Limited Efficacy of α-Conopeptides, Vc1.1 and RgIA, To Inhibit Sensory Neuron CaV Current,,

Better analgesic drugs are desperately needed to help physicians to treat pain. While many preclinical studies support the analgesic effects of α-conopeptides, Vc1.1 and RgIA, the mechanism is controversial.


Introduction
Severe pain reduces the quality of life of millions of people each year (Cousins et al., 2004). Conventional treatment for chronic pain includes opiates and nonsteroidal antiinflammatory agents. However, the therapeutic potential of these treatment options for chronic pain are often limited by the development of serious adverse effects and tolerance. Thus, the discovery of improved drug therapies is of great importance.
␣-conopeptides are small, disulfide-rich peptides that are isolated from the venom of the Conus genus of carnivorous marine snails and that block nicotinic acetylcholine receptors (nAChRs) (McIntosh et al., 2009). Two ␣-conopeptides, Vc1.1 and RgIA, have been shown to display antinociceptive effects in animal models; however, the mechanism responsible for analgesia remains debated (Vincler et al., 2006;McIntosh et al., 2009;Napier et al., 2012). Early studies found these ␣-conopeptides to be potent antagonists of heterologously expressed and native ␣9␣10 nAChRs Vincler et al., 2006). Other studies have found that Vc1.1, but not RgIA, also weakly antagonizes nAChRs subtypes expressed in the periphery containing the ␣3 subunit (Clark et al., 2006;Ellison et al., 2006). Analogs of Vc1.1 that retain their specificity for ␣9␣10 nAChRs, but not nAChRs with the ␣3 subunit, are devoid of analgesic effects in animal pain models (Nevin et al., 2007). These findings indicate the possible involvement of off-target effects being responsible for analgesia. However, mice lacking ␣9 nAChRs have reduced mechanical hyperalgesia in both neuropathic and inflammatory pain models, supporting a role for ␣9␣10 nAChRs as a target for treatment of chronic pain (Mohammadi and Christie, 2014).
One group has proposed that the antinociceptive effects of Vc1.1 and RgIA are elicited by inhibition of N-type Ca V (Ca V 2.2) channels via activation of GABA B receptors (Callaghan et al., 2008;Callaghan and Adams, 2010;Klimis et al., 2011;Adams et al., 2012;Mohammadi and Christie, 2014). The analgesic effects of GABA B receptor activation by the specific GABA B receptor agonist baclofen have been previously shown (Franek et al., 2004). Furthermore, GABA B receptor activation inhibits the activity of N-type Ca V channels (Ca V 2.2) and inhibition of N-type channels expressed by nociceptors in the spinal cord dorsal horn is analgesic (Raingo et al., 2007). Pain relief comes from the reduction of excitatory neurotransmitter release (e.g., glutamate) from nociceptive nerve terminals when presynaptic N-channels are blocked (Elmslie, 2004;Miljanich, 2004;McIntosh et al., 2009). The inhibition of N-type Ca V current by Vc1.1 and RgIA requires functional GABA B receptors since the effect can be blocked by either application of a GABA B receptor antagonist (Callaghan et al., 2008) or the knockdown of GABA B receptors by siRNA (Cuny et al., 2012).
While inhibition of N-type Ca V channel activity is a potential mechanism for Vc1.1-or RgIA-induced analgesia, this hypothesis is controversial (McIntosh et al., 2009). Neither Vc1.1 or RgIA were able to prevent the binding of a specific competitive antagonist to the human GABA B receptor and both failed to activate GABA B receptors expressed in Xenopus laevis oocytes (McIntosh et al., 2009). In addition, Vc1.1 failed to affect excitatory postsynaptic currents (eEPSCs) in the dorsal horn of rat spinal cord, which were almost completely blocked by baclofen (Napier et al., 2012). These findings are inconsistent with GABA B receptor-induced inhibition of N-type Ca V channels as the mechanism for analgesia produced by Vc1.1 and RgIA. Given these findings, there is a question of whether the Ca V current inhibition in sensory neurons can be independently reproduced. The data presented here shows that the inhibition of Ca V current in sensory neurons is on average either small (Vc1.1) or insignificant (RgIA), and that activation of GABA B receptors is not consistent with the small inhibition induced by Vc1.1.

