Table 1.

Affinity of modes at the αδ site

Agonist	Mode	E1obs	E1corr	Kd	ΔG
				µM	kcal/mol
TMA	H	0.7	2.6 × 10−3	25.8	−6.2
	L	0.004	1.5 × 10−5	4,470	−3.2
ACh	HHH	3.7	0.09	0.74	−8.3
	HH	0.9	0.02	3.4	−7.4
	H	0.2	5 × 10−3	13.4	−6.6
	L	0.01	2.5 × 10−4	268	−4.8
ACha	−	0.1	6.3 × 10−3	10.6	−6.7

All AChRs had αP197A and the αε knockout mutation εP121R (70 mV; [TMA] = 5 mM and [ACh] = 10 mM). Mode, H for high P_O and L for low P_O. E₁^obs, observed gating equilibrium constant with one agonist at the αδ site; E₁^corr, gating equilibrium constant after correction for the background mutations; K_d = (E₀^WT/E₁^corr), where E₀^WT = 6.7 × 10⁻⁸ at 70 mV (Nayak et al., 2012); ΔG, αδ-binding free energy (+0.59 lnK_d). The adult WT, αδ K_d values are: TMA (580 µM; −4.4 kcal/mol) and ACh (175 µM; −5.1 kcal/mol). Additional background mutations (total fold increase in E₀): TMA, εL269F + εS450A (271); ACh, βV266A (40), ACh^a, εL269F (16).

^aδW57A added to the background (WT value is K_d^ACh = 80 µM; −5.6 kcal/mol (Nayak et al., 2014).