Tryptophan scanning mutagenesis reveals distortions in the helical structure of the δM4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

Channels (Austin). 2012 Mar-Apr;6(2):111-23. doi: 10.4161/chan.19540. Epub 2012 Mar 1.

Abstract

The lipid-protein interface is an important domain of the nicotinic acetylcholine receptor (nAChR) that has recently garnered increased relevance. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, there is still a need to gain insight into the mechanism by which lipid-protein interactions regulate the function and conformational transitions of the nAChR. In this study, we extended the tryptophan scanning mutagenesis (TrpScanM) approach to dissect secondary structure and monitor the conformational changes experienced by the δM4 transmembrane domain (TMD) of the Torpedo californica nAChR, and to identify which positions on this domain are potentially linked to the regulation of ion channel kinetics. The difference in oscillation patterns between the closed- and open-channel states suggests a substantial conformational change along this domain as a consequence of channel activation. Furthermore, TrpScanM revealed distortions along the helical structure of this TMD that are not present on current models of the nAChR. Our results show that a Thr-Pro motif at positions 462-463 markedly bends the helical structure of the TMD, consistent with the recent crystallographic structure of the GluCl Cys-loop receptor which reveals a highly bent TMD4 in each subunit. This Thr-Pro motif acts as a molecular hinge that delineates two gating blocks in the δM4 TMD. These results suggest a model in which a hinge-bending motion that tilts the helical structure is combined with a spring-like motion during transition between the closed- and open-channel states of the δM4 TMD.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Bungarotoxins / pharmacology
  • Ion Channel Gating / drug effects
  • Ion Channel Gating / genetics*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oocytes
  • Patch-Clamp Techniques
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Receptors, Nicotinic / chemistry*
  • Receptors, Nicotinic / genetics
  • Receptors, Nicotinic / metabolism
  • Structure-Activity Relationship
  • Torpedo
  • Tryptophan / chemistry
  • Xenopus laevis

Substances

  • Bungarotoxins
  • Receptors, Nicotinic
  • Tryptophan