Mutagenesis primers were designed with PrimerX software (http://www.bioinformatics.org/primerx) and were 20–30 base pairs in length with a five to eight base pair overhang on the 3′ end. Appropriate melting temperatures were calculated with NEB Tm Calculator (https://tmcalculator.neb.com/main). The primers were synthesized by ThermoFisher and were diluted to 10 μM from 100 μM stocks for polymerase chain reaction (PCR). NEB Phusion High-Fidelity DNA polymerase (ThermoFisher) was used to amplify the template DNA (10–100 ng). PCR cycling conditions were as follows: initial denaturation at 98°C for 30 seconds; 35 cycles of denaturation at 98°C for 10 seconds, annealing at 48–68°C for 30 seconds, and extension at 72°C for 150 seconds; and final extension at 72°C for 10 minutes. One percent DMSO was added to the PCR reactions. After the PCR, the DNA was digested with DpnI (New England Biolabs, Ipswich, MA) overnight at 37°C. Purity of product was assessed on 1% agarose gels. Transformations were performed with 1–5 μl of PCR product for each mutant and 100 μl MAX Efficiency DH10B competent Escherichia coli using heat shock according to the manufacturer’s protocol. Successfully transformed cells gave visible colonies on either ampicillin or kanamycin selection plates. Mutant sequences were confirmed via sequencing using ABI 3730 Capillary Electrophoresis Genetic Analyzers (University of California, Davis DNA Sequencing Facility). Mutants were deemed functional if they produced at least 200 pA of current in response to 100 µM GABA and were sensitive to positive modulation by diazepam. The following mutants did not produce functional currents in our hands: α2T6′F, α2V2′A, β3T6′F, β3T6′W, and γ2T6′Y.

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