Expression and Pharmacology of Endogenous Cav Channels in SH-SY5Y Human Neuroblastoma Cells

SH-SY5Y human neuroblastoma cells provide a useful in vitro model to study the mechanisms underlying neurotransmission and nociception. These cells are derived from human sympathetic neuronal tissue and thus, express a number of the Cav channel subtypes essential for regulation of important physiological functions, such as heart contraction and nociception, including the clinically validated pain target Cav2.2. We have detected mRNA transcripts for a range of endogenous expressed subtypes Cav1.3, Cav2.2 (including two Cav1.3, and three Cav2.2 splice variant isoforms) and Cav3.1 in SH-SY5Y cells; as well as Cav auxiliary subunits α2δ1–3, β1, β3, β4, γ1, γ4–5, and γ7. Both high- and low-voltage activated Cav channels generated calcium signals in SH-SY5Y cells. Pharmacological characterisation using ω-conotoxins CVID and MVIIA revealed significantly (∼ 10-fold) higher affinity at human versus rat Cav2.2, while GVIA, which interacts with Cav2.2 through a distinct pharmacophore had similar affinity for both species. CVID, GVIA and MVIIA affinity was higher for SH-SY5Y membranes vs whole cells in the binding assays and functional assays, suggesting auxiliary subunits expressed endogenously in native systems can strongly influence Cav2.2 channels pharmacology. These results may have implications for strategies used to identify therapeutic leads at Cav2.2 channels.


Introduction
Voltage-gated Ca 2+ channels (Ca v ) are membrane proteins essential for the control of calcium signaling events, such as muscle contraction, gene expression, and neurotransmitter and hormone release. Dysfunction of Ca v channels is related to a variety of heart, circulatory and neurological diseases; including arrhythmias, hypertension, some forms of epilepsy, migraine and other chronic diseases such as cancer, diabetes, ischemic brain injury and neuropathic pain [1,2]. The Ca v a subunit contains the voltage sensor and gating machinery and is the binding site for most inhibitors. This subunit comprises 4 domains each with six transmembrane segments. The pore is formed by the S5/S6 segments and the connecting pore loop, with channel opening gated by bending of the S6 segments at a hinge glycine or proline residue [3,4]. The voltage sensor domain consists of the S1-S4 segments, with positively charged residues in S4 serving as gating charges [5] (for review see: [3,6,7]).
Based on the distinct pharmacological and electrophysiological properties of Ca v channels, ten different gene subfamilies have been identified in vertebrates and classified as high voltage activated (HVA) Ca v 1.1-4 (L-type), Ca v 2.1 (P/Q-type), Ca v 2.2 (Ntype), Ca v 2.3 (R-type); and low voltage activated (LVA) Ca v 3.1-3 (T-type). The a subunit includes channels containing a 1S , a 1C , a 1D , and a 1F , which mediate L-type Ca 2+ currents. The Ca v 2 subfamily (Ca v 2.1 to Ca v 2.3) includes a subunits a 1A , a 1B , and a 1E , which mediate P/Q-type, N-type, and R-type Ca 2+ currents, respectively. The Ca v 3 subfamily (Ca v 3.1 to Ca v 3.3) includes a subunits a 1G , a 1H , and a 1L , which mediate T-type Ca 2+ currents ( Table 1) (for reviews see: [3,8,9]). Of these, Ca v 2.2 has been of particular interest as a therapeutic target given the central role it plays mediating neurotransmitter release in nociceptive pathways such as presynaptic nerve terminals and dendrites [10]. Ca v a subunits are co-expressed in native systems together with two or three auxiliary subunits (b, a 2 d and c), which undergo alternative splicing (for review see: [9]) and dramatically influence Ca v channel function, intracellular trafficking and posttranslational modifications [11]. Indeed, when expressed alone in recombinant system, the a 1B subunit, for example, encodes a voltage-dependent calcium channel with kinetic properties different from those of native Ca v 2.2 channels [9,12]. In contrast, when co-expressed with auxiliary b and a 2 d, increased current amplitudes are observed and the kinetics of activation and inactivation are closer to those of native channels [12].
