Table 1.

Overview of methods for heteromeric TRP channels analyses.

Method	Application	Limitation	Example
AFM (atomic force microscopy) [36]	for stoichiometric determination in cells expressing tagged channels; allows for detection at the single channel level as it yields three dimensional structures in an Ångström (Å) range	not applicable for electrophysiological analysis	TRPP2/V4 [34]
Co-IP (co-immuno- precipitation) [37]	proof of interactions between TRP channel subunits in cells expressing tagged channels; allows for differentiation of heteromeric or homomeric assemblies	not applicable for structural and electrophysiological analysis	TRPV1/4 [38]
cross-linking in combination with mass spectrometry [39]	proof of interactions between TRP channel subunits and for structural and stoichiometric determination in native cells; provides a 3D-structure of interacting proteins, the distance between TRP channel subunits in the cross-linking reaction depends on the length of the crosslinker	not applicable for electrophysiological analysis	no applications made for heteromeric TRP channels yet
cryo-electron microscopy [40]	for structural analysis in cells overexpressing channels; allows for detection at the single channel level as it shows three dimensional structures, but sensitivity depends on the investigated protein’s electron irradiation and scattering	not applicable for electrophysiological analysis	no applications made for heteromeric TRP channels yet
FRET (Förster resonance energy transfer) [41]	proof of interactions between fluorescently tagged TRP channel subunits by live cell imaging; quantifies interaction over short distances (10–100 Å), yet fluorophores can alter binding properties	not applicable for structural, stoichiometric and electrophysiological analysis	TRPV1/4 [38]
molecular docking [31]	for prediction of structural domains that stabilize the TRP heteromer; method is based on acquired input data	not applicable for electrophysiological analysis	no applications made for heteromeric TRP channels yet
PLA (proximity ligation assay) [42]	proof of interactions between TRP channel subunits in native cells; quantifies interactions in a 30-nm range, allows quantification of heteromeric channel assemblies, but not at the single channel level; strongly depends on antibody specificity	not applicable for structural, stoichiometric and electrophysiological analysis	TRPV1/4 [27]
whole-cell patch-clamp recordings [43]	for electrophysiological analysis in native cells or cells expressing tagged channels; measures single ion channel current in a picoampere range	not applicable for structural and stoichiometric determinations	TRPC1/4 [44]
SC-SMD (single channel single molecule determinations) [45]	proof of interactions between TRP channel subunits and for structural and stoichiometric analysis in cells expressing tagged channels; detection at the single channel level as it yields three dimensional structures of proteins in a nanometer range	not applicable for electrophysiological analysis	no applications made for heteromeric TRP channels yet
x-ray crystallography [46]	for structural and stoichiometric analysis in cells overexpressing channels; detection at the single channel level as it yields three dimensional structures of molecules in an (Å) range, but is difficult to perform for transmembrane proteins	not applicable for electrophysiological analysis	no applications made for heteromeric TRP channels
Y2H (yeast two-hybrid technique) [47]	proof of interactions between TRP channel subunits in living native cells; detection of protein interactions by yeast reproduction, but not on a single channel level, difficult application for transmembrane proteins, and high rate of false positive results	not applicable for structural, stoichiometric and electrophysiological analysis	Drosophila retinal-specific TRP/L [29]