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Zhu MX, editor. TRP Channels. Boca Raton (FL): CRC Press/Taylor & Francis; 2011.

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TRP Channels.

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Chapter 19Studying TRP Channels in Intracellular Membranes

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19.1. INTRODUCTION

Advances in modern cell biology and physiological techniques have dramatically improved our understanding of basic cellular functions. Together with classical genetic and biochemical approaches, these technical advances have allowed us to uncover the novel functions of a variety of proteins inside the cell. For example, recent studies have revealed intracellular functions of several TRP proteins (reviewed in Ref. 1), a family of cation non-selective ion channels that were initially thought to operate exclusively at the plasma membrane to regulate the transmembrane flux of Na+, K+, Ca2+, and Mg2+.2,3 Initially discovered in the Drosophila melanogaster photoreceptor, TRPs are also found in vertebrates, widely expressed in most tissues and cell types.2 The TRP superfamily can be divided into six subfamilies: canonical (TRPC), vanilloid (TRPV), melastatin (TRPM), polycystin (TRPP), mucolipin (TRPML), and ankyrin transmembrane proteins (TRPA). Currently, the most well-defined TRP functions are serving as cellular sensors for detecting an array of environmental stimuli including temperature, mechanical forces, and pain.2–4 However, the list of nonsensory functions for TRPs also has expanded rapidly in recent years.3

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 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+ flux pathways, i.e., channels or transporters, must be tightly regulated to ensure the functional specificity 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+ influx.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-1,4,5-trisphosphate 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 trafficking, 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.

19.2. STUDYING INTRACELLULAR LOCALIZATION OF TRP CHANNELS

Subcellular localization studies have revealed that many TRPs are localized on the membranes of intercellular organelles.1 Among these, TRPV2, TRPM2, TRPML1, TRPML2, and TRPML3 are found in endosomal and/or lysosomal (endolysosomal, collectively) compartments.1 Two strategies are commonly used to study intracellular localization of TRPs: fluorescent fusion proteins and antibody-mediated immunochemical approaches.

19.2.1. Fluorescent Fusion Proteins

Reporter genes usually encode fluorescent proteins (FP) that can emit fluorescence at specific wavelengths.9 As such, they are often fused in-frame to the target proteins to monitor protein localization in live cells.9 There are a number of these reporter proteins, each emitting at specific wavelengths; for example, red (RFP/ mCherry/DsRed), green (GFP), and yellow (YFP). A combination of reporter genes allows monitoring of multiple proteins simultaneously. Studies using overexpressed TRPML proteins fused with various reporter genes have revealed that TRPMLs primarily localize to a population of membrane-bound vesicles along the endosomal/ lysosomal pathways in a variety of host cells.10–14 GFP-fused TRPML (TRPML1-GFP) is mainly colocalized with Lamp-1, a late endosome and lysosome marker (Figure 19.1). Both Anti-Lamp1 and Lamp1-FP fusion proteins can be used to define late endosomes and lysosomes (LELs). The colocalization index of TRPML1 and Lamp-1 is more than 80%.15 Thus, TRPML1 is a TRP channel specifically localized in the LEL. Using a similar approach, TRPML2, TRPML3, and TRPM2 are also found in Lamp-1-labeled compartments.11,13,14,16–18 TRPML3 and TRPV2 also are present in compartments that are positive for Rab5, an early endosome-specific small GTPase,13,14,19 TRPML2 is also present in Arf6-positive recycling endosomes.17 By tagging TRPMLs to different reporter genes, recent studies suggest that although TRPMLs are localized in endolysosomes, the colocalization between them is rather limited.13,20

FIGURE 19.1. (See color insert.

FIGURE 19.1

(See color insert.) While wild-type TRPML1 is localized mainly in the late endosomal and lysosomal compartments, gain of function proline-substituted TRPML1 channels are present at the plasma membrane. (a) Confocal images showing the colocalization of (more...)