Cell-based systems are desirable in the field of high-throughput screening assays due to their similarity to in vivo environment. SH-SY5Y human neuroblastoma cells are derived from human sympathetic neuronal tissue. This cell line maintains in culture many of the properties of nerve cells, providing a useful model for the characterisation of molecules affecting human neuronal function, including endogenously expressed Ca v channels [13][14][15]. In particular, SH-SY5Y cells have been an attractive model system for the study of Ca v 2.2 function [13]. Although heterologous expression models provide control of subunit expression, native systems provide potentially more complex models which, when characterized, can help to determine the pharmacology of drugs in a native context and the physiology and pathophysiology of endogenously expressed receptors and channels. However, little is known about the Ca v subtypes and auxiliary subunits endogenously expressed in SH-SY5Y cells, limiting the interpretation of pharmacological data. Here we report a detailed characterisation of endogenously expressed Ca v channels expressed in SH-SY5Y cells using PCR and pharmacological approaches, with particular emphasis on the nociceptive target Ca v 2.2.

SH-SY5Y Cells Endogenously Express Multiple Ca v Subtypes, Ca v 2.2 Isoforms and Auxiliary Subunits
We assessed expression of mRNA transcripts for Ca v subtypes and auxiliary a 2 d, b and c subunits isoforms in SH-SY5Y cells by performing RT-PCR using specific primers ( Fig. 1A 1A). In addition, bands of expected sizes for ( Table 2) b 1 , b 3 , b 4 , a 2 d 1-3 , c 1 , c 4 , c 5 and c 7 auxiliary subunits ( Fig. 1C-D) were also identified. Since splice variants can be generated by alternative RNA processing, which can influence function and pharmacology [16], we also investigated the expression of some human splice variants [16,17]. PCR bands with the predicted sizes for Ca v 1.3 isoforms 1 and 2; full length Ca v 2.2, a 1B1 (Gene bank accession number M94172.1), shorter a 1B variant, a 1B2 (Gene bank accession number M94173.1) [17]; and D 1 (but not D 2 ) [16,18] were detected for the first time in the SH-SY5Y cells (Fig. 1A, Table 1).
The best annealing temperature for each gene analysed (see Table 1-2) was determined using a gradient PCR protocol in rounds of control experiments prior to testing each Ca v genespecific primer. Target-specific primers for the housekeeping gene GAPDH were designed as previously described [19] and GAPDH was detected in all PCRs, indicating amplifications were cDNA specific. PCR master mix using random primers without cDNA was used as negative gDNA control in all PCRs. Specificity of primers was demonstrated in a range of control experiments (data not shown), including detection of Ca v 2.2 plasmid but not other Ca v subtypes by Ca v 2.2 primers; and absence of Ca v 2.2 in HEK cells. b 1 and a 2 d 1 primers were positive for b 1 and a 2 d 1 plasmids, while the same primers were negative for b 2-4 and a 2 d 2-4 (data not shown), indicating primers were selective for b 1 and a 2 d 1 auxiliary subunits. The identity of each of these PCR products, including c 1 , c 4 , c 5 and c 7 , was confirmed by sequencing analysis (data not shown).

Displacement of 125 I-GVIA Binding from SH-SY5Y Cell Membranes
GVIA is a highly selective Ca v 2.2 blocker [20] and 125 I-GVIA binding assays have been well established using rat brain membranes [21][22][23]. We performed binding assays and confirmed SH-SY5Y cells contain 125 I-GVIA binding sites which can be fully displaced by Ca v 2.2 selective inhibitors v-conotoxins CVID, GVIA and MVIIA. Affinities of v-conotoxins for human and rat Ca v 2.2 channels were next compared using these assays. CVID, GVIA and MVIIA each fully displaced 125 I-GVIA binding to crude rat brain membranes with similar affinities (pIC 50 Table 3), consistent with earlier studies [21]. Intriguingly, the affinity of GVIA (pIC 50 10.5560.15) to displace 125 I-GVIA binding to SH-SY5Y membranes was similar to that shown in rat brain, while both CVID and MVIIA had significant higher affinity for the human cell membranes (pIC 50 s of 11.5160.12 and 11.2960.23, respectively) than for rat brain membranes ( Fig. 2A, B and D, Table 3). In addition, the affinities of these v-conotoxins dramatically decreased when determined on the intact SH-SY5Y cells instead of membranes, with GVIA affinity shifted ,10-fold, and CVID and MVIIA affinity shifted ,100-fold (see Fig. 2C, Table 3).

