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The Journal of Neuroscience, February 11, 2009, 29(6):1626-1635; doi:10.1523/JNEUROSCI.4121-08.2009

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Cellular/Molecular
Probing Protein Packing Surrounding the Residues in and Flanking the Nicotinic Acetylcholine Receptor M2M3 Loop

Roger Ernest Wiltfong¹ and Michaela Jansen^1,2

¹Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, and ²Department of Cell Physiology and Molecular Biophyics, Texas Tech University Health Sciences Center, Lubbock, Texas 79420-6551

Correspondence should be addressed to Dr. Michaela Jansen, Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, 3601 4th Street, STOP 6551, Lubbock, TX 79430-6551. Email: Michaela.Jansen{at}TTUHSC.edu

Nicotinic acetylcholine receptors (nAChR) are cation-selective, ligand-gated ion channels of the cysteine (Cys)-loop gene superfamily. The recent crystal structure of a bacterial homolog from Erwinia chrysanthemi (ELIC) agrees with previous structures of the N-terminal domain of AChBP (acetylcholine-binding protein) and of the electron-microscopy-derived Torpedo nAChR structure. However, the ELIC transmembrane domain is significantly more tightly packed than the corresponding region of the Torpedo nAChR. We investigated the tightness of protein packing surrounding the extracellular end of the M2 transmembrane segment and around the loop connecting the M2 and M3 segments using the substituted cysteine accessibility method. The M2 20' to 27' residues were highly water accessible and the variation in reaction rates were consistent with this region being -helical. At all positions tested, the presence of ACh changed methanethiosulfonate ethylammonium (MTSEA) modification rates by <10-fold. In the presence of ACh, reaction rates for residues in the last extracellular -helical turn of M2 and in the M2M3 loop increased, whereas rates in the penultimate -helical turn of M2 decreased. Only three of eight M2M3 loop residues were accessible to MTSEA in both the presence and absence of ACh. We infer that the protein packing around the M2M3 loop is tight, consistent with its location at the interdomain interface where it is involved in the transduction of ligand binding in the extracellular domain to gating in the transmembrane domain. Our data indicate that the Torpedo nAChR transmembrane domain structure is a better model than the ELIC structure for eukaryotic Cys-loop receptors.

Key words: acetylcholine; nicotine; serotonin; Cys loop; ion channel; gating

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Received Aug. 28, 2008; revised Jan. 6, 2009; accepted Jan. 8, 2009.

Correspondence should be addressed to Dr. Michaela Jansen, Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, 3601 4th Street, STOP 6551, Lubbock, TX 79430-6551. Email: Michaela.Jansen{at}TTUHSC.edu

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