Differential regulation of Cav2.2 channel exon 37 variants by alternatively spliced μ-opioid receptors
We have examined the regulation of mutually exclusive Cav2.2 exon 37a and b variants by the mouse μ-opioid receptor (mMOR) C-terminal splice variants 1, 1C and 1O in tsA-201 cells. Electrophysiological analyses revealed that both channel isoforms exhibit DAMGO-induced voltage-dependent (Gβγ-mediated) inhibition and its recovery by voltage pre-pulses, as well as a voltage-independent component. However, the two channel isoforms differ in their relative extent of voltage-dependent and independent inhibition, with Cav2.2-37b showing significantly more voltage-dependent inhibition upon activation of the three mMOR receptors studied. In addition, coexpression of either mMOR1 or mMOR1C results in an agonist-independent reduction in the peak current density of Cav2.2-37a channels, whereas the peak current density of Cav2.2-37b does not appear to be affected. Interestingly, this decrease is not due to an effect on channel expression at the plasma membrane, as demonstrated by biotinylation experiments. We further examined the mechanism underlying the agonist-independent modulation of Cav2.2-37a by mMOR1C. Incubation of cells with pertussis toxin did not affect the mMOR1C mediated inhibition of Cav2.2-37a currents, indicating a lack of involvement of Gi/o signaling. However, when a Src tyrosine kinase inhibitor was applied, the effect of mMOR1C was lost. Moreover, when we recorded currents using a Cav2.2-37a mutant in which tyrosine 1747 was replaced with phenylalanine (Y1747F), the agonist independent effects of mMOR1C were abolished. Altogether our findings show that Cav2.2-37a and Cav2.2-37b isoforms are subject to differential regulation by C-terminal splice variants of mMORs, and that constitutive mMOR1C activity and downstream tyrosine kinase activity exert a selective inhibition of the Cav2.2-37a splice variant, an N-type channel isoform that is highly enriched in nociceptors. Our study provides new insights into the roles of the MOR full-length C-terminal variants in modulating Cav2.2 channel isoform activities.
KeywordsN-type Calcium channel Cav2.2 Opioid receptor Splicing G proteins
Voltage-gated calcium channels trigger depolarization-mediated calcium influx into electrically excitable cells in nerve, muscle and heart (for review see ). The nervous system expresses multiple types of calcium channels encompassing three distinct families (Cav1, Cav2 and Cav3) with specialized functional roles. Calcium entry via voltage gated calcium channels can be further tuned by a number of factors, including phosphorylation [2, 3, 4, 5], post-translational modifications such as glycosylation [6, 7, 8] and ubiquitination [9, 10, 11, 12], interactions with adaptor proteins [13, 14, 15, 16, 17], and association with synaptic proteins  (for review see ). These examples highlight the vast spectrum of regulatory mechanisms that fine tune calcium entry into neurons.
MOR full-length C-terminal splice variants, produced by 3′ alternative pre-mRNA splicing of the OPRM1 gene, have been reported in different species. These variants have identical receptor structures, but contain a unique intracellular C-terminal tail sequence, and are known to exhibit different cellular and regional distributions [34, 35, 36]. The original mMOR1 includes a C-terminal tail sequence encoded by exon 4 with 12 amino acids. Both mMOR1C and mMOR1O have an alternative C-terminal tail encoded by exon 7a with a unique 30 amino acid sequence, while mMOR1C contains additional exons 8/9 with an extra 22 amino acids (Fig. 1b). These variants exhibit different signalling bias and differentially contribute to various morphine actions including morphine tolerance, physical dependence, reward behavior and locomotor activity profile without affecting morphine analgesia . We thus wondered whether these receptor variants may couple differentially to Cav2.2 channels, and if so, whether this may occur in a Cav2.2 splice isoform specific manner. Here, we report that different combinations of mMOR1, mMOR1C and mMOR1O and rat Cav2.2 exon 37 isoforms exhibit distinct voltage dependent and independent modulation.
Materials and methods
tsA-201 cells were transfected with 3 μg of each plasmid encoding Cav2.2α1 (WT or Y1747F mutant), Cavβ1 and Cavα2δ-1, respectively, in the presence of empty vector, or mMOR1, mMOR1C or mMOR1O using the calcium phosphate method as described previously . In addition, 0.5 μg of cDNA encoding green fluorescent protein was added to the transfection mixture to identify and select transfected cells. Cells used for electrophysiology experiments were moved to 30 °C after transfection, whereas those used for Western blotting were maintained at 37 °C.