Pharmacology of the Endogenously Expressed Ca v Channels
To investigate if the Ca v channels endogenously expressed in SH-SY5Y cells were functional, and to further study the pharmacology of these channels, we assessed KCl-evoked Ca v responses using a fluorescent high-throughput Ca 2+ imaging assay  Table 4), similar to previously described values [24][25][26]. To assess the contribution of each Ca v channel expressed in SH-SY5Y cells to the KCl-evoked Ca 2+ responses, we determined concentrationresponse curves for KCl/Ca 2+ stimulation in the presence of subtype-specific inhibitors. The Ca v 1 (L-type) inhibitor nifedipine was used at a concentration (10 mM) that does not affect responses of other Ca v subtypes (N, R, P/Q or T-type) [27], to isolate non-Ltype responses. The KCl concentration-response curve was shifted  Table 4). Conversely, the Ca v 2.2 (N-Type) inhibitor v-conotoxin CVID was used at a concentration that does not affect responses of other Ca v s (up to 3 mM) [21,28], to isolate non-N-type responses. Compared to responses in the presence of nifedipine, the KCl concentrationresponse curve was shifted to the left in the presence of CVID (EC 50 of 18.6 mM, pEC 50 1.8660.10, Hill slope of 3.5) (Fig. 3A, Table 4). These differences can be accounted for by the electrophysiological properties of each Ca v channel subtype identified, since L-type requires a larger depolarization than Ntype to be activated [28], and the control KCl responses is a result of activation of both channel types. These results confirm that SH-SY5Y cells express functional Ca v subtypes, including Ca v 1 and Ca v 2.2, which can be pharmacologically isolated using selective inhibitors. The observed pharmacology is consistent with the subtypes identified in our PCR experiments and with previous reported electrophysiological data [14,15]. Since 90 mM KCl/5 mM CaCl 2 elicits maximal Ca v 1 and Ca v 2.2 responses (Fig. 3A), we used this combination to further characterize the Ca v channel subtypes expressed in SH-SY5Y cells. Concentration-response curves for nifedipine at Ca v 1 channels were generated in the presence of saturating concentration of CVID (3 mM). Under these conditions, nifedipine inhibited KCl evoked Ca 2+ responses with an IC 50 of 0.28 mM (pIC 50 6.560.052) (Fig. 3C, Table 4), consistent with reports for nifedipine block of L-type responses in neuronal cells [29]. To characterize Ca v 2.2 pharmacology, inhibition by v-conotoxins was determined in the presence of a near saturating concentration of nifedipine (10 mM). Under these conditions, the potency of CVID was IC 50 0.16 mM (pIC 50 6.8760.078), GVIA 0.15 mM (pIC 50 6.8460.06) and MVIIA 0.024 mM (pIC 50 7.760.13) (Fig. 3D, Table 4). These results are consistent with previous studies on MVIIA [22][23][24]26] and CVID [22][23][24] inhibition of Ntype responses in native and recombinant systems, when Ca v 2.2 was co-expressed with b and a 2 d subunits [22][23][24]. In contrast, GVIA potency at Ca v 2.2 expressed in SH-SY5Y cells (IC 50 of 0.15 mM; pIC 50 6.860.072) was consistently lower than previously described for heterologous expressed rat [24,26] and human [25] a 1B co-expressed with a 2 d 1 and b 3 , but similar to data obtained using native expression systems such as dissociated rat DRG cells [30] and chicken synaptosomes [31].
A small portion (5-15%) of the KCl-evoked responses was insensitive to block by co-application of 10 mM nifedipine and 3 mM CVID ( Fig.3C-D). To pharmacologically characterize these remaining responses, we assessed the effects of Ca v 2.1 and Ca v 2.3 subtype-specific inhibitors, as well as of compounds with activity at Ca v 3, on the Ca 2+ responses evoked by 90 mM KCl/ 5 mM CaCl 2 , in the presence of both CVID and nifedipine. The Ca v 2.1 blockers v-agatoxin IVA (data not shown) and v-agatoxin TK did not significantly affect KCl-evoked Ca 2+ responses at concentrations up to 10 mM ( Fig. 4A-B, Table 4). The Ca v 2.3 antagonist SNX 482 also had no significant inhibitory effect at concentrations up to 10 mM ( Fig. 4A and C, Table 4). On the other hand, mibefradil (30 mM), a benzimidazolyl-substituted tetraline reported to inhibit Ca v 3 responses in different systems with weak affinity [32,33] fully inhibited these remaining responses with an IC 50 of 3 mM (pIC 50 5.360.035) ( Fig. 4A and D, Table 4). Similar IC 50 values for mibefradil block of T-type responses in native systems have been previously reported (see [32][33][34][35]). In addition, another Ca v 3 inhibitor, the antipsychotic pimozide, also fully inhibited the remaining responses with an IC 50 of 1.3 mM (pIC 50 5.260.097) (Fig. 4A, Table 4), similar to previously reported literature values [34]. These findings are in agreement with our PCR (Fig. 1A-B) which detected mRNA transcripts for Ca v 3.1, but neither Ca v 2.1 nor Ca v 2.3 was identified.

