Generating Na_V_Ab Mutant Plasmids

The pFastBac-Na_VAbΔ28 plasmid carrying a C-terminal 28-residue truncation (residues 1–239) of Na_VAb gene (Gamal El-Din et al., 2019 (link)) was used as a template for site-directed mutagenesis. To generate a desired plasmid of a Na_VAb mutant, overlapping oligonucleotide primers with a codon changed to a specific mutation were synthesized (Integrated DNA Technologies) and used for site-directed mutagenesis PCR with PfuUltra II Fusion HotStart DNA Polymerase (Agilent). The PCR products were treated with DpnI (New England Biolabs) and transformed into E. coli GC10 competent cells (Genesee Scientific). Plasmid DNAs were isolated from transformed colonies using the QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer’s protocol and sequenced to confirm the mutation. Plasmids containing Na_VAbΔ28 with the IEM mutation were used to transform E. coli DH10Bac competent cells for bacmid production, and baculoviruses were prepared with Sf9 insect cells using the Bac-to-Bac protocol according to the manufacturer (Life Technologies). All baculoviruses were tested for protein expression in Hi5 insect cells and the expression levels were confirmed by Western blot analysis to be consistent for all mutants. The same batches of baculoviruses were used for both electrophysiology and protein purification experiments.