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Differential Neuronal Targeting of a New and Two Known Calcium

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Differential Neuronal Targeting of a New and Two Known Calcium 1446 • The Journal of Neuroscience, January 22, 2014 • 34(4):1446–1461 Cellular/Molecular Differential Neuronal Targeting of a New and Two Known ␤ Calcium Channel 4 Subunit Splice Variants Correlates with Their Regulation of Gene Expression Solmaz Etemad,1 Gerald J. Obermair,1 Daniel Bindreither,3 Ariane Benedetti,1 Ruslan Stanika,1 Valentina Di Biase,1 Verena Burtscher,2 Alexandra Koschak,2 Reinhard Kofler,3 Stephan Geley,3 Alexandra Wille,4 Alexandra Lusser,4 Veit Flockerzi,5 and Bernhard E. Flucher1 1Department of Physiology and Medical Physics, Medical University Innsbruck, 6020 Innsbruck, Austria; 2Center of Physiology and Pharmacology, Department of Neurophysiology and Pharmacology, Medical University Vienna, 1090 Vienna, Austria; 3Division of Molecular Pathophysiology, Biocenter; 4Division of Molecular Biology; Biocenter; Medical University Innsbruck, 6020 Innsbruck, Austria; and 5Experimental and Clinical Pharmacology and Toxicology, University of Saarland, 66421 Homburg, Germany ␤ ␣ The subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain ␤ subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of ␤ isoforms derived from four genes and abundant alternative splicing, little is ␤ ␤ known about the roles of individual variants in specific neuronal functions. In the present study, an alternatively spliced 4 subunit ␤ lackingthevariableNterminus( 4e )isidentified.Itishighlyexpressedinmousecerebellumandculturedcerebellargranulecells(CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known ␤ ␤ ␤ ␤ full-length 4 variants ( 4a and 4b ), 4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To ␤ determine the importance of nuclear targeting of 4 subunits for transcriptional regulation, we performed whole-genome expression ␤ ␤ ␤ ␤ profiling of CGCs from lethargic ( 4-null) mice individually reconstituted with 4a , 4b , and 4e. Notably, the number of genes regulated ␤ ␤ Ͼ ␤ Ͼ ␤ by each 4 splice variant correlated with the rank order of their nuclear-targeting properties ( 4b 4a 4e ). Together, these findings ␤ ␤ support isoform-specific functions of 4 splice variants in neurons, with 4b playing a dual role in channel modulation and gene ␤ regulation, whereas the newly detected 4e variant serves exclusively in calcium-channel-dependent functions. 2ϩ Key words: Ca channel; Cacnb4; CaV2.1; cerebellar granule cells; hippocampal neurons; lethargic mice Introduction rent density and modulate their gating properties (Buraei and Voltage-activated calcium channels control multiple neuronal Yang, 2010; Dolphin, 2012). Mammalian genomes encode four ␤ ␤ ␤ ␤ ␤ functions including excitability, synaptic transmission and plas- isoforms ( 1, 2, 3, and 4), all of which are expressed in the ticity, and activity-dependent gene regulation (Catterall and Few, brain. Whereas coexpression in heterologous cells demonstrated ␣ ␤ 2008). The cytoplasmic ␤ subunits are essential components of promiscuous interactions of all examined 1- combinations, it is generally accepted that different ␤ subunits endow calcium high-voltage-activated calcium channels (CaV1 and CaV2). They regulate surface expression of the channel and thus calcium cur- channels with specific properties and that particular neuronal functions require specific subunit combinations. Accordingly, the lack of specific ␤ isoforms causes distinct neurological phe- Received Sept. 13, 2013; revised Nov. 27, 2013; accepted Dec. 14, 2013. notypes (Arikkath and Campbell, 2003). For example, mutations Authorcontributions:S.E.,G.J.O.,V.D.B.,A.K.,R.K.,A.L.,andB.E.F.designedresearch;S.E.,G.J.O.,D.B.,A.B.,R.S., ␤ V.B., and A.W. performed research; S.G. and V.F. contributed unpublished reagents/analytic tools; S.E., G.J.O., D.B., of the 4 gene cause ataxia and epilepsy in humans and mice A.B., V.D.B., V.B., A.K., R.K., A.L., and B.E.F. analyzed data; S.E. and B.E.F. wrote the paper. (Burgess et al., 1997; Barclay and Rees, 1999; Hosford et al., 1999; This work was supported by the Austrian Science Fund (Grants P23479, P24079, W1101, F4406, and F4408). We Escayg et al., 2000). thank Anita Kofler and Barbara Gschirr for excellent technical support with the gene chip analysis and Benedikt Structurally, calcium channel ␤ subunits resemble mem- Nimmervoll for help with the FM dye experiments. The authors declare no competing financial interests. brane-associated guanylate kinase (GK) proteins, with conserved This article is freely available online through the J Neurosci Author