Discussion
Ca v 2.2 channels play a key role in regulating nociception. Inhibition of Ca v 2.2 at the spinal cord produces analgesia in animal models of pain [23,36] and in humans [37], with direct (eg. Prialt) and indirect (eg. gabapentin) inhibitors among some of the  [13][14][15]38]. In contrast, we detected mRNA transcripts for the N-type (Ca v 2.2), two L-type (Ca v 1.3 isoform 1 and 2) and one T-type isoform (Ca v 3.1). In addition, we also detected mRNA transcripts for Ca v 2.2 splice variants, including a 1B2 (74 amino acid shorter) [16,17,39] and the splice a 1B D 1 (382 amino acid shorter) [16].
Functional Ca v responses elicited by addition of KCl/CaCl 2 were assessed using a fluorescent high-throughput Ca 2+ imaging assay on the FLIPR Tetra . KCl has been used extensively to activate Ca v responses in a diversity of functional assays ( [24,26,40]). Addition of high concentrations of KCl causes a change in membrane potential, which in turn leads to opening of Ca v channels, influx of Ca 2+ and a resultant increase in intracellular fluorescence. While the change in membrane potential elicited by addition of KCl at the concentrations used here is approximately linear, accumulation of intracellular Ca 2+ is saturable and fits a sigmoidal concentration-response curve because a change in membrane potential leads to a finite change in channel open probability and thus Ca 2+ influx.
Ca v 2.2 channels expressed in SH-SY5Y cells were functional and generated KCl activated responses that were inhibited by vconotoxins CVID, GVIA and MVIIA in the presence of saturating concentrations of nifedipine. As expected, when L-type responses were isolated by addition of saturating concentrations of CVID, nifedipine concentration-dependently blocked KCl responses. However, a small response remained (5-15%) in the presence of specific inhibitors of T-type currents and also inhibit L-type channels [32,41], inhibition of the residual Ca 2+ response was also observed in the presence of saturating concentrations of nifedipine, suggesting that activity of these compounds at L-type channels did not contribute to inhibition of residual response. Based on our observations that this resistant response was not blocked by inhibitors of L-type (nifedipine), N-type (CVID), R-type (SNX 482) or P/Q-type channels (v-agatoxin), but was completely abolished by compounds with known activity at T-type channels, it seems plausible that this response may be mediated by Ca v 3.1, which mRNA expression was detected in SH-SY5Y. Alternatively, it is known that the D 1 splice variant, which mRNA expression was detected in SH-SY5Y cells, is significantly more resistant to the blockade by MVIIA and GVIA [42]. While inhibition by CVID of the Ca v 2.2 splice variants detected in SH-SY5Y cells has not been characterised, it is possible that, akin to inhibition of Na v channels by the m-conotoxin GIIIA, complete current inhibition by CVID cannot be achieved for these splice variants. Alternatively, the response remaining in the presence of nifedipine and CVID could represent another undefined resistant current, or an artifact of the KCl/Ca 2+ activation buffer used in this study.
Development of non-electrophysiological HTS Ca v 3 channel assays has been hampered by some of the properties of this channel, including their low voltage threshold for activation and inactivation and rapid inactivation kinetics. However, although Ttype currents inactive rapidly, fluorescence Ca 2+ assays detect accumulation of intracellular Ca 2+ rather than currents, and are thus not subject to the same temporal resolution constraints. In addition, compared to heterologous systems, SH-SY5Y cells have a relatively hyperpolarised resting membrane potential [43], which would be conducive to channels being present in the resting state. Accordingly, Ca 2+ assays at Ca v 3 channels using the FLIPR have been successfully developed [40] and it is clearly conceivable that functional responses of Ca v 3.1 expressed in SH-SY5Y cells could be elicited using KCl/Ca 2+ stimulation.
