Table 3.

Functional effects of mutating αM4–αM1/αM3 interfacial aromatics

Mutation	Dose responsea
	TMD α-helix	EC50 (μm)	Hill slope	n
WT		7.61 ± 1.25	1.70 ± 0.47	50
F225A	αM1	0.71 ± 0.12b	1.57 ± 0.39	8
F227A	αM1	6.51 ± 1.47	1.43 ± 0.16	8
F233A	αM1	2.63 ± 0.31b	1.63 ± 0.20	9
Y234A	αM1	No currentc	No currentc	8
Y234L	αM1	5.79 ± 0.72	2.20 ± 0.33	8
Y234F	αM1	8.43 ± 2.03	1.66 ± 0.27	8
Y277A	αM3	11.2 ± 1.97b	1.78 ± 0.58	8
F280A	αM3	4.25 ± 1.00b	1.56 ± 0.29	8
F284A	αM3	4.80 ± 0.75b	1.33 ± 0.21	9
F414A	αM4	4.47 ± 0.82b	1.76 ± 0.34	10
F225A+F280A	αM1+αM3	No currentc		8
F225A+F284A	αM1+αM3	No currentc		8
F280A+F284A	αM3+αM3	8.42 ± 2.98	1.45 ± 0.08	9
F225A+F280A+F284A	αM1+αM3+αM3	No currentc		8
F233A+F414A	αM1+αM4	1.08 ± 0.10b	2.21 ± 0.36	8

^a Measurements were performed 1–4 days after cRNA injection (V_hold ranging from −20 to −80 mV). Error values are represented as standard deviations.

^b p < 0.001 relative to the WT via one-way ANOVA followed by Dunnett's post hoc test.

^c No significant current was observed up to 4 days after cRNA injection.