Considering the heterologous nature and overexpression of the FP-fused TRPs (FP-TRPs), results obtained from fusion protein studies should be interpreted with caution and, ideally, verified by other independent approaches (see below). For example, substantial overexpression of TRPs might lead to accumulation of the proteins in intracellular compartments involved in their biosynthetic pathway, i.e., the ER and Golgi. In addition, TRPs are localized differently in different cell types. TRPV2-GFP is localized in the ER of pancreatic beta-cells,21,22 but in J774 macrophage cells and HEK293 cells, TRPV2 is localized in early endosomes.19,23 Thus, the intracellular localization pattern of TRPV2-GFP depends on the host cell type. Because FPs are relatively large proteins (e.g., GFP is 27 kDa), it is possible that tagging strategies could alter the localization of TRPs. For example, GFP tagged to the C-terminus of TRPML2 or TRPML3 appeared to function differently compared with the N-terminal tagged counterparts.11,13,16 Therefore, although FPs are useful tools to investigate protein localization, conclusions drawn from these studies should be tested by other means.

19.2.2. Immunostaining and Cellular Fractionation Studies

Because heterologously expressed FP-TRPs could potentially localize and function differently from endogenous proteins, TRP localization is analyzed using immunocytochemical techniques. However, there are only few effective and specific TRP antibodies available for this purpose, partially owing to the low endogenous expression levels of most TRPs.24 In cases where the specificity of TRP antibody is not clear, immunocytochemical approaches could still yield useful information if they are used in combination with fluorescent fusion proteins. For example, if used in combination, reasonable confidence could be achieved if substantial overlapping was observed between TRP-specific immunofluorescence and FP fluorescence. By using antibodies against endogenous TRPMLs, endogenous TRPML1 was shown to be localized to the late endosomal/lysosomal vacuoles,25 a conclusion drawn from previous studies based on FP fusion proteins.10–14 Similarly, endogenous TRPML2 and TRPML3 exhibit a more unrestrained vesicular expression pattern,25 consistent with observations that TRPML2-GFP and TRPML3-GFP also are found in recycling and early endosomes, respectively.13,17,26

Cellular fractionation has been used successfully to identify TRPML1- and TRPML3-resident vesicles with the aid of specific antibodies.13 Cell fractionation refers to the separation of homogeneous organelles from total cell lysates by using centrifugation at different speeds. Fractionation of different cellular compartments can be achieved by filtering cell lysates on a Percoll gradient.13 Using antibodies specific for TRPMLs and fusion tags, TRPML1 is found in the heavy fractions containing Lamp-1.13 Unlike TRPML1, TRPML3 was found to be associated with both early endosomes and LELs.13 These results are consistent with fluorescence studies of FP-TRPMLs.13,14 TRPP1 (previously known as polycystin-2, PC2, or APKD2) is another intercellular TRP channel whose localization was originally studied using density gradient fractionation.27 By using TRPP1-specific antibody on cell lysates from kidney tissues, TRPP1 was found mainly in the ER membranes, a result consistent with several other expression studies.1,27 These studies validate the use of fluorescent fusion proteins in the heterologous systems for studying subcellular localization of TRPs, while demonstrating the need for verification by other methods.

19.3. STUDYING CHANNEL PROPERTIES OF INTRACELLULAR TRPS

19.3.1. Electrophysiological Characterization of Intracellular TRP Channels at the Plasma Membrane

A subset of TRPs, such as TRPML3, TRPV2, and TRPM2, have double lives; these channels are able to traffic between the plasma membrane and intracellular organelles.1 This unique property makes it possible to characterize the channel properties of these TRPs using whole-cell recordings. Other intracellular TRPs, such as TRPML1 and TRPML2, however, are mainly localized in the intercellular organelles.7 Fortunately, recent studies suggest that gating and/or trafficking mutations of TRPML1 and TRPML2 are present at the plasma membrane (Figure 19.1), allowing electrophysiological characterization of the pore properties of these channels.1,7,15,20 Furthermore, TRPML2 was shown to be present at the plasma membrane of specific cell types.28