In addition to functional characterization, we also confirmed Ca v 2.2 expression at the protein level using 125 I-GVIA binding assays. The v-conotoxins CVID, GVIA and MVIIA each fully displaced 125 I-GVIA binding to SH-SY5Y cell membranes with high affinity. Interestingly, while the affinity of GVIA was not significantly different between species, CVID and MVIIA affinities were ,10-fold higher in human SH-SY5Y membranes compared to rat brain membranes. These results support the findings that MVIIA and CVID interacts with Ca v 2.2 human channels through a different pharmacophore, as compared with GVIA [44].
Variation in the affinity of v-conotoxins between species is likely influenced by Ca v a splice variants, with differences in toxin sensitivity, time course and voltage-dependence of inactivation, single channels conductance, gating behavior and sensitivity to Gprotein-mediated modulation reported for splice isoforms endogenously expressed in neuronal cells of rat, mouse, rabbit and humans [16,17,39,42,[45][46][47][48] (for review see: [46]). In pain, the Ca v 2.2 splice variant 37a replaces the usual variant 37b in a specific subset of nociceptive neurons, and thus may represent a potential therapeutic target [42,46,49]. However, this variant has to date only been described in rat dorsal root ganglion neurons, and is not known to be present in human tissue.
Additional human splice variants include two a 1B isoforms that have long or short C-termini [17], and two human forms that lack large parts of the domain II-III linker region, including the synaptic protein interaction site. These splice variants, termed D 1 and D 2 , have been previously isolated from IMR32 human neuroblastoma cell line and human brain cDNA libraries [16]. We have identified mRNA transcripts for the full length a 1B1 , a 1B2 (74 amino acid shorter) [16,17,39] and the splice variant D 1 (382 amino acid shorter) [16] in SH-SY5Y cells. The a 1B1 is an axonal/ synaptic isoform, while a 1B2 is restricted to neuronal soma and Table 3. v-Conotoxin affinities (IC 50 6 SEM) to displace 125 I-GVIA binding. dendrites [39,50], however, apart from differential susceptibility to Gai/Gao-versus Gaq-mediated inhibition, little is known regarding its biophysical and pharmacological properties. On the other hand, the D 1 splice variant has lost part of the synaptic protein interaction (synprint) site and is thus unlikely to play a role in fast synaptic transmission, with shifts in the voltage dependence of steady-state inactivation and a more rapid recovery from inactivation compared to full length a 1B1 [16]. Importantly and clinically relevant, D 1 variant was significantly more resistant to the blockade by MVIIA and GVIA; however the degree of effect varied for each toxin [16]. Thus, expression of the D 1 variant in SH-SY5Y cells may contribute to the reduced v-conotoxin affinity observed. While expression of these splice variants in SH-SY5Y cells was detected using gene specific primers, which have been extensively validated in the literature [17], further confirmation of expression at the protein level is warranted.
Ca v channel auxiliary subunits can also influence the pharmacology of Ca v inhibitors, with v-conotoxins displaying reduced affinity in the presence of the a 2 d subunit [11,[22][23][24]27,51].
Specifically v-conotoxins GVIA, MVIIA and CVID had reduced affinity when a 2 d 1 subunit was co-expressed with the Ca v a 1B [23]. a 2 d up-regulation has been associated with chronic pain and epilepsy, with gabapentin and pregalin binding to a 2 d reducing Ca v 2.2 trafficking and the symptoms of pain [11]. The a 2 d 1 -3 , b 1 , b 3 and b 4 , c 1 , c 4-5 and c 7 subunits were detected in SH-SY5Y cells and potentially contribute to the differences in v-conotoxins potency in whole cell vs. membrane assays.
