ABSTRACT
Most TRPs are Ca2+-permeable nonselective cation channels, generally believed to regulate intracellular Ca2+ ([Ca2+]i) levels. [Ca2+]i is usually low at rest (approximately
19.1 Introduction .................................................................................................. 411 19.2 Studying Intracellular Localization of TRP Channels ................................. 412
19.2.1 Fluorescent Fusion Proteins .............................................................. 412 19.2.2 Immunostaining and Cellular Fractionation Studies ........................ 414
19.3 Studying Channel Properties of Intracellular TRPs ..................................... 415 19.3.1 Electrophysiological Characterization of Intracellular TRP
Channels at the Plasma Membrane ................................................... 415 19.3.2 Endolysosome Patch Clamp ............................................................. 416 19.3.3 Planar Lipid Bilayer Studies ............................................................. 417 19.3.4 Monitoring Ca2+ Release from Intracellular Ca2+ Stores .................. 418
19.4 Conclusion .................................................................................................... 419 Acknowledgments .................................................................................................. 419 References .............................................................................................................. 419
100 nM) but may increase 10-to 100-fold to the micromolar range upon cellular stimulation.5 Ca2+ is a ubiquitous second messenger and is reportedly involved in almost every single biological process.5 Therefore, the Ca2+ Ÿux pathways, i.e., channels or transporters, must be tightly regulated to ensure the functional speci—city of each cellular stimulus.5 Despite the apparent importance of TRPs, their activation mechanisms are largely unknown. Nevertheless, TRP channel dysfunction can cause human diseases, such as polycystic kidney disease and mucolipidosis type IV (MLIV), which result from mutations of human TRPP2 and TRPML1 genes, respectively.6,7 One source for [Ca2+]i increase is the Ca2+ in extracellular space, which is approximately 20,000 times (2 mM) more concentrated than resting [Ca2+] i. Hence, plasma membrane TRPs are natural candidates to mediate Ca2+ inŸux.3 However, because most intracellular organelles also contain Ca2+ (at concentrations from hundreds of micromolar to millimolar),1 activation of TRPs localized in these compartments could result in elevation of [Ca2+]i. The best studied example of Ca2+ release from intracellular membranes is the phospholipase C (PLC)-inositol-1,4,5trisphosphate receptor (IP3R) system.8 Receptor-mediated activation of PLC leads to the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into IP3, which binds to the IP3R in the membranes of endoplasmic reticulum (ER), and releases Ca2+
into the cytoplasm.8 Unlike plasma membrane TRPs, the activation mechanisms of TRPs localized in intracellular organelles1 are largely unknown. Many plasma membrane TRPs are activated or regulated by protein kinases or lipid signaling.3 It is not clear whether intracellular TRPs are also regulated by these mechanisms and, if so, whether they exhibit electrophysiological characteristics similar to plasma membrane TRPs. Although Ca2+ release from intracellular compartments is important for signal transduction and membrane traf—cking, our knowledge of ion channels involved in Ca2+ release remains very limited.1 Because TRPs are Ca2+ permeable, and some localize to intracellular organelles, they are natural candidates for Ca2+ release from intracellular organelles. In this chapter, we will discuss techniques employed to identify and characterize TRPs localized in intracellular compartments.
