Fig. 3.

Mapping of BTX-binding sites on GABA_ARs. HEK 293 cells transfected with cDNAs encoding indicated subunits were stained with BTX after (a, b, and d) or before (c) fixation and permeabilization. (a) Rat (r) GABA_AR β3 binds BTX, but rat GABA_AR β1, β2, α2, and γ2 do not. (b) Human (h) GABA_AR β3 binds BTX, but human GABA_AR β1 does not. (c) A GABA_AR β1 subunit that had been mutated to enhance its ability to form pentamers (β1*) failed to bind BTX. The subunit bore an amino-terminal “flag” epitope tag; antibody to the tag stained live cells, confirming that the subunit reached the cell surface. A similarly tagged wild-type β3 subunit did bind BTX. (d) Chimeric receptors containing the amino-terminal extracellular domain of GABA_AR β3 fused to the remainder of GABA_AR β1 bind BTX, but chimeras containing the amino-terminal extracellular domain of GABA_AR β1 fused to the remainder of GABA_AR β3 do not. (Insets) Portions of receptors derived from β3 (gray) and β1 (black); fusions are at amino acid 314 in the first and third chimeras and at amino acid 213 in the second and fourth chimera (11). (Scale bars, 5 μm.)