Panap plasmid

The tsA-201 cells were cultured, maintained and transfected as previously reported (Dai et al., 2016 (link)). The mBACE1 construct was obtained from Addgene. The fluorescent L10 and S15 constructs were made in the lab of Dr. Byung-Chang Suh at Daegu Gyeongbuk Institute of Science and Technology (Myeong et al., 2021 (link)). The caveolin 1 construct was from Suzanne F. Scarlata (Worcester Polytechnic Institute, Worcester, MA). Point mutations were made using Quickchange II XL Site-Directed Mutagenesis kit (Agilent Technologies). The human KCNQ2 and human KCNQ3 constructs were fused with sequence of enhanced YFP at the carboxyl-terminal end. The sequences of the DNA constructs were confirmed by fluorescence-based DNA sequencing (Genewiz). The pANAP plasmid (Addgene) contained the orthogonal tRNA/aminoacyl-tRNA synthetase specific to L-Anap. L-Anap (AsisChem) was dissolved in ethanol as a 10-mM stock, stored at −20°C, and diluted 500-fold into the culture medium. 2-Bromopalmitate (2-BP) was delivered with fatty acid–free BSA (A6003; Sigma-Aldrich) at a stock concentration of 1 mM albumin with 5 mM 2-BP, which was diluted 50-fold into the culture medium, generating a final concentration of 0.1 mM 2-BP.

All VSP mutants were constructed from WT Ci-VSP in pSD64TF (8 (link)). To generate fluorescence-tagged Ci-VSP constructs, mCherry was fused at the C-terminal via a glycine-serine linker. Amino acid mutations were generated by using PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.) and were confirmed by DNA sequencing. Mouse K_ir3.2d (GIRK2d) (56 (link)) was a kind gift from Yoshihisa Kurachi (Osaka University, Osaka, Japan). Bovine G protein β and γ (Gβ and Gγ) (57 (link)) were kindly provided by Toshihide Nukada (retired). The pAnap plasmid (Addgene ID: 48696) (58 (link)) encoding transfer RNA (tRNA) and aminoacyl-tRNA synthetase was obtained from Addgene and was identical to that used in our previous studies (17 (link), 21 (link)).