TRPML3 was the first TRPML family member to be characterized electrophysiologically, owing to its limited plasma membrane expression and its ability to generate whole-cell currents.13,14,29–32 Much larger whole-cell TRPML3-mediated currents are recorded in the presence of small molecule activators.20 A unique mutation at amino acid 419 (A419P) of TRPML3 causes pigmentation and hearing defects in Varitint-Waddler (Va) mice.29,33,34 Interestingly, the Va mutation could dramatically increase the plasma membrane expression and whole-cell currents of TRPML3, facilitating its electrophysiological characterization.30–32,35 Whole-cell patch clamping studies revealed that TRPML3/TRPML3Va is an inwardly rectifying non-selective cation channel.30–32,36 Unlike TRPML3, determining the functional characteristics of plasma membrane localized TRPV2 and TRPM2 was less challenging owing to their high levels of plasma membrane expression. Whole-cell recordings revealed that TRPV2 is an outwardly rectifying nonselective cation channel activated by heat and other chemical agonists.37 TRPM2 is a linear current channel activated by free cytosolic adenosine diphosphate ribose (ADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and Ca2+.18,38,39

Although no significant whole-cell current can be recorded from TRPML1- and TRPML2-expressing cells, equivalent Va mutations of TRPML1 and TRPML2 exhibit large inwardly rectifying Ca2+-permeable currents.32,40 In addition, mutation of the dileucine lysosome-targeting motifs of TRPML1 (TRPML1-NC) results in significant plasma membrane expression of TRPML1.11,12 A small molecule activator of TRPML3 can induce Ca2+ influx in cells expressing TRPML1-NC but not wild-type TRPML1.20 These results suggest that like TRPML3, wild-type TRPML1 is likely to be a Ca2+-permeable inwardly rectifying channel.

19.3.2. Endolysosome Patch Clamp

Because basic properties of intracellular membranes differ significantly from the plasma membrane,1 it is necessary to characterize the functions of intracellular TRPs in their native environment. One of the biggest hurdles to characterizing intracellular TRPs in their native membranes is the relatively small size of intracellular vesicles. For example, most endosomes and lysosomes are usually less than 0.5 μm in diameter, which is suboptimal for patch-clamping studies. However, recent studies suggest that endolysosomes can be enlarged by disrupting membrane trafficking using genetic and pharmacological approaches.19,40

Endosomes can be enlarged by introducing a hydrolysis-deficient mutant form of the AAA ATPase, SKD1/VPS4, into HEK293 cells.19 Such a maneuver could lead to the formation of large endosomes (3–6 μm in diameter) in HEK293 cells. TRPV2 is localized in the early endosomes of macrophages23 and other cell types.19 TRPV2 could act as an endosomal Ca2+ channel, potentially facilitating endosomal fusion and fission.19 The endosomal enlargement allowed Saito and colleagues to test this hypothesis by directly patch-clamping isolated endosomes.19 Enlarged endosomes are identified by GFP-tagged endososomal markers and isolated by slicing the cell membrane using electrodes.19 Because the endosomal current had similar pharmacological characteristics to whole-cell TRPV2 current, Saito and colleagues proposed that TRPV2 is an endosomal Ca2+ channel.

Dong and colleagues40 used a chemical approach to enlarge the endosomes and lysosomes (Figure 19.2). Vacuolin-1 is a small molecule that induces the formation of enlarged endosomes and lysosomes by an unknown mechanism.41 After exposure to 1 μM vacuolin-1 for 1 hour, large endosomes and lysosomes (up to 3–5 μm in diameter) are observed.40 Enlarged LELs that are positive for both mCherry-Lamp1 and TRPML1-GFP are isolated by slicing the cell membrane using a patch electrode (Figure 19.2). Similar to whole-cell recordings, four distinct configurations can be used for lysosome recordings: lysosome-attached, lysosome luminal-side-out, lysosome cytoplasmic-side-out, and whole lysosome (Figure 19.2). In the whole-lysosome configurations, the extracellular solution in the patch pipette (electrode) was adjusted to pH 4.6 in order to mimic the acidic condition of the LEL.40 On the LEL membrane, TRPML1 is positioned such that its large intraluminal loop, between trans-membrane domains one and two, faces the luminal side of the LEL, while its short N- and C-termini face the cytoplasm (Figure 19.2). Whole lysosome recordings of TRPML1-positive enlarged LELs revealed that TRPML1 and TRPML1Va give rise to inwardly rectifying cationic currents similar to the whole-cell TRPML1Va currents.40 In this case, inward is defined as cations flowing out of the LEL lumen (Figure 19.2). These results demonstrate that the Va-activating mutation is a valid approach to studying TRPML1 channels at the plasma membrane. Because Dong and colleagues were also able to occasionally isolate enlarged LELs and measure TRPML1 current without any vacuolin-1 treatment,40 they have confirmed that vacuolin-1 is unlikely to alter TRPML1 channel pore properties significantly. Similar approaches can be used to characterize the channel functions of TRPML2, TRPML3, and TRPM2.