The c 1 subunit was originally identified in skeletal muscle in complex with Ca v 1 channels [52], but effects of this subunit on the v-conotoxins affinity at Ca v 2.2 have not been determined. In contrast, co-expression of the c 7 subunit almost abolished the functional expression of Ca V 2.2 in either Xenopus oocytes or COS-7 cells [53,54]. The neuronal c 2 is associated with epileptic and ataxic phenotypes of stargazer mouse [55], but was not detected in SH-SY5Y cells. The c 5 and c 7 subunits represent a distinct subdivision of the c subunit family of proteins identified by structural and sequence homology to stargazing. The c 4 subunit affected only the Ca v 2.1 channel [55,56]. The c 5 subunit may be a regulatory subunit of Ca v 3.1 channels (for review see: [57]). These subunits may also potentially contribute to differences in vconotoxins binding affinities observed in whole cell vs. membrane assays.
While auxiliary subunits affect v-conotoxin affinity in functional studies, this quaternary complex is likely to be disrupted upon preparation of homogenized membranes for the binding assays [58]. To examine this possibility, we studied the ability of GVIA to displace 125 I-GVIA from whole SH-SY5Y cells compared to homogenized membranes. Interestingly, v-conotoxins CVID, MVIIA and GVIA had higher affinity to displace 125 I-GVIA from the homogenized membranes compared to the whole cells, an effect that was most pronounced for CVID and MVIIA (,100fold) compared to GVIA (,10-fold). We have previously reported a similar trend for both CVID and MVIIA in heterologous expression system with and without the a 2 d subunit [22]. Potency estimates obtained with the functional assays were significantly lower than estimates obtained in whole cell radioligand binding assays. The relatively high level of Ca 2+ in the physiological saline used traditionally for functional assays compared to binding assays could contribute to these differences, since Ca 2+ non-competitively inhibits v-conotoxin binding [21]. However, our whole cell data was also obtained by incubating v-conotoxins in a Ca 2+ -free physiological saline solution and the origin of these differences is unclear. Interestingly, this effect was most marked for GVIA, intermediate for CVID and insignificant for MVIIA.
In summary, we have characterized functional Ca v channels expressed in SH-SY5Y human neuroblastoma cell line. Our studies have shown expression of different Ca v a splice variants, in conjunction with auxiliary subunits in a native context, can modulate the pharmacology of Ca v 2.2 channel inhibitors. SH-SY5Y cell line provides a useful model for the investigation of novel human Ca v 2.2 inhibitors and is amenable to the establishment of high-throughput assays [59], which can be adapted to detect endogenously expressed human Ca v 1.3, Ca v 2.2 and possibly Ca v 3.1, in the presence of appropriate inhibitors. These assays are expected to prove useful for the discovery and pharmacological characterization of novel Ca v channel modulators targeting human Ca v related diseases.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Ca v channel subtype and auxiliary subunits mRNA expression profiles were investigated in SH-SY5Y cells using standard RT-PCR and specific primers. The primers were designed using The Basic Local Alignment Search Tool (BLAST) [60,61], or otherwise specified as, previously described in the literature. Primer sequences, Gene Bank reference numbers, predicted PCR product sizes, and optimum annealing temperatures are shown in Table 1.
The primers used to identify Ca v subtypes and auxiliary subunits were designed so that all splice variants of specific isoforms would be amplified. On the other hand, primers to amplify Ca v 2.2 splice variants isoform were designed to be specific to each isoform. PCR conditions to detect splice variants were set as previously described [16,17], with gradient PCR performed for all sets of primers, allowing the identification of optimal annealing temperatures. Different sets of primers were used to identify the full length and isoforms D 1 and D 2 (see table 1) [16]. These primers were designed based on the region of the domain II-III linker of Ca v 2.2 channels, as previously described [16]. Primers used to identify the full length a 1B1 and short a 1B2 isoforms were designed based on the Cterminus region [17]. Data is representative of at least three independent experiments. SH-SY5Y cells (1610 6 ) were harvested and total RNA isolated using TrizolH Reagent (Invitrogen, Carlsbad, CA). The isolated RNA was subsequently treated with RNase-free DNase to remove any genomic DNA contamination. RNA concentration was determined by absorbance measurements at 260 nm and its purity/integrity was