Biochemical methods to study the interactions between integrins and ion channels.

Protein-protein interactions between integrins and ion channels consist in a complicated bidirectional talk, not yet understood in detail, which triggers a downstream signaling network. Such a coordinated process occurs in discrete, localized microcompartments and involves different membrane and cytoplasmic proteins. Since the early nineties, when the first functional association between integrins and ion channels was characterized, the number of similar examples is constantly increasing. Identifying the components of this pathway has general importance for cell physiology and will eventually lead to fully understand the role of ion channels in the physiological processes typically controlled by integrin receptors, such as cell adhesion, migration and proliferation. Here, we detail the main experimental methods currently available to study these processes and discuss their advantages and disadvantages. Biochemical copurification and genetic interaction studies, as well as high-throughput screening, can be performed to initially identify the interacting proteins. Successively, in vitro binding assays such as pull-down and immunoprecipitation-based techniques allow to verify and better characterize these partnerships, possibly in combination with mass spectrometry methods. When transient interactions are involved, more sophisticated techniques, such as photoaffinity labelingprocedures, are necessary to detect the multiprotein complexes by having them covalently bound together as they interact. To provide even more thorough analyses of the formation, function and composition of protein complexes, other technologies such as confocal microscopy, fluorescence resonance energy transfer microscopy and site directed mutagenesis (possibly in murine models) have to be performed. The progressive accumulation of data defining novel protein-protein interactions has been considerably accelerated by the identification of specific sequence motifs that regulate integrin binding to other proteins as well as integrin recognition sequences in the ligand. Moreover, the availability of protein tagging strategies and the increased sensitivity of mass spectrometry-based methods for protein identification have also contributed important tools. In the near future, the coupling of traditional techniques with proteomic approaches is likely to offer invaluable help in unraveling integrin-ion channel interactions, thus elucidating the biological implication of these complexes.