Whole cell patch-clamp recordings were performed at room temperature (22–24 °C). Currents were recorded using an Axopatch 200B amplifier linked to a computer with pCLAMP9.2 software. The external recording solution contained (in mM): 2 CaCl2, 137 CsCl, 1 MgCl2, 10 HEPES, 10 glucose (pH 7.4 adjusted with CsOH). The pipette solution contained (in mM): 130 CsCl, 2.5 MgCl2, 10 HEPES, 10 EGTA, 3 ATP, 0.5 GTP (pH 7.4 adjusted with CsOH). I peak was obtained by dividing the peak current by the whole cell capacitance. Current-voltage relations were fitted using the Boltzmann equation to obtain the half activation voltage. Time constants of activation were obtained by mono-exponential fits to the late rising phase of the current. The effects of receptor coexpression or pharmacological treatments on Cav2.2 current densities were always assessed in the same batch of cells. G protein modulation induced by μ-opioid receptor activation was assessed as described in the results section. Cells expressing Cav2.2-37a and mMOR1C were incubated overnight with 500 ng/ml of PTX (Tocris 3097) or with 2 μM of Src inhibitor for 4 h (PP1, Millipore 567,809).
Cell surface biotinylation
Cell surface biotinylation experiments were performed as previously described . Briefly, surface proteins from transfected cells were biotinylated for 1 h on ice with 1 mg/ml of EZ- Link Sulfo-NHS-SS-Biotin (Thermo Scientific, 21,331). The reaction was quenched with 100 mM glycine for 15 min, and cells were lysed in modified RIPA buffer (in mM: 50 Tris, 150 NaCl, 5 EDTA, 1% Triton X-100, 1% NP-40, 0.2% SDS, pH 7.4) for 45 min. Two mg of lysates were incubated with 100 μl of Neutravidin beads (Thermo Scientific 29,200) for 1.5 h at 4 °C. Beads were washed and proteins eluted with 2× Laemmli sample buffer. Biotinylated proteins and lysates were resolved by SDS-PAGE and analyzed by western blot using the antibodies anti-Cav2.2 (1:500, Alomone ACC-002) and anti-Na/K ATPase (1:5000, Abcam AB 7671).
All error bars reflect standard errors. All data were analyzed for normality using D’Agostino and Pearson tests. Normal data were statistically analyzed using Student’s t-tests or One-way Analysis of variance (ANOVA) for multiple comparisons. Pre-pulse facilitation was analyzed using a Wilcoxon matched-pairs test. Non normally distributed data were analyzed via a Mann-Whitney test, or a Kruskal-Wallis test for multiple comparisons. Significance was set at 0.05. Asterisks denote significance as follows: * p < 0.05, ** p < 0.01, ***p < 0.001. Unless stated otherwise, data are presented as means plus standard errors.
Results and discussion
Cav2.2-37a channels are subject to agonist-independent modulation by mMOR variants
Exon 37 Cav2.2 variants exhibit differential degrees of DAMGO-induced voltage-dependent and independent modulation
Agonist-independent modulation of Cav2.2-37a by mMOR1C involves tyrosine kinases
MORs are expressed in both the afferent pain pathway and within the brain’s pain matrix and are the target of nearly all the opioids used to treat pain . In the afferent pain pathway, the activation of MORs leads to the activation of GIRK channels and the inhibition of N-type channel mediated calcium entry, thus attenuating neuronal excitability and synaptic communication in the spinal dorsal horn . Opioid receptors are G protein coupled receptors that feature 7 transmembrane helices and a large cytoplasmic C-terminal region [45, 46]. The C-terminus is important for G protein activation, as well as phosphorylation-dependent recruitment of β-arrestins [47, 48]. Hence, alternative splicing of the MOR C-terminal region has the propensity to alter MOR signaling, as demonstrated clearly by Xu and colleagues  that various MOR full-length C-terminal variants can have differences in biased signaling between β- arrestin recruitment and G protein activation. Our data suggest novel roles of MOR full-length C-terminal variants in the regulation of Cav2.2 channel isoform activity. We show that three C-terminal variants, mMOR1, mMOR1C and mMOR1O, can differentially regulate Cav2.2-37a activity at the basal level (i.e., agonist-independent state), with a limited effect on Cav2.2-37b. The mere coexpression of mMOR1C and mMOR1 significantly suppressed Cav2.2-37a current densities. This regulation seems unrelated to the expression level of Cav2.2-37a. Rather, the constitutive receptor variant activities, particularly those of mMOR1C, produced a form of tonic voltage-independent modulation that appears to preferentially target exon 37a containing channels. Furthermore, the voltage-independent inhibition in absence of receptor agonist could be attributed to selective tyrosine kinase phosphorylation of Cav2.2-37a by Src acting on its Y1747F residue. This effect appears to be due to constitutive receptor activity, since the effects of MOR1C were abolished upon incubation with the Src inhibitor PP1. The notion that the effects of MOR1C coexpression were insensitive to PTX indicates that such constitutive activity does not involve Gi/o signaling. Although we do not discount the possibility that other phosphorylation sites on the channel might also play a role, the Y1747F mutant was entirely resistant to the agonist-independent effects of mMOR1C on peak current density, thus suggesting that the agonist independent receptor signaling affects current densities by primarily targeting residue Y1747. Interestingly, Raingo et al.  showed that the Y1747F mutant exhibited a drastic reduction in agonist-induced voltage-independent inhibition of Cav2.2-37a, resulting in a regulation that more closely approximates that of the Cav2.2-37b isoform, suggesting that our observations presented in Fig. 5 (i.e., largest voltage-independent modulation with the mMOR1/Cav2.2-37a combination) may also be due to tyrosine kinase regulation. Given that Cav2.2-37a also showed agonist mediated voltage-independent modulation despite being constitutively modulated by Src would then suggest an additional Src/Y1747 independent component of voltage-independent modulation in the presence of DAMGO. This is supported by the notion that Cav2.2-37b channels still exhibit agonist-mediated voltage-independent modulation despite the absence of a C-terminal Src site. This indicates that this type of inhibition involves additional mechanisms common to both splice isoforms which require further investigation.
mMOR1, mMOR1C and mMOR1O share same receptor sequences except for the differences in their intracellular C-terminal tails. mMOR1 has a C-terminal tail that contains 12 amino acids encoded by exon 4, while both mMOR-1C and mMOR1O have 30 amino acids encoded by exon 7a with an additional 22 amino acids encoded by exons 8/9 in mMOR1C. The agonist-independent inhibition of Cav2.2-37a by mMOR1C and mMOR1, but not by mMOR1O, raises interesting questions how these different C-terminal sequences influence Cav2.2-37a channel activity. We also demonstrated that all three receptor variants can modulate DAMGO-induced changes in pre-pulse facilitation ratio for both Cav2.2-37a and Cav2.2-37b. Exon-4 associated mMOR1 and exon 7-associated mMOR1C and mMOR-1O are differentially expressed in various brain regions at both mRNA and protein levels [34, 35, 37]. This suggests a potentially specific function of each individual receptor variant in modulating Cav2.2 isoforms in various brain regions.
Even though there were no significant differences in pre-pulse facilitation among three variants in the presence of DAMGO, a more detailed analysis reveals differences in the relative proportion of voltage-dependent and voltage-independent modulation of the Cav2.2 isoforms by the different mMOR variants, further highlighting the importance of the C-terminal sequences in Cav2.2 activity. A distinct opioid receptor mediated modulation of the two Cav2.2 exon 37 variants is consistent with earlier findings of Raingo et al. . These authors showed that both exon 37 variants underwent a similar degree of total agonist mediated current inhibition, but the relative proportion of voltage-independent to voltage dependent modulation was larger in the Cav2.2-37a variant, and this fits with the data presented in Fig. 5.
It is now well established that MORs couple differentially to different members of the Cav2 channel family [49, 50], which is consistent with observations that these channels are differentially modulated by Gβγ subunits [51, 52, 53]. Moreover, different members of the extended opioid receptor family including NOP receptors types produce differential effects on Cav2 calcium channels and this is further modulated by receptor heteromerization [40, 54]. As we show here, this functional diversity of opioid receptor family signaling to various calcium channels is further expanded by MOR and Cav2.2 splice variation. While we have focused on only a subset of known mMOR splice variants, mouse, human and rat MORs have a much richer complement of possible variants  and it will be interesting to examine how these different isoforms of the MOR receptor affect N-type channel function and perhaps trafficking. Suffice it to say, our findings along with those published in previous literature highlight the importance of selecting the correct variants for expression studies when attempting to correlate findings between recombinant and native systems.
We thank Lina Chen for providing tissue culture support, and Dr. Diane Lipscombe for the Cav2.2-37a, Cav2.2-37b constructs and the tyrosine mutant channel, and Dr. Terry Snutch for the ancillary calcium channel subunit clones.
MAG and IAS co-designed study and performed biochemical and electrophysiological measurements. DR contributed electrophysiological recordings. YXP and JX provided mMOR cDNAs. YXP and GWZ co-designed the study. GWZ supervised the study and wrote manuscript. All authors read and approved the final manuscript.
This work was supported by a grant to GWZ from the Canadian Institutes of Health Research (CIHR) and by funds to YXP from the National Institutes of Health National Institute on Drug Abuse [Grants DA07242, DA042888 and DA046714], the Mayday Foundation, the Peter F. McManus Charitable Trust, and a core grant from the National Institutes of Health National Cancer Institute [Grant CA008748] to the Memorial Sloan-Kettering Cancer Center. GWZ holds a Canada Research Chair. MAG was supported by a Fellowship from Alberta Innovates and holds a CIHR Postdoctoral Fellowship.
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YXP is a co-scientific founder of Sparian Biosciences.
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