collection, IHMRI-UOW) were cultured according to the manufacturer’s instructions and detached using TrypLE. Cells were resuspended in cold external recording solution and kept in suspension by automatic pipetting at 4°C. The extracellular solution used for Kv1, Kv4.3, hERG, and Nav1 recordings contained (in mM): 140 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 10 glucose, and 10 HEPES (pH 7.4 with NaOH, 298 mOsmol/kg). Kv1, Kv4.3 and hERG intracellular solution (in mM): 60 KF, 70 KCl, 10 EGTA, 10 glucose, and 10 HEPES (pH 7.2 with KOH, 285 mOsmol/kg). Peak I_K currents for Kv1.1–3 and Kv4.3: 500 ms test pulse to 20 mV (Vh = −120 mV; 0.1 Hz). hERG I_K: 1-second prepulse to +40 mV was followed by 200 ms test pulse to −40 mV (Vh = −80 mV; 0.1 Hz). Nav1 intracellular solution (mM): 60 CsF, 60 CsCl, 10 NaCl, 10 EGTA, and 10 HEPES (pH 7.2 with CsOH, 285 mOsmol/kg). Nav currents were elicited by 10 ms test pulses to −10 mV with a 1-second prepulse to −120 mV (Vh = −90 mV; 0.1 Hz). APC of Cav1 and Cav2: extracellular solution (in mM): 135 NaCl, 4 KCl, 10 BaCl₂, 1 MgCl₂, 5 glucose, and 10 HEPES (pH 7.4 with NaOH, 298 mOsmol/kg) and intracellular solution (in mM): 90 CsSO₄, 10 CsCl, 10 NaCl, 10 EGTA, and 10 HEPES (pH 7.2 with CsOH, 285 mOsmol/kg). Peak calcium currents were measured upon 50 ms step depolarization to +10 mV (Vh = −80 mV; 0.1 Hz). Recordings where seal resistance (SR) was >500 MΩ and access resistance was <3xSR were considered acceptable. Chip and whole-cell capacitance were fully compensated, and series resistance compensation (70%) was applied via Auto Rs Comp function. Recordings were acquired with PatchMaster (HEKA Elektronik, Lambrecht/Pfalz, Germany) and stored on a computer running PatchControlHT software (Nanion Technologies GmbH, Munich, Germany).

Manual patch clamp (MPC) was performed on human embryonic kidney (HEK293T) cells containing the SV40 Large T-antigen cultured and transiently transfected by calcium phosphate method as reported previously [80]. In brief, cells were cultured at 37°C, 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen Life Technologies, Victoria, Australia), supplemented with 10% fetal bovine serum (Bovigen, Victoria, Australia), 1% GlutaMAX and penicillin–streptomycin (Invitrogen). The human orthologues of Cav3.1, Cav3.2, and Cav3.3 channels were cotransfected with GFP for identification of positive transfectants. cDNAs encoding hCav3.1 (kindly provided by G. Zamponi, University of Calgary), hCav3.2 (a1Ha-pcDNA3, Addgene #45809), and hCav3.3 (a1Ic-HE3-pcDNA3, Addgene #45810) were a kind gift from E. Perez-Reyes (University of Virginia). MPC experiments employed a MultiClamp 700B amplifier, digitalized with a DigiData1440, and controlled using Clampex11.1 software (Molecular Devices, California, USA). Recordings of I_Ca through hCav3.1–3 were performed using an extracellular solution containing (in mM): 110 NaCl, 10 CaCl₂, 1 MgCl₂, 5 CsCl, 30 TEA-Cl, 10 D-Glucose, and 10 HEPES (pH 7.35 with TEA-OH, 305 mOsmol/kg). Pipettes were pulled from borosilicate glass capillaries (GC150F-15, Harvard Apparatus, Massachusetts, USA), fire polished to a final resistance of 1 to 3 MΩ, and filled with intracellular solution (in mM): 140 KGluconate, 5 NaCl, 2 MgCl₂, 5 EGTA, and 10 HEPES (pH 7.2 with KOH, 295 mOsmol/kg). Peak currents were measured upon stimulation using 50 ms test pulses to −20 mV from a holding potential (Vh) of −90 mV and pulsed at 0.2 Hz. Whole-cell currents were sampled at 100 kHz and filtered to 10 kHz, with leak and capacitive currents subtracted using a P/4 protocol, and 60% to 80% series resistance compensation.

Data analysis of electrophysiology experiments

APC analysis was performed using Igor Pro-6.37 (WaveMetrics). Cav2.3 peak currents measured in the presence of increasing Mu8.1 concentrations (I_Mu8.1) were divided by the current in control conditions (I_Ctr) to generate a concentration–response curve that was fit with a Hill equation of the form: where IC₅₀ is the half-maximal inhibitory concentration, and h is the Hill coefficient (nH).

For ease of comparison, IC₅₀ values were calculated from fractional inhibition for the other voltage-gated channels that were screened at a single Mu8.1 concentration according to the following equation: