Cell Calcium 46 (2009) 282–292 Contents lists available at ScienceDirect Cell Calcium journal homepage: www.elsevier.com/locate/ceca The ␣2␦ subunit augments functional expression and modifies the pharmacology of CaV1.3 L-type channels Arturo Andrade a,1, Alejandro Sandoval b,c, Ricardo González-Ramírez b, Diane Lipscombe d, Kevin P. Campbell e, Ricardo Felix b,∗ a Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies of the National Polytechnic Institute, Cinvestav-IPN, Mexico City, Mexico b Department of Cell Biology, Cinvestav-IPN, Mexico City, Mexico c School of Medicine FES Iztacala, National Autonomous University of Mexico, Tlalnepantla, Mexico d Department of Neuroscience, Brown University, Providence, RI, USA e Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA article info abstract 2+ Article history: The auxiliary CaV␣2␦-1 subunit is an important component of voltage-gated Ca (CaV) channel complexes Received 12 May 2009 in many tissues and of great interest as a drug target. Nevertheless, its exact role in specific cell functions Received in revised form 27 August 2009 is still unknown. This is particularly important in the case of the neuronal L-type CaV channels where Accepted 28 August 2009 these proteins play a key role in the secretion of neurotransmitters and hormones, gene expression, and Available online 30 September 2009 the activation of other ion channels. Therefore, using a combined approach of patch-clamp recordings and molecular biology, we studied the role of the CaV␣2␦-1 subunit on the functional expression and Keywords: the pharmacology of recombinant L-type Ca 1.3 channels in HEK-293 cells. Co-expression of Ca ␣ ␦- Ca2+ channels V V 2 ␣ ␤ Ca ␣ ␦ subunit 1 significantly increased macroscopic currents and conferred the CaV1.3 1/CaV 3 channels sensitivity V 2 ␣ ␦ L-type calcium channels to the antiepileptic/analgesic drugs gabapentin and AdGABA. In contrast, CaV 2 -1 subunits harboring Gabapentin point mutations in N-glycosylation consensus sequences or the proteolytic site as well as in conserved cysteines in the transmembrane ␦ domain of the protein, reduced functionality in terms of enhancement of CaV1.3␣1/CaV␤3 currents. In addition, co-expression of the ␦ domain drastically inhibited macroscopic currents through recombinant CaV1.3 channels possibly by affecting channel synthesis. Together these results provide several lines of evidence that the CaV␣2␦-1 auxiliary subunit may interact with CaV1.3 channels and regulate their functional expression. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Four mammalian genes encode L-type CaV channel ␣1 sub- units: CaV1.1 (also known as ␣1S), CaV1.2 (␣1C), CaV1.3 (␣1D), 2+ L-type voltage-gated Ca (CaV) channels are expressed in and CaV1.4 (␣1F). CaV1.1␣1 and CaV1.4␣1 subunits are enriched many different cell types and tissues. In myocytes they are in skeletal muscle and retina, respectively, whereas CaV1.2␣1 and essential for excitation–contraction coupling, whereas in neurons CaV1.3␣1 subunits are expressed in many cells including neurons and endocrine cells they regulate neurotransmitter and hormone and endocrine cells [1,2].CaV1.2 and CaV1.3 channels underlie the release, gene expression, and the activity of other ion channels [1,2]. majority of L-type currents in neuronal, endocrine, and cardio- Biochemical evidence suggests that L-type CaV channels are com- vascular systems. Ca 1.3 channels have relatively low activation ␣ V prised of five subunits. A principal transmembrane 1 subunit is thresholds, are less sensitive to dihydropyridine antagonists, and predicted to associate with a disulfide-linked ␣ ␦ dimer, an intra- 2 activate with fast kinetics when compared to CaV1.2 L-type cur- cellular ␤ subunit, and a transmembrane ␥ subunit [2,3]. The ␣ 1 rents [1]. There is growing interest in CaV1.2 and CaV1.3 channels subunit is the ion-conducting element in the channel protein com- because of their link to neurodegenerative disorders including plex [2,3]. autism, bipolar disorder, and Parkinson’s disease [4–8]. Under- standing the mechanisms that regulate L-type calcium channel activity and surface expression is of major importance. ∗ 2+ Corresponding author at: Departamento de Biología Celular, Cinvestav-IPN, In the central nervous system, L-type Ca channels (CaV1.2 Avenida IPN #2508, Colonia Zacatenco, México D.F., CP 07300, Mexico. and CaV1.3) apparently do not support synaptic transmission, but Tel.: +52 55 50 61 39 88; fax: +52 55 50 61 33 93. seem to play an important role in the excitation–transcription cou- E-mail address: rfelix@fisio.cinvestav.mx (R. Felix). 