Animals
All animal procedures were performed in accordance with the authors' university animal care committee's regulations and were consistent with the National Research Council Guide for the Care and Use of Laboratory Animals. Adult male Sprague Dawley rats (200 Ϫ 400 g; Hilltop Lab Animals) were used in these experiments. The rats were housed in a U.S Department of Agricultureapproved, Association for Assessment and Accreditation of Laboratory Animal Care-certified animal care facility at a constant temperature 24 Ϯ 1°C, under controlled 12:12 h light-dark cycles, and fed a standard rat chow diet and tap water ad libitum.
Ionic currents were recorded using the whole-cell configuration of the patch-clamp technique with an Axopatch 200B amplifier (Molecular Devices) and digitized with an ITC-18 A/D converter (Instrutech Corp). Microelectrodes with a resistance of 2 Ϫ 5 M⍀ were pulled from Schott 8250 glass (King Precision Glass) on a Sutter P-97 puller (Sutter Instruments). Series resistance was compensated by at least 80% using the electronic circuits of the Axopatch 200A amplifier. Neurons were voltage clamped at a holding potential of Ϫ80 mV and Ca V currents were assessed using a three-step voltage protocol that tests for voltage-dependence of Ca V channel inhibition (Elmslie et al., 1990;Ikeda, 1991;Ehrlich and Elmslie, 1995).
Experiments were controlled by a Power Macintosh computer (Apple Computer) running S5 data acquisition software written by Dr. Stephen Ikeda (NIH, NIAAA, Bethesda, MD). Leak current was subtracted from the step current using a ϪP/4 protocol. All experiments were conducted at room temperature (Ramachandra et al., 2012).
Data were analyzed with IgorPro (WaveMetrics) running on a Macintosh computer. Cell diameter was calculated from the cell capacitance as measured by the Axopatch circuitry, assuming a specific membrane capacitance of 1 F/cm 2 and that the neuron was spherical (Ramachandra et al., 2012).
Membrane currents were recorded from oocytes with a two-electrode voltage-clamp amplifier (TEV-200; Dagan) at room temperature (ϳ22°C). Electrodes had resistances of 0.5 to 2 M⍀ when filled with 3 M KCl. The membrane potential was clamped at Ϫ70 mV. Data acquisition utilized an analog-to-digital converter (Digidata 1320A; Molecular Devices), and data acquisition as well as analysis was done on a Pentium-based microcomputer using pCLAMP software. Data were low-pass filtered (5 Hz cutoff) and digitized at a sampling frequency of 20 Hz.
To apply a pulse of ACh to the oocyte, the perfusion fluid was switched to one containing 10 M ACh for 1 s. This was done at intervals of ϳ6 min and has previously been shown to allow reproducible control responses without substantial desensitization (Vincler et al., 2006). To measure block by ␣-conopeptides, the perfusion system was stopped, the solution from around the oocyte was removed via a mechanical pipetter, and the bath was filled with 200 l of a solution containing one of the peptides (either 1 M Vc1.1 or 100 nM RgIA). The oocyte was incubated with the conopeptide for 5 min in the static bath. The perfusion system was then restarted with a 1 s pulse of ACh. The conopeptide dwell time was sufficiently long-lasting that the majority of ␣9␣10 nAChR were still blocked (Ͻ2 s of wash time) when the ACh pulse arrived at the oocyte (Vincler et al., 2006). Control ACh responses prior to peptide application were exposed to the same procedure except that control Frog Ringers was used instead of peptide-containing solution. Control ACh responses were measured from the average of two preceding responses and the first response following recovery from conopeptide block (ϳ6 min of washout).

