FIGURE 7.

GB2 intrasubunit FRET. A, three types of GB2, having Cerulean fixed at Asp⁷⁶⁹ and EYFP at one of different intracellular loops, paired with GB1a Ser⁹²³ stop mutant. No sign of response to GABA or when the agonist was coapplied with CGP7930. Plots of mean ± S.E. of individual recordings from GB1a Ser⁹²³ stop and GB2-i1EYFP-D769Cer (n = 8), GB2-i2EYFP-D769Cer (n = 7), or GB2-i3EYFP-D769Cer (n = 7) are shown. Application profile was the same as in Fig. 6. Hereafter, thin horizontal lines indicate the initial base-line levels. Schematic drawing of GB1a Ser⁹²³ stop and GB2-i3EYFP-D769Cer is shown on the right as a representative. B, three types of GB2, the same as shown in A, were paired with GB1a Ile⁸⁶⁰ stop mutant. No sign of response to GABA or when the agonist coapplied with CGP7930 except GB2-i3EYFP-D769Cer showing a subtle decrease while GABA was applied. Plots of mean ± S.E. of individual recordings from GB1a Ile⁸⁶⁰ stop and GB2-i1EYFP-D769Cer (n = 6) or GB2-i2EYFP-D769Cer (n = 4) or GB2-i3EYFP-D769Cer (n = 4) are shown. Application profile was the same as in Fig. 6. Schematic drawing of GB1a Ile⁸⁶⁰ stop and GB2-i3EYFP-D769Cer is shown on the right as a representative. C, mAChR M₁ construct for detection of intrasubunit structural change exhibited agonist-induced FRET decrease. Left, plots of mean ± S.E., of individual FRET traces from M₁-i3EYFP-Cer (n = 5). 10 μm oxotremorine M was applied for 60 s, washed for 60 s, and repeated again for checking the reproducibility. Right, schematic drawing of the M₁ construct. nF, net FRET.