Determining stoichiometry of ion channel complexes using single subunit counting

Abstract Most membrane proteins, and ion channels in particular, assemble to multimeric biological complexes. This starts with the quarternary structure and continues with the recruitment of auxiliary subunits and oligomerization or clustering of the complexes. While the quarternary structure is best determined by atomic-scale structures, stoichiometry of heteromers and dynamic changes in the assembly cannot necessarily be investigated with structural methods. Here, single subunit counting has proven a powerful method to study the composition of these complexes. Single subunit counting uses the irreversible photodestruction of fluorescent tags as means to directly count a labeled subunit and thereby derive the composition of the assemblies. In this chapter, we discuss single subunit counting and its limitations. We present alternative methods and provide a detailed protocol for recording and analysis of single subunit counting data.