Generating Cav3.1 channel constructs

Wild-type human Cav3.1b cDNA was donated by T. Snutch (Vancouver, BC, Canada) (GenBank accession number AF134986.1), HA-Cav3.1 and Kir2.1 cDNA from E. Bourinet (Institut de Génomique Fonctionnelle, Montpellier, FR), and CaM cDNAs by J. Adelman (Vollum Institute, OR, USA). GFP-Cav3.1 and GFP-αCaMKII were subcloned into a GFP mammalian vector (pCMV6-AN-GFP) (OriGene Technologies, Rockville, MD, USA) and mKate-CaM prepared from a pmKate2-N mammalian expression vector (Axxora, Farmingdale, NY, USA). CaMKIIN was amplified from a mouse hippocampal cDNA library (RIKEN) and cloned into the pcDNA3.1 vector (Addgene, Cambridge, MA, USA) [24 (link)]. To create a deletion of the C-terminus, PCR with specific primers was performed on Cav3.1 outside of the C-terminus region, followed by sequencing and subcloning into the pCMV6-AN-GFP vector (OriGene Technologies, Rockville, MD, USA) or the pcDNA3.1 vector (Addgene, Cambridge, MA, USA) and sequenced for final confirmation. The GFP and mKate tags were attached to the N-terminal regions of target proteins. To create the Cav3.1 pore mutant, a single point mutation PCR was performed on E354K in the pore region with the primer sequences: 5′-ACAGGTCGTTGAGCCGC-3′, followed by sequencing and subcloning into the pcDNA3.1 vector (Addgene, Cambridge, MA, USA) and sequenced for final confirmation.