Examining the Structure of the Neuronal a4b2 nAChR Transmembrane Domain by Photoaffinity Labeling

Abstract

The ability to purify neuronal nAChRs in large quantities allows the use of photoaffinity labeling to study their structure. To characterize the structure of the transmembrane domain of the α4β2 nAChR, we used [3H]chlorpromazine, which has been used to identify amino acids in the Torpedo nAChR ion channel, and [3H]TDBzl-etomidate, which acts as a Torpedo nAChR positive allosteric modulator by binding at a novel site within the transmembrane domain at the interface between the γ and α subunits. In the presence of agonist, [3H]chlorpromazine and [3H]TDBzl-etomidate incorporated into α4 and β2 subunits with >70% of the labeling in the β2 subunit. [3H]chlorpromazine subunit labeling was inhibited (∼40%) by the non-competitive antagonist PCP (Fig 1).View Large Image | View Hi-Res Image | Download PowerPoint SlideWhen HPLC-purified EndoLys-C digests of [3H]chlorpromazine-labeled β2 subunit was sequenced, a PCP-inhibitable 3H release at cycle 6 was evident, consistent with labeling of β2Ser246 (M2-6), the position photolabeled by [3H]chlorpromazine in the Torpedo nAChR ion channel. Sequence analyses of HPLC-purified EndoLys-C/V8 protease digests of α4 and β2 subunits photolabeled by [3H]TDBzl-etomidate indicated the presence of multiple sites of 3H incorporation. Studies are in progress to identify amino acids labeled by [3H]TDBzl-Etomidate and [3H]chlorpromazine.