Materials and Methods 

Unnatural Hydroxy Acid Suppression  

  Synthetic α‐hydroxy acids were conjugated to the dinucleotide dCA and ligated to 

truncated 74‐nucleotide tRNA as previously described.29 Typically, 25 ng of tRNA was injected 

per oocyte along with mRNA in a total volume of 50 nL/cell. mRNA was prepared by in vitro 

runoff transcription using the Ambion (Austin, TX) T7 mMessage mMachine kit. The site of 

interest was mutated to the amber stop codon by standard means, verified by sequencing 

through both strands. Mouse muscle embryonic nAChR in the pAMV vector was used. A total of 

4.0 ng of mRNA was injected in an α:β:γ:δ subunit ratio of 10:1:1:1. The ratio of tRNA to mRNA 

was typically 1:1.  In addition, the α‐subunits contain an HA epitope in the M3‐M4 cytoplasmic 

loop for Western blot studies. Control experiments show that this epitope does not detectably 

alter EC50. As a negative control for suppression, truncated 74‐nucleotide tRNA or truncated  tRNA ligated to dCA to give a 76mer was co injected with mRNA in the same manner as fully 

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than 10% of the experimental were excluded. The positive control for suppression involved wild‐

type recovery by co‐injection with 74‐nucleotide tRNA ligated to wild type amino acid. For 

suppression at multiple sites, the tRNA:mRNA ratio was increased to 2:1. 

Electrophysiology 

  Stage V‐VI oocytes of Xenopus laevis were employed. Oocyte recordings were made 12‐

48 h post‐injection in two‐electrode voltage clamp mode using the OpusXpress 6000A 

instrument (Axon Instruments, Union City, CA). Oocytes were superfused with a Ca2+‐free ND96 

solution at flow rates of 1 mL/min before application, (15 second) 4 mL/min during drug 

application, and 3 mL/min during wash. Holding potentials were ‐60 mV. Data were sampled at 

125 Hz and filtered at 50 Hz. Acetylcholine chloride was purchased from Sigma/Aldrich/RBI. 

Solutions ranging from 0.01 to 5000 mM were prepared in Ca2+‐free ND96 from a 1 M stock 

solution.  Dose‐response data were obtained for a minimum of 8 concentrations of agonists and 

for a minimum of five cells. Dose‐response relations were fitted to the Hill equation to 

determine EC50 and Hill coefficient values.  The dose‐response relations of individual oocytes  were examined and used to determine outliers.  The reported EC50 values are from the curve fit  of the averaged data.   

Total Internal Reflection Fluorescence Microscopy 

  Xenopus oocytes were prepared for single molecule TIRF microscopy as follows.  24‐48 

hours after injection of mRNA and tRNA, oocytes were incubated in a 0.5 ng/μL solution of 

biotin conjugated to α‐bungarotoxin (Molecular Probes, Invitrogen, Eugene, OR) for 4‐ 12 hours.  

This was followed by two wash steps with a 5 mg/mL bovine serum albumin solution and one 

wash with Ca2+‐free ND96.   Subsequently, oocytes were incubated in a 0.2 ng/μL solution 

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for 15‐ 30 minutes, followed by two additional wash steps each with BSA and Ca2+‐free ND96.  

The vitelline membrane of labeled oocytes was removed with forceps following a 5‐10 minute 

incubation in a hypertonic solution (220 mM sodium aspartate, 10 mM HEPES, 10 mM EDTA,       

2 mM MgCl2, pH 7.38). Devitellinized oocytes were transferred to an imaging chamber with a 

clean glass coverslip mounted on the microscope stage with the animal pole oriented toward 

the coverslip.  The TIRF microscope used to collect the data consists of a Melles‐Griot Argon (Ar) 

ion laser coupled to a fiber optic that extends to an Olympus TIRF illuminator adapted to a 

standard inverted IX‐71 Olympus microscope (Center Valley, PA).  A wavelength of 488 nm was 

used to excite and detect the Alexa488 fluorophores, by using the Z488 filter cube from Chroma 

Technology Corporation (Rockingham, VT).  A 100X 1.45 NA Olympus TIRF objective was used. 

Images were captured with a Photometrics Cascade front illuminated CCD camera from 

Princeton Instruments (Trenton, NJ).  Andor iQ from Andor Technology (South Windsor, CT) was 

used to acquire the data.  The data were subsequently processed and analyzed with ImageJ 

(National Institutes of Health).  Puncta were manually counted and densities from ≥6 images 

and ≥2 oocytes were compiled and averaged.  Expected current sizes were calculated as 

(#puncta/image frame area)*average oocyte area*single channel conductance. 

Acknowledgements 

We thank Dr. Rigo Pantoja for advice and assistance with TIRF measurements.  This work was 

supported by the NIH (NS 34407; NS 11756).  K. R. G. was partially supported by an NSF 

Graduate Research Fellowship. 

III‐17 3.6 References 

1. Corringer, P.J., Le Novere, N. & Changeux, J.P. Nicotinic receptors at the amino acid level. Annu Rev Pharmacol Toxicol 40, 431-58 (2000).

2. Grutter, T. & Changeux, J.P. Nicotinic receptors in wonderland. Trends Biochem Sci 26, 459-63 (2001).

3. Connolly, C.N. & Wafford, K.A. The Cys-loop superfamily of ligand-gated ion channels: the impact of receptor structure on function. Biochemical Society Transactions 32, 529- 534 (2004).