Statistics
All data are presented as mean Ϯ SD. Two-tailed onesample t tests (Excel) were used to determine significant differences (p Ͻ 0.05) versus zero of normally distributed data, while a Wilcoxon rank-sum analysis (IgorPro) was used to determine significant differences for data deviating from a normal distribution. The Pearson correlation test (Excel) was used to test for significant correlations between data sets.

Drugs and chemicals
MEM, FBS, and penicillin-streptomycin were purchased from Life Technologies. Liberase Blendzyme and collagenase were from Roche Molecular Biochemicals, and trypsin was from Worthington. ␣-conopeptides Vc1.1 and RgIA were synthesized as reported previously (Cartier et al., 1996;Ellison et al., 2008). All other chemicals were obtained from Sigma-Aldrich.

Results
Inhibition of Ca V current is one mechanism that has been proposed for analgesia induced by the ␣-conopeptides Vc1.1 and RgIA (Callaghan et al., 2008;Cuny et al., 2012). The effects of Vc1.1 and RgIA on Ca V current were studied in sensory neurons dissociated from adult rats. Since the Ca V current inhibition by these conopeptides has been reported to be mediated by GABA B receptors, the specific GABA B receptor agonist, baclofen, was used to test for functional presence of GABA B receptors by measuring Ca V current inhibition (Tosetti et al., 2002). Ca V current was tested using a triple-pulse voltage protocol to examine the voltage dependence of inhibition (Elmslie et al., 1990), which results from transient disrup-tion of G protein ␤␥ subunits binding to Ca V 2 channels (Ikeda, 1996). Thirty micromolar baclofen significantly inhibited prepulse currents by 19.2 Ϯ 6.8% (mean Ϯ SD, n ϭ 21; Fig. 1). As expected, this inhibition was voltagedependent since the postpulse current was inhibited by only 12.0 Ϯ 5.5%, which was significantly smaller than the prepulse inhibition (p ϭ 0.005). Ca V current in 20/21 (95%) sensory neurons was inhibited by baclofen.
While the effect of Vc1.1 and RgIA was on average small or insignificant, there were a few neurons that responded with inhibitions Ͼ10%. This includes 4/21 (19%) neurons tested with Vc1.1 and 2/12 (17%) neurons tested with RgIA. This contrasts with the previous report showing Failure to Replicate that Ca V current in ϳ75% of sensory neurons was inhibited by 100 nM Vc1.1 (Callaghan et al., 2008). We wanted to further investigate the peptide-induced inhibition to determine if the properties were similar to those reported previously (Callaghan et al., 2008). It was previously reported that Ca V current block by Vc1.1 was irreversible, but we found that the block by Vc1.1 was readily reversible with an average recovery ϭ 0.5 Ϯ 0.2 min (n ϭ 4; Fig. 2). Thus, this inhibition appears to be distinct from that previously reported (Callaghan et al., 2008).
Another question was if a particular group of neurons exhibited conopeptide sensitivity. One possibility is that the sensitive neurons were nociceptors, which would predict that the somal diameter of these neurons would be smaller (Ͻ35 m) than the unresponsive, non-nociceptive neurons (Djouhri et al., 2003). This possibility was investigated by plotting the somal diameter versus percentage prepulse inhibition (Fig. 3A). Against this prediction, the "responsive" neurons spanned the size range with large neurons (Ͼ35 m) just as likely (n ϭ 2) to respond to the conopeptides than small neurons (Ͻ30 m, n ϭ 2; Fig.  3A). Thus, the evidence suggests that the conopeptideinduced Ca V current inhibition is not a marker for nociceptive sensory neurons.
Surprisingly, the baclofen-induced prepulse inhibition did not appear to correlate with that induced by Vc1.1 (Fig. 3A), as expected if GABA B receptors mediate Vc1.1induced inhibition. This relationship was more fully investigated by plotting the Vc1.1-induced Ca V current inhibition versus that induced by baclofen (Fig 3B). Cal-culation of the Pearson correlation yielded R ϭ 0.27 (n.s.). For completeness, the RgIA data are also plotted (Fig. 3B) and the Pearson correlation was R ϭ 0.29 (n.s.). Thus, no correlation was found between the responses induced by either Vc1.1 or RgIA versus baclofen. Notably, the neuron with the largest baclofen response (34%) showed only a 3.5% Ca V current inhibition by Vc1.1 (Fig. 3B). The neuron with the largest Vc1.1 (41%) and RgIA (35%) showed a 22% inhibition by baclofen. However, seven other neurons with baclofen responses ranging from 20 Ϫ 24% responded to Vc1.1 with an average 4.3 Ϯ 6.6% inhibition of Ca V current, while the four neurons also tested with RgIA responded with a 0.0 Ϯ 7.6% effect. It appears that GABA B receptors do not mediate the small Ca V current inhibition induced by Vc1.1.
Interestingly, there was a significant correlation found between the Ca V inhibitions induced by Vc1.1 and RgIA (R ϭ 0.88, p Ͻ 0.05). This result suggests a common inhibitory mechanism for both Vc1.1 and RgIA, but as mentioned above, we could find no evidence that GABA B receptors mediate this Ca V current inhibition.