2+ 1 Present address: Department of Neuroscience, Brown University, Providence, pling. It has been reported that Ca entry via postsynaptic L-type RI, USA. channels activates transcription factors pCREB [9,10] and NFATc4 0143-4160/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2009.08.006 A. Andrade et al. / Cell Calcium 46 (2009) 282–292 283 [11]. Phosphorylation of CREB, acting in conjunction with nuclear subunit, which was made by assembling a PCR fragment after the translocation and co-activator proteins promotes transcription of CaV␣2␦-1b signal sequence [22], and the CD8 surface marker were multiple genes [12,13]. Likewise, CaV1.3 channels play a unique role cloned in the pcDNA3 expression plasmid (Invitrogen). The PERK for hearing. Inner hair cells (IHCs), the primary sensory receptors of mutant constructs [34] were cloned into the pcDNAI/Amp vector the mature mammalian cochlea, are responsible for relaying acous- (Invitrogen). tic information transduced by mechano-sensitive channels to the central nervous system via afferent auditory nerve fibres. This is 2.3. Site-directed mutagenesis 2+ driven by Ca entering IHCs through L-type channels of the CaV1.3 class [14] activated in response to depolarizing receptor potentials The Pfu DNA polymerase was used in all PCRs to generate the initiated by hair bundle deflection. In addition, it has been reported CaV␣2␦-1 mutations and all constructs were verified by sequencing. that CaV1.3 L-type channels are important for the sinoatrial node The glycosylation and proteolysis mutant constructs were gener- ␣ function. Using gene-targeted deletion of the CaV1.3 1 subunit, ated following standard procedures in use in the laboratory [27,33]. Zhang et al. [15] found a decrease in the rate of firing associated The CaV␣2␦-1 subunit in which all cysteines in the extracellular with a diminished rate of diastolic depolarization. Last, CaV1.3 L- region of ␦ were mutated to serines, was made using the pIRES- type channels are expressed at high density and play a role in the hrGFP-1a-based construct encoding the rat CaV␣2␦-1 [27] as a control of hormone secretion in a variety of endocrine cells includ- template and standard PCR techniques. In all cases, the mutations ␤ ing pancreatic - and adrenal chromaffin cells where they control were introduced with 40-mer synthetic oligonucleotides using the insulin and catecholamine release [2,16]. Quick-Change XL II mutagenesis kit (Stratagene). In contrast to the functional studies, the molecular architec- ture of the L-type CaV1.3 channels is largely unknown. Though 2.4. Cell culture and transfection a role for the CaV␤ subunits in determining a functional inter- action with protein kinase A [17] and arachidonic acid [18] has been reported, little is known regarding the role of the Ca2+ chan- Human embryonic kidney (HEK)-293 cells (American Type Cul- ture Collection, ATCC; Manassas, VA) were grown in Dulbecco’s nel auxiliary subunits in the regulation of the CaV1.3 channel activity. On the other hand, it has been reported that Ca ␣ ␦ pro- modified Eagle’s medium (DMEM)-high glucose supplemented V 2 l motes surface expression of different Ca ␣ subunits and it speeds with 10% horse serum, 2 mM -glutamine, 110 mg/l sodium pyru- V 1 ␮ ◦ channel activation and inactivation kinetics [19–30]. However, vate, 100 U/ml penicillin and 100 g/ml streptomycin at 37 Cina 5% CO2/95% air humidified atmosphere. After splitting on the pre- investigations of the CaV␣2␦ subunit effects on CaV1.3 channels are vious day and seeding at ∼60% confluence, cells were transfected lacking. Here, we show that CaV␣2␦ augments surface expression of with the cDNA clones mentioned earlier using the Lipofectamine recombinant CaV1.3 channels in HEK-293cells. We also show that Plus reagent (Invitrogen) according to the manufacturer’s instruc- co-expression of the CaV␣2␦ subunit renders the CaV1.3 channels sensitive to antiepileptic/analgesic gabapentinoid drugs (GBP and tions. After DNA–lipid complexes were allowed to form, cells were ␣ ␮ AdGABA) and that co-expression of the transmembrane ␦ domain transfected with either cDNAs encoding CaV1.3 1 alone (1 g 2+ DNA/35-mm culture dish) or co-transfected with cDNAs for CaV␤, alone, together with the CaV1.3/CaV␤3 Ca channel combination CaV␣2␦-1 and CaV␦ subunits in a 1:1 molar ratio (except where in absence or presence of CaV␣2␦, results in an important inhibition of the whole-cell current. indicated). The HEK-293 cell line stably expressing the CaV3.2 chan- nel (GenBank accession number AF051946 [35]) was grown as 2. Materials and methods previously described [36]. Likewise, the RIN-m5F insulinoma ␤- cells (ATCC) were cultured in RPMI-1620 medium supplemented 2.1. Materials with 10% fetal bovine serum, 100 U/ml penicillin and 100 ␮g/ml streptomycin. Both cell lines were plated on poly-l-lysine (0.05%)- Chloroquine (C-6628) and Fillipin III (F-4767) were obtained precoated glass coverslips placed into 35-mm culture plates, and from Sigma–Aldrich (St.