4. Lester, H.A., Dibas, M.I., Dahan, D.S., Leite, J.F. & Dougherty, D.A. Cys-loop receptors: new twists and turns. Trends in Neurosciences 27, 329-336 (2004).

5. Sine, S.M. & Engel, A.G. Recent advances in Cys-loop receptor structure and function.

Nature 440, 448-455 (2006).

6. Unwin, N. Refined structure of the nicotinic acetylcholine receptor at 4 angstrom resolution. Journal of Molecular Biology 346, 967-989 (2005).

7. Changeux, J.P. & Edelstein, S.J. Allosteric mechanisms of signal transduction. Science

308, 1424-1428 (2005).

8. Cui, Q. & Karplus, M. Allostery and cooperativity revisited. Protein Sci 17, 1295-307 (2008).

9. Grosman, C., Zhou, M. & Auerbach, A. Mapping the conformational wave of acetylcholine receptor channel gating. Nature 403, 773-6 (2000).

10. Lee, W.Y. & Sine, S.M. Principal pathway coupling agonist binding to channel gating in nicotinic receptors. Nature 438, 243-7 (2005).

11. Mukhtasimova, N., Free, C. & Sine, S.M. Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor. J Gen Physiol 126, 23-39 (2005). 12. Xiu, X., Hanek, A.P., Wang, J., Lester, H.A. & Dougherty, D.A. A unified view of the

role of electrostatic interactions in modulating the gating of Cys loop receptors. J Biol Chem 280, 41655-66 (2005).

13. Deechongkit, S., Dawson, P.E. & Kelly, J.W. Toward assessing the position-dependent contributions of backbone hydrogen bonding to beta-sheet folding thermodynamics employing amide-to-ester perturbations. J Am Chem Soc 126, 16762-71 (2004).

14. Zhong, W. et al. From ab initio quantum mechanics to molecular neurobiology: a cation- pi binding site in the nicotinic receptor. Proc Natl Acad Sci U S A 95, 12088-93 (1998). 15. Brejc, K. et al. Crystal structure of an ACh-binding protein reveals the ligand-binding

domain of nicotinic receptors. Nature 411, 269-76 (2001).

16. Sixma, T.K. & Smit, A.B. Acetylcholine binding protein (AChBP): a secreted glial protein that provides a high-resolution model for the extracellular domain of pentameric ligand-gated ion channels. Annu Rev Biophys Biomol Struct 32, 311-34 (2003).

17. Law, R.J., Henchman, R.H. & McCammon, J.A. A gating mechanism proposed from a simulation of a human alpha7 nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A

102, 6813-8 (2005).

18. McLaughlin, J.T., Fu, J., Sproul, A.D. & Rosenberg, R.L. Role of the outer beta-sheet in divalent cation modulation of alpha7 nicotinic receptors. Mol Pharmacol 70, 16-22 (2006).

19. Yi, M., Tjong, H. & Zhou, H.X. Spontaneous conformational change and toxin binding in alpha7 acetylcholine receptor: insight into channel activation and inhibition. Proc Natl

Acad Sci U S A 105, 8280-5 (2008).

20. England, P.M., Zhang, Y., Dougherty, D.A. & Lester, H.A. Backbone mutations in transmembrane domains of a ligand-gated ion channel: implications for the mechanism of gating. Cell 96, 89-98 (1999).

III‐18

21. Lu, T. et al. Probing ion permeation and gating in a K+ channel with backbone mutations in the selectivity filter. Nat Neurosci 4, 239-46 (2001).

22. Nagaoka, Y., Shang, L., Banerjee, A., Bayley, H. & Tucker, S.J. Peptide backbone mutagenesis of putative gating hinges in a potassium ion channel. Chembiochem 9, 1725- 8 (2008).

23. Gleitsman, K.R., Shanata, J.A., Frazier, S.J., Lester, H.A. & Dougherty, D.A. Long-range coupling in an allosteric receptor revealed by mutant cycle analysis. Biophys J 96, 3168- 78 (2009).

24. Pantoja, R., Rodriguez, E.A., Dibas, M.I., Dougherty, D.A. & Lester, H.A. Single- molecule imaging of a fluorescent unnatural amino acid incorporated into nicotinic receptors. Biophys J 96, 226-37 (2009).

25. Nirthanan, S. & Gwee, M.C. Three-finger alpha-neurotoxins and the nicotinic acetylcholine receptor, forty years on. J Pharmacol Sci 94, 1-17 (2004).

26. Miyazawa, A., Fujiyoshi, Y. & Unwin, N. Structure and gating mechanism of the acetylcholine receptor pore. Nature 423, 949-55 (2003).

27. Dellisanti, C.D., Yao, Y., Stroud, J.C., Wang, Z.Z. & Chen, L. Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution.

Nat Neurosci 10, 953-62 (2007).

28. Cheng, X., Wang, H., Grant, B., Sine, S.M. & McCammon, J.A. Targeted molecular dynamics study of C-loop closure and channel gating in nicotinic receptors. PLoS

Comput Biol 2, e134 (2006).

29. Nowak, M.W. et al. In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system. Methods Enzymol 293, 504-29 (1998).

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