accessed by analyzing the ratio 260/280 nm with a NanodropH (Thermo Scientific). Synthesis of first strand cDNA was performed using 1 mg of the extracted RNA and the Omniscript Reverse Transcription Kit (Qiagen), according to the manufacturer's instructions. cDNA amplifications were performed using Taq Polymerase (New England Biolabs, US). The reaction mix (total 25 mL) included (mL): 1 cDNA (100 ng), 0.125 of the enzyme, 0.5 reverse and 0.5 forward primers (10 mM), 0.5 dNTPs (10 mM), 2.5 Thermopol reaction buffer (106) and nuclease free water. RT-PCR was carried through as an initial denaturation step at 95uC for 3 min followed by 35 cycles of the steps: 95uC for 30 s, optimal annealing temperature as previously determined ( Table 1) for 60 s, 68uC extension for 60 s, plus an extra 5 min elongation step at 68uC. PCR products were analyzed by 1% agarose gel and predicted sizes estimated by comparison with DNA molecular weight makers (50 and 100 bp ladder, New England Biolabs). Target-specific primers for the housekeeping gene GAPDH were designed as previously described [19]. PCR master mix using random primers without cDNA was used as negative gDNA control in all PCRs. Specificity of primers was demonstrated in a range of control experiments (data not shown), including detection of Ca v 2.2 plasmid but no other Ca v subtypes by Ca v 2.2 primers; and absence of detectable levels of Ca v 2.2 in HEK cells. b 1 and a 2 d 1 primers were positive for b 1 and a 2 d 1 plasmids, while the same primers were negative for b 2-4 and a 2 d 2-3 (data not shown), indicating primers were selective for b 1 and a 2 d 1 auxiliary subunits. In addition, identity of PCR products was further confirmed by sequencing analysis (data not shown). Sequencing PCR amplicons were first separated on agarose gels and bands of expected sizes identified. PCR products were purified using the Wizard SV Gel and PCR clean-up system (Promega), and a sample of each purified PCR product was sent for sequencing at the Australian Genome Research Facility. cDNA sequences of human Ca v subtypes and auxiliary subunits were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/Entrez/) and BLASTn [62] was used for confirmation of the identity of human Ca v subtypes and auxiliary subunits.

Cell Culture
The human neuroblastoma SH-SY5Y cells (Victor Diaz, Goettingen, Germany) were cultured and routinely maintained at 37uC and 5% CO 2 in RPMI 1640 antibiotic-free medium (Invitrogen) supplemented with 10% heat-inactivated FBS and 2 mM GlutaMAX TM (Invitrogen). Trypsin/EDTA was used to detach the cells from the T-75 or T175 flasks and cells were split in a ratio of 1:5-1:10 every 3-4 days or when ,80% confluent.

Membrane Preparation for the Radioligand Binding Assay
Radioligand binding assays were performed using rat brain or SH-SY5Y cell membranes prepared as described by Wagner, et al., 1988 [63] with slight modification. For rat brain membranes, male Wistar rats weighing 175-250 g were sacrificed by cervical dislocation and the whole brain was rapidly removed and dissected on ice. At 4uC, tissue was re-suspended in 50 mM HEPES, pH 7.4 (50 mg wet weight tissue/ml buffer), homogenized using a Brinkmann Polytron homogenizer and centrifuged for 15 min at 40,0006g. The pellet was re-suspended in 50 mM HEPES and 10 mM EDTA at pH 7.4, incubated on ice for 30 min and centrifuged at 40,0006g for 10 min. The pellet was then resuspended in 50 mM HEPES pH 7.4 containing 10% glycerol, aliquots were made and kept at -80uC prior to use. Bicinchoninic acid (BCA) assay reagent (Pierce Rockford, IL) was used for protein quantification.
SH-SY5Y cell membranes were harvested using trypsin/EDTA, washed once with DPBS, and centrifuged for 4 min at 5006g. After centrifugation, the supernatant was discarded and the pellet re-suspended in 10 ml binding assay buffer at pH 7.2 containing (mM): 20 HEPES, 75 NaCl, 0.2 EDTA, 0.2 EGTA and complete protease inhibitor (Roche Diagnostics, AU) and sonicated. The homogenates were then centrifuged for 30 min at 40,0006g and 4uC. The supernatant was discarded and the pellet dissolved in aliquots of binding assay buffer containing 10% glycerol stored at -80uC prior to use. BCA was used for protein quantification.

Whole Cell Preparation for the Radioligand Binding Assay
Whole cells were prepared as described for SH-SY5Y cell membranes with the following modifications: after cells were harvested and centrifuged, the supernatant was discarded and the pellet re-suspended in sufficient volume of binding buffer to plate 50 mL/well in triplicates in 96 well plates. Specific v-conotoxins binding was determined using the same concentration of protein as used for SH-SY5Y cell membranes (20 mg/50 mL), corresponds to 600.000 cells per well.

