Identifying TRP Channel Subunit Stoichiometry Using Combined Single Channel Single Molecule Determinations (SC-SMD)

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 [1–3]. This was suggested by dominant negative effects of nonfunctional TRP fragments or loss-of-function mutations, as well as by the generation of other properties by the coexpression of different subunits. Thus, electrophysiological experiments using defined expression of pore proteins provide information on the stoichiometry within ion channel complexes. Experimental approaches to analyze stoichiometry include both the independent coexpression of potential subunits and the coexpression of combinations of subunits. Solid understanding of the functional properties of individual subunits is a prerequisite for such strategies. One classical strategy to confirm interactions between TRP channel proteins is to test whether loss-of-function mutations of a particular species (e.g., proteins that lack a functional pore structure) are able to prevent currents through the potential heteromerization partner. 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 use of mutant channels fused to fluorescent proteins is helpful to test and confirm proper expression and targeting of the proteins. This allows for subunit stoichiometry determination by Förster resonance energy transfer (FRET) [4]. Recent advances in imaging and the use of very sensitive cameras in combination with surface-selective procedures, such as total internal reflection fluorescence microscopy (TIRFM), have facilitated the observation of single molecules [5,6]. There are several types of single molecule determinations: in this report we focus on identifying single molecules based on photobleaching steps. Single molecule detection (SMD) studies, however, provide no information about the functionality of the protein observed. On the other hand, patch clamp (PC) is a powerful technique that allows the observation in real time of single channel kinetics [7]. Unfortunately, PC electrophysiology cannot provide any information about the molecular identity of the channels studied. In an attempt to overcome the limitations imposed by SMD or PC alone, we developed a novel method, which combines both into an integrated procedure to simultaneously detect channel stoichiometry and assess single-channel gating [8]. We have named this new method single channel single molecule detection (SC-SMD) system. As an example of the power of this new method, we have recently obtained stoichiometric information on six members of the transient receptor potential canonical (TRPC) family of cation channels, a task that using biochemistry and crystallographic studies would have taken several years, with SC-SMD was accomplished in several weeks [8].