Neuronal CaV channels convert activity into intracellular calcium signals, which control a vast number of cellular functions. CaV channels located in the presynaptic region of neurons couple calcium influx to the release of neurotransmitters. At postsynpatic sites, CaV channels can couple membrane depolarization to activity-dependent gene expression. Each CaV channel has unique physiological and/or pharmacological properties. Within each CaV channel sub-family, additional diversity arises from alternate start sites and alternative pre-mRNA splicing.
Alternative splicing is a feature of all 10 mammalian CaV channel alpha-subunit genes and underlies the expression of hundreds of splice isoforms. Each cell-type expresses a unique pattern of alternative splice isoforms and this additionally influenced by development, neuronal activity, injury, and disease. CaV channel splice isoforms have different biophysical properties, pharmacological sensitivities, and they can have uniquely different interaction with other proteins. Close to 95 percent of all multi-exon mammalian genes contain alternatively spliced exons and aberrant alternative splicing is known to underlie an ever expanding list of diseases.
To investigate the molecular and cellular mechanisms that regulate CaV channel function, we employ a range of methodologies including mouse genetics, molecular, biochemical, electrophysiological and behavioral approaches. We use electrophysiological methods to assess ion channel function in specific neural circuits, and also to characterize the pharmacological properties of ion channels, which are key drug targets and are expressed in different regions of the nervous system.
Our work has contributed to our understanding of the role of CaV channels in control of basic cellular processes, in disease, and as therapeutic drug targets. Below is a select list of publications that highlight our areas of research.
Voltage-gated CaV2.2 channels control release of neurotransmitter at many synapses in mammalian central and peripheral nervous systems. We showed that highly selective tissue-specific expression of key alternatively spliced exons of Cacna1b influence CaV2.2 channel gating, expression and sensitivity to G protein modulation.
Tong XJ, Lopez-Soto EJ, Li L, Liu L, Nedelcu D, Lipscombe D, Hu Z, Kaplan JM. (2017) Retrograde Synaptic Inhibition Is Mediated by α-Neurexin Binding to the α2δ Subunits of N-Type Calcium Channels. Neuron 95(2):326-340.e5. PMC5548138.
Manarangoudakis S, Andrade A, Helton TD, Denome S, Lipscombe D. (2012) Differential Ubiquitination and Proteasomal Regulation of CaV2.2 Splice Isoforms. Journal of Neuroscience 32(30):10365-10369. PMC3428229.
Raingo J, Castiglioni AC, Lipscombe D. (2007) Alternative splicing controls G protein-dependent inhibition of N-type calcium channels in nociceptors. Nature Neuroscience 10(3):285-292. PMC3027493.
Bell TJ, Thaler C, Castiglioni AJ, Helton TD, Lipscombe D. (2004) Cell-specific alternative splicing increases calcium channel current density in the pain pathway. Neuron 41(1):127-138. PMID:14715140.
Cell-specific control of alternative splicing of Cacna1b impacts animal behavior in vivo. We demonstrated this by exon substitution in Cacna1b and limiting exon choice in mice. Our studies show that nociceptor-specific exon selection during processing of Cacna1b pre mRNA influences CaV2.2 trafficking to the cell surface and the analgesic efficacy of morphine at the level of the spinal cord.
Jiang Y-Q, Andrade A, Lipscombe D. (2013) Spinal morphine but not ziconotide or gabapentin analgesia is affected by alternative splicing of voltage-gated calcium channel CaV2.2 pre-mRNA. Molecular Pain 9:67. PMC3916075.
Andrade A, Denome S, Jiang Y-Q, Marangoudakis S, Lipscombe D. (2010) Opioid receptor inhibition of N-type calcium channels and spinal morphine analgesia couple to cell-specific alternative splicing. Nature Neuroscience. PMC2956429.
Altier C, Dale CS, Kisilevsky AE, Chapman K, Castiglioni AJ, Matthews EA, Evans RM, Dickenson AH, Lipscombe D, Vergnolle N, Zamponi GW. (2007) Differential role of N-type calcium channel splice isoforms in pain. Journal of Neuroscience 27(24):6363-6373. PMID:17567797.
A set of splicing factors are critical for controlling exon inclusion/exclusion during alternative splicing of calcium ion channel pre mRNAs. We have shown that RNA binding proteins Rbfox2 and Nova2 control the selection of cassette exons according to cell-type and developmental stage.
Lipscombe D, Lopez-Soto EJ. (2018). Protected by a Fox. Neuron. 98(1):3-5. PMID:29621488.
Allen SE, Toro CP, Andrade A, Denome S, Lipscombe D. (2017) Cell-specific RNA binding protein Rbfox2 regulates CaV2.2 mRNA exon composition and overall CaV2.2 current size. eNeuro. 4(5):eneuro.0332-16.2017. PMC5633781.
Allen SE, Lipscombe D. (2010) The neuronal splicing factor Nova controls alternative splicing in N-type and P-type CaV2 calcium channels. Channels 4(6):483-489. PMC3047467.
In collaboration, we characterized rare missense variations in two different CACNA1 genes. A rare variation in CACNA1B identified in a 3-generation family with a myoclonus dystonia-like syndrome alters properties of the CaV2.2 channel. CACNA1B encode the pore-forming subunit of presynaptic CaV2.2 channels which control transmitter release from presynaptic terminals at mammalian synapses. We found altered ion flow through CaV2.2 channels containing MIM 601012 using single channel analyses. We also analyzed the functional consequences of a de novo rare variant in CACNA1I that is linked to schizophrenia. In collaboration with colleagues at the Broad Institute, we showed disrupted trafficking of CaV3.3 to the cell surface which impacts the ability of CaV3.3 current to mediate burst firing in thalamic relay neurons.
Andrade A, Hope J, Allen A, Yorgan V, Lipscombe D, Pan JQ. (2016) A rare schizophrenia risk variant of CACNA1I disrupts CaV3.3 channel activity. Scientific Reports. 6:34233. PMC5069464.
Groen JL, Andrade A, Ritz K, Jalalzadeh H, Haagmans M, Bradley TE, Jongejan A, Verbeek DS, Nürnberg P, Denome S, Hennekam RC, Lipscombe D, Baas F, Tijssen MA. (2015) CACNA1B mutation is linked to unique myoclonus-dystonia syndrome. Human Molecular Genetics. 24(4):987-993. PMC4817404.
In 2001 we demonstrated novel features of a neuronal CaV1.3 channel with important implications for its contribution to control of neuronal function. Compared to the widely studied CaV1.2 channel, CaV1.3 channels opened at membrane voltages significantly more hyperpolarized. Previous studies of CaV1.3 clones were performed using non-physiological conditions that distorted channel properties. We provided our clones to a large number of laboratories and facilitated many exciting studies. CaV1.3 is now implicated in Parkinson Disease and it is known to drive pacemaking in several excitable cells.
Helton TD, Xu W, Lipscombe D. (2005) Neuronal L-type calcium channels open quickly and are blocked slowly. Journal of Neuroscience. 25(44):10247-10251. PMID:16267232.
Lipscombe D, Xu W, Helton TD. (2004) Neuronal L-type calcium channels: The low down. Journal of Neurophysiology 92(5):2633-2641. PMID:15486420.
Xu W, Lipscombe D. (2001) Neuronal CaV1.3a1 L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines. Journal of Neuroscience 21(16):5944-5951. PMID:11487617.