Open Choice option. SH3 and GK domains linked by a variable hook region and Correspondence should be addressed to Bernhard E. Flucher, Medical University Innsbruck, Department of Phys- flanked by variable N- and C-terminal sequences (Hanlon et al., iology and Medical Physics, Division of Physiology, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria. E-mail: 1999). The GK domain forms the high-affinity binding pocket for [email protected]. ␣ ␣ Valentina Di Biase’s present address: Institute of Biophysics, Medical University of Graz, 8010 Graz, Austria. the cytoplasmic interaction domain in the CaV1 and CaV2 1 DOI:10.1523/JNEUROSCI.3935-13.2014 subunits (Chen et al., 2004; Opatowsky et al., 2004; Van Petegem Copyright © 2014 the authors 0270-6474/14/341446-16$15.00/0 et al., 2004). Alternative splicing of the exons encoding the vari- • ␤ Etemad et al. CaV 4 Subunit Splice Variants J. Neurosci., January 22, 2014 • 34(4):1446–1461 • 1447 ␤ ␤ ␤ ␤ able regions gives rise to multiple variants of each isoform, p A- 4e-V5, the N terminus of 4e was generated with specific primers ␤ which differ in their endogenous membrane-targeting properties and PCR amplified using 4a as a template, thereby introducing an arti- and protein interactions (Buraei and Yang, 2010). These specific ficial, 5Ј HindIII site and a new Kozak sequence, as well as the codons for ␤ Ј properties result in distinct stabilities of channel complexes methionine and alanine in the forward primer ( 4eV5-F, 5 -aagcttccta Ј ␤ Ј Ј ccatggctgggtcagcagattcctat-3 and 4eV5-R, 5 -tgtctttatcagctgtgctgg-3 , (Campiglio et al., 2013), different subcellular targeting properties ␤ ␤ 850-bp PCR product) and cloned accordingly into p A- 4a-V5 using (Xie et al., 2007; Subramanyam et al., 2009; Obermair et al., ␤ ␤ ␤ HindIII/EcoRV. For generating the viral vectors pHR- A- 4a, pHR- A- 2010), and differential functional modulation of calcium cur- ␤ ␤ ␤ 4b, and pHR- A- 4e, the following constructs were used as templates: rents (Helton and Horne, 2002). Nevertheless, the full comple- ␤ ␤ ␤ ␤ ␤ ␤ p A- 4a-V5, p A- 4b-V5, and p A- 4e-V5. The V5 tag was removed by ment of ␤ subunits expressed in the brain is still not completely digestion with BglII/SalI. To this end, a stop codon and a SalI site was Ј ␤ ␤ ␤ ␤ ␤ ␤ known and our understanding of their specific targeting proper- introduced to the 3 -end of the p A- a, p A- b, and p A- e coding ␤ ␤ ␤ ␤ ␤ ties or their specific involvement in particular neuronal functions sequence. Genes of interest (p A-4a,p A- 4b,orp A- 4e) were intro- is still rudimentary. duced with HindIII/SacI into the pENTRI (Invitrogen) vector and in- Moreover, calcium-channel-independent functions of ␤ sub- serted into a custom-built destination vector, pHR-␤A-DEST, using LR units have been reported. For example, several studies demon- Clonase II enzyme mixture (GATEWAY; Invitrogen). strated targeting of ␤ subunits into the nucleus and suggested a Lentiviral transduction and reconstitution of lethargic neurons. Lentivi- 4 ruses were produced by transient transfection of confluent 293T cells direct function in activity-dependent gene regulation (Colecraft ␤ ␤ with the lentiviral expression vectors containing pHR-p A- 4a, pHR- et al., 2002; Hibino et al., 2003; Subramanyam et al., 2009; Xu et ␤ ␤ ␤ ␤ p A- 4b, or pHR-p A- 4e in combination with psPAX2 (packaging al., 2011; Tadmouri et al., 2012). A lack of this nonconventional plasmid) and the pVSV (envelope plamid) using Metafectene (Biontex ␤ function of the 4 subunit might contribute to the ataxic pheno- Laboratories). The following day, medium was changed to neuronal plat- ␤ type in patients and mice with mutations in the 4 gene. How- ing medium and, after 24 and 48 h, supernatants containing the viruses ever, many aspects of the regulation and function of this new were harvested, sterile filtered (0.20 ␮m), aliquoted, and stored at signaling pathway are still controversial. Ϫ80°C. Cultured hippocampal neurons and cultured CGCs from lethar- Here, we report the discovery and characterization of a hith- gic mice were transfected with the lentiviral constructs immediately after ␤ ␤ plating for 4 h. Reconstituted neurons were used for experiments from erto unknown 4 splice variant. Our data demonstrate that 4e is ␤ DIV 1 on. the second most abundant 4 variant in cerebellum and that it can interact functionally with Ca 2.1 in tsA201 cells and cultured Transfection of hippocampal neurons. Expression plasmids were intro- V duced into neurons on day 6 using Lipofectamine 2000-mediated trans- neurons. The three known full-length ␤ variants show differen- 4 fection reagent (Invitrogen)
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