Discussion
In previous work, the maximum inhibition of Ca V current in sensory neurons isolated from either rat or mouse DRG was 40 Ϫ 50% by 1 M of either Vc1.1 or RgIA (Callaghan et al., 2008;Callaghan and Adams, 2010). The mechanism of this inhibition was concluded to be mediated by GABA B receptor activation. We also utilized 1 M Vc1.1 and RgIA, but found on average only small (7%, Vc1.1) and insignificant (RgIA) effects on Ca V current in rat sensory neurons. The role of GABA B receptors was also assessed, but there was no correlation between the magnitudes of baclofen-and Vc1.1-induced inhibitions. These same ␣-conopeptides that minimally affected Ca V current strongly (Ͼ80%) inhibited ␣9␣10 nAChRs expressed in Xenopus oocytes, which demonstrates the expected potency of these ␣9␣10 nAChR antagonists. While our results fail to reproduce results reported in some previous publications (Callaghan et al., 2008;Callaghan and Adams, 2010), they do support other publications showing that RgIA and Vc1.1 do not activate GABA B receptors expressed in Xenopus oocytes (McIntosh et al., 2009) and showing that Vc1.1 does not affect excitatory neurotransmitter release from sensory nerve terminals that express GABA B receptors (Napier et al., 2012).

Different effects
While the overall inhibition was small, we found that these conopeptides could inhibit Ca V current (Ͼ10%) in a minority of sensory neurons (Ͻ20%). This fraction of sensitive neurons is much smaller than that previously reported (75%) (Callaghan et al., 2008). Other differences include a relative fast recovery from block versus irreversible block, and the apparent lack of GABA B receptor involvement (Callaghan et al., 2008). We were unable to identify a single neuronal group as conopeptide sensitive, since a few small (Ͻ30 m), medium, and large (Ͼ40 m) diameter neurons were found to be sensitive, while other neurons within the same size range were insensitive. As a result, it seems unlikely that nociceptors define the conopeptide-sensitive population.
We have no data to explain why we cannot reproduce the previously published Ca V current inhibitions (Callaghan et al., 2008;Callaghan and Adams, 2010). However, we can exclude some possibilities. First, the ␣-conopeptides used here were potent inhibitors of ␣9␣10 nAChRs, which demonstrated that they were functional peptides. Second, the previous publications demonstrated that N-type Ca V channels were the Ca V channel type inhibited by Vc1.1 and RgIA (Callaghan et al., 2008), and we have demonstrated N-type channels comprise approximately half of the total Ca V current in rat sensory neurons (Ramachandra et al., 2012). Thus, the absence of the target channel cannot explain the differences. Finally, the GABA B receptors were functional in these neurons since the specific agonist, baclofen, inhibited Ca V current in 95% of neurons tested, which confirms the presence of the putative receptor that mediates the Ca V current inhibition induced by these ␣-conopeptides.
Differences among species have been proposed as a possible reason for differing results. McIntosh et al. (2009) demonstrated that both Vc1.1 and RgIA failed to block binding of [ 3 H]CGP-54626, a specific competitive antagonist, to human GABA B receptors, and suggested that the human receptors were not a target for these conopeptides. However, Ca V current inhibition by Vc1.1 and RgIA has been shown in both rat and mouse sensory neurons (Callaghan et al., 2008;Callaghan and Adams, 2010), and our results from rat sensory neurons fit well with the human data. Thus, species differences are unlikely to explain these differences. The sources of the conopeptide are different, but it is not clear how that would explain the different results. These peptides are synthesized by manual solid-phase synthesis. Disulfide bond formation is by directed synthesis and/or verified by NMR analysis. The peptides are purified in a similar manner between labs with reversedphase high-performance liquid chromatography using trifluoracetic acid and acetonitrile buffer systems.

