Automated analysis of the architecture of receptors, imaged by atomic force microscopy

Fast neurotransmission involves the operation of ionotropic receptors, which are multi-subunit proteins that respond to activation by opening an integral ion channel. Examples of such channels include the GABA(A) receptor, the 5-HT(3) receptor and the P2X receptor for ATP. These receptors contain more than one type of subunit, although the exact subunit stoichiometry and arrangement around the receptor rosette is often unknown. We are using atomic force microscopy (AFM) of purified receptors to address these issues. Measurement of the molecular volume of the receptor permits the determination of the number of subunits that it contains. Furthermore, analysis of the geometry of complexes between receptors and subunit-specific antibodies reveals the subunit arrangement. Our AFM-based approach has so far been dependent on manual data processing, which is both time-consuming and prone to operator bias. In this study, we set out to develop a novel method capable of automatic segmentation and quantitative analysis of both single receptor particles and receptor-antibody complexes. The method was validated using images of wild type and mutant forms of the P2X(6) receptor. We suggest that the automated method will greatly facilitate further progress in the use of AFM for the determination of receptor and multi-protein architecture.