FIGURE 19.2. Lysosome patch-clamp configurations.

FIGURE 19.2

Lysosome patch-clamp configurations. (a) Colocalization of mCherry–TRPML1 and EGFP–Lamp-1 at the membrane of an isolated enlarged LEL. The patch pipette is filled with rhodamine B dye. (b) Diagrams of four distinct patch-clamp configurations (more...)

19.3.3. Planar Lipid Bilayer Studies

An alternative way to study intracellular TRPs is to reconstitute TRP proteins or TRP-resident intracellular membranes into a planar lipid bilayer. This technique is used commonly to study the electrophysiological properties of single ion channels in a defined and artificial lipid bilayer.42 Two intracellular TRPs (TRPP1 and TRPML1) have been studied using planar lipid bilayers.27,43 NAADP is a second messenger that induces Ca2+ release from acidic organelles in a variety of cell types.44 By reconstituting lysosomal membranes into a planar lipid bilayer, NAADP activated a linear current that could be blocked by antibodies against TRPML1.45 As this NAADP-activated current differed significantly from the aforementioned inwardly rectifying current, the significance of this study is not clear, especially because there is now compelling evidence that two-pore channels (TPCs) are likely to be the NAADP receptors.46 In addition, ER membrane TRPP1 has been studied using planar lipid bilayers.27,47 TRPP1 can be activated by cytoplasmic Ca2+ and is regulated by SNARE proteins.27,47

19.3.4. Monitoring Ca2+ Release from Intracellular Ca2+ Stores

Ca2+ imaging has been used extensively to study TRP channel function. There are two different types of Ca2+ indicators that are commonly used to detect intracellular Ca2+ levels: Ca2+-sensitive fluorescent dyes or genetically encoded Ca2+ sensors. Fura-2 is the most commonly used fluorescent dye that can be easily loaded into most cell types.48 Fura-2 is excited at two different wavelengths (340 and 380 nm); the ratio of light emission at these two wavelengths (F340/F380), which increases dramatically with increased Ca2+, is used to estimate Ca2+ levels independent of dye loading.48 Not only has Fura-2, or other Ca2+-sensitive dyes such as Fluo4, been used extensively to study plasma membrane TRPs such as TRPV1,49 but they also are frequently used to evaluate Ca2+ release from intracellular compartments, with modifications.1 As many TRPs are present in both plasma membrane and intracellular membranes, to exclude the possibility of extracellular Ca2+ entry, experiments should be performed in a Ca2+-free bath solution.15 For instance, when TRPV1 is present in the ER or Golgi membranes;50 in the absence of extracellular Ca2+, application of the TRPV1-specific, membrane-permeable agonist capsaicin induces Ca2+ release from ER membranes.50 The ER/Golgi Ca2+ source is confirmed using a SERCA pump inhibitor to deplete ER Ca2+ stores. Using a similar approach, TRPM2 was shown to mediate ADPR-induced Ca2+ release from LELs.18 The LEL source of Ca2+ can be confirmed using the proton pump inhibitor bafilomycin A to disrupt lysosomal pH gradients and, consequently, the Ca2+ storage.18 Because ADPR is a membrane-impermeable molecule, it can be delivered by a patch electrode or through photolysis of pre-injected caged-ADPR.18 Photo un-caging has also been used to study NAADP-activated TPCs in the LEL.46

Genetically encoded Ca2+ indicators (GECIs) are a new generation of research tools for Ca2+ signaling that use luminescent and/or fluorescent reporter genes, whose spectral properties are modified upon binding to Ca2+.51 On the basis of their structure, there are at least three different types of GECIs. Aequorins are naturally bioluminescent reporters, whose Ca2+-dependent activation requires a co