ABSTRACT
The assembly of transient receptor potential (TRP) proteins into multimers and the existence of heteromeric TRP pores of defined subunit composition were recognized early on in TRP channel research. The dominant negative suppression of channel function and transfer of mutant properties to a heteromeric channel complex allow the determination of subunit stoichiometry and testing of certain concepts relating to pore properties and stoichiometry. The chapter focus on the reagents, procedures, and description of a typical single channel single molecule detection (SC-SMD) experiment, including cell culture and transfection of the plasmids of interest, patch clamp pipette preparation and mounting of the SC-SMD device. Even though Single molecule detection can provide information about the stoichiometry of the channel of interest, it does not provide any information about the functional state of the channel being studied. SC-SMD provides very high signal-to-noise ratios because excitation only occurs at the tip of the pipette.