Analgesic mechanisms
Many experiments have demonstrated the analgesic properties of ␣-conopeptides that block ␣9␣10 nAChR, including Vc1.1 and RgIA (Satkunanathan et al., 2005;Vincler et al., 2006;Napier et al., 2012;Di Cesare Mannelli et al., 2014). However, post-translational modifications of Vc1.1 that preserved ␣9␣10 nAChR block eliminated the analgesic effects (Nevin et al., 2007). This suggested that the analgesic effect of Vc1.1 did not result from ␣9␣10 nAChR block. Interestingly, the effect of Vc1.1 to inhibit Ca V current was lost by these same posttranslational modifications, which supported Ca V current inhibition as an analgesic mechanism for Vc1.1 (Callaghan et al., 2008). However, the pharamacokinetic properties of this analog were not investigated, leaving open the possibility that the compound did not reach its in vivo target in adequate concentration. In the present study, we found, on average, little to no Ca V current inhibition by Vc1.1 or RgIA in sensory neurons, in contrast to prior reports. These overall findings agree with a recent publication that demonstrated no inhibition of EPSPs in secondary sensory neurons in the dorsal horn by Vc1.1, even though the EPSP was strongly inhibited by baclofen (Napier et al., 2012).
The analgesic mechanism of the -conopeptide, ziconitide, involves the direct block of presynpatic Ca V 2.2 channels to decrease glutamate release and the resulting EPSP in secondary nociceptors (Elmslie, 2004), which blocks pain transmission between primary nociceptors and second-order neurons in the dorsal horn of the spinal cord (Vanegas and Schaible, 2000;Elmslie, 2004). RgIA and Vc1.1 are unlikely to cross the bloodϪbrain barrier and reach spinal neuron synapses, which further suggests that RgIA and Vc1.1 induced analgesia may not involve Ca V channels. In addition, the highly selective N-type Ca V antagonist, ziconotide, did not decrease neuropathic pain when given peripherally by intravenous injection (Chaplan et al., 1994). This FDA-approved drug must be delivered by intrathecal administration for therapeutic effect (Sanford, 2013). Furthermore, N-type channel expression has been reported to be reduced in peripheral sensory neurons after nerve injury (McCallum et al., 2011). Together, these findings suggest non-Ca V channel mechanisms are important for RgIA-and Vc1.1-induced analgesia. Recent work has demonstrated a possible role of ␣9 nAChR in pain, since mechanical hyperalgesia was reduced in ␣9 nAChR knockout mice following chronic nerve constriction and in an inflammatory pain model (Mohammadi and Christie, 2014). In addition, a major effect of RgIA appears to be on the glial/immunological response to chronic nerve injury to prevent pathological changes within the nervous system that are thought to result in neuropathic pain (Di Cesare Mannelli et al., 2014). There are also small molecule antagonists of ␣9␣10 nAChRs. These compounds have also been shown to be analgesic, lending further support for the importance of this mechanism (Holtman et al., 2011;Zheng et al., 2011;Wala et al., 2012). While the present study does not allow us to identify the mechanism by which Vc1.1 or RgIA produce analgesia, our findings do not support a role for Ca V channel inhibition in sensory neurons as one of those mechanisms.