Radioligand Binding Assay
Tyr22-[ 125 I]-GVIA, was prepared using IODOGEN, as previously described by Ahmad [64], purified using reverse phase HPLC and stored at 4uC for use within 3 weeks. On the day of the assay, membranes were thawed on ice and reconstituted to 10 mg/ 50 mL (rat) or 10-20 mg/50 mL (SH-SY5Y) in binding assay buffer containing 2% complete protease inhibitor and 0.1% bovine serum albumin. Stock [ 125 I]-GVIA was diluted to 20000 cpm/ 50 mL or 30 pM. For displacement studies, [ 125 I]-GVIA was incubated with rat brain or SH-SY5Y membranes or whole cells and varying concentrations of the competing ligand in triplicates in 96 well plate formats. The plates were incubated with shaking for 1 h at room temperature and vacuum filtered through a glass fiber filter pre-soaked in 0.6% polyethyleneimine (PEI), to reduce non-specific binding and washed with buffer containing (mM) 20 HEPES and 125 NaCl at pH 7.2 using a vacuum system (Tomtec harvester). The filters were then dried at 37uC before being placed in sample bags and soaked in liquid scintillant. Radioactivity was counted using a Microbeta Jet (Wallac, Finland). The non-specific binding was determined in the presence of 50 mL of unlabeled peptides.
Intracellular Ca 2+ Response Measurement Using the FLIPR SH-SY5Y cells were seeded onto 96-well or 384-well flat, clear bottom, black-walled imaging plates (Corning, Lowell, MA, US) at a density of 160,000 or 40,000 cells/well, respectively, resulting in 90-95% confluent monolayer after 48 h. On the day of the Ca 2+ imaging assays, cells were loaded for 30 min in the dark at 37uC with 5 mM Fluo-4 acetomethoxyester (Fluo-4-AM), in physiological salt solution (PSS composition: NaCl 140 mM, glucose 11.5 mM, KCl 5.9 mM, MgCl 2 1.4 mM, NaH 2 PO 4 1.2 mM, NaHCO 3 5 mM, CaCl 2 1.8 mM, HEPES 10 mM, pH 7.4) containing in addition 0.3% BSA and 10 mM nifedipine. After the incubation period, the cells were washed once with 100 mL assay buffer (no Fluo-4-AM or BSA), and replaced with 100 mL of the same buffer. Plates were then transferred to the FLIPR TETRA (Molecular Devices, Sunnyvale, CA) fluorescent plate image reader, camera gain and intensity were adjusted for each plate to yield between 800-1000 arbitrary fluorescence units (AFU) baseline fluorescence, and Ca 2+ responses measured using a cooled CCD camera with excitation at 470-495 nM, and emission at 515-575 nM. Ten baseline fluorescence readings were taken prior to the addition of antagonists, and then fluorescent readings every 2 s for 300 s before 90 mM KCl/5 mM CaCl 2 buffer was added and fluorescence readings again recorded each second for further 300 s. To ensure full inhibition of Ca v 1 responses, the cells were pre-incubated for 40 min with 10 mM nifedipine. To ensure full inhibition of Ca v 2.2 responses, the cells were pre-incubated for 10 min with 1-3 mM CVID.

Statistical Analysis
Concentration-response curves were determined following nonlinear regression analysis using a 4-parameter Hill equation, with variable Hill slope fit to the functional assays data and one site fit to the radioligand binding assays; and normalized using GraphPad Prism (Version 5.00, San Diego, California). Negative and positive controls (PSS buffer and KCl 90 mM +5 mM CaCl 2 , respectively) were used to normalize functional data. All data is presented as mean 6 SEM of 6-10 independent experiments performed in triplicate, unless otherwise stated. Statistical significance was determined using analysis of variance (ANOVA) or student's t-test, with statistical significance defined as p,0.05, unless otherwise stated.

Author Contributions
Conceived and designed the experiments: IV RJL. Performed the experiments: SRS IV LR. Analyzed the data: SRS IV LR RJL. Contributed reagents/materials/analysis tools: RJL. Wrote the paper: SRS IV LR RJL.