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Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing

Abstract

Alternative pre-mRNA splicing occurs extensively in the nervous systems of complex organisms, including humans, considerably expanding the potential size of the proteome. Cell-specific alternative pre-mRNA splicing is thought to optimize protein function for specialized cellular tasks, but direct evidence for this is limited. Transmission of noxious thermal stimuli relies on the activity of N-type CaV2.2 calcium channels in nociceptors. Using an exon-replacement strategy in mice, we show that mutually exclusive splicing patterns in the CaV2.2 gene modulate N-type channel function in nociceptors, leading to a change in morphine analgesia. Exon 37a (e37a) enhances μ-opioid receptor–mediated inhibition of N-type calcium channels by promoting activity-independent inhibition. In the absence of e37a, spinal morphine analgesia is weakened in vivo but the basal response to noxious thermal stimuli is not altered. Our data suggest that highly specialized, discrete cellular responsiveness in vivo can be attributed to alternative splicing events regulated at the level of individual neurons.

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Figure 1: Exon 37 substitution in Cacna1b and resultant mRNAs in wild-type and mutant DRG.
Figure 2: Whole-cell calcium current densities in DRG neurons of Cacna1bb*b/b*b mice are indistinguishable from wild-type DRG neurons.
Figure 3: N-type and non–N-type current densities in sensory neurons of Cacna1bb*b/b*b mice are indistinguishable from those in wild-type mice.
Figure 4: DAMGO inhibits N-type currents similarly in nociceptors from all three genotypes.
Figure 5: Voltage-independent inhibition by DAMGO is reduced in nociceptors that only express e37b and not e37a.
Figure 6: Anti-CaV2.2, anti-CGRP and FITC-IB4 signals in superficial layers of dorsal spinal horn are similar among genotypes.
Figure 7: Morphine's spinal-level analgesia is reduced in mice that lack e37a.

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Acknowledgements

We thank J. Kysik and E. Paul in the mouse transgenic facility at Brown University, R. Burwell for advice on animal behavior, T.D. Helton for help with image analyses and C.G. Phillips and S.E. Allen for helpful comments on the manuscript. This work was supported by US National Institutes of Health grants RO1NS055251 (D.L.), F31NS066702 (S.M.) and P20RR015578 (Transgenic Facility at Brown University).

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All authors contributed to writing this manuscript. A.A. carried out electrophysiological studies, S.D. designed targeting constructs and generated all exon-substituted mice, Y.-Q.J. performed the behavioral studies and immunohistochemistry, S.M. performed western analyses, and D.L. directed the project and oversaw all analyses.

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Correspondence to Diane Lipscombe.

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The authors declare no competing financial interests.

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Andrade, A., Denome, S., Jiang, YQ. et al. Opioid inhibition of N-type Ca2+ channels and spinal analgesia couple to alternative splicing. Nat Neurosci 13, 1249–1256 (2010). https://doi.org/10.1038/nn.2643

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