Truncation of the M3/M4 loop does not perturb receptor assembly or function in oocytes.

(A) Cartoon of subunit construct design for α1 and β2 subunits shows locations for GFPuv fusion and loop truncation. A polypeptide linker (red line) connects GFPuv to the N-terminus of the α1 subunit. (B) Expression of GFPuv-α1/β2 increases receptor homogeneity relative to α1-EGFP/β2. However, an increase in cleaved GFPuv fluorescence signal is observed (arrow). (C) Replacement of the native M3/M4 loop in both subunits with a tri-peptide induces a right shift in the elution profile, consistent with a smaller hydrodynamic radius. Absolute fluorescence intensities are shown on the same scale. The FSEC traces shown in (B) and (C) were obtained from the same batch of oocytes. (D) Two-electrode voltage clamp of GFPuv-α1-LT/β2-LT demonstrating that the receptor retains gating activity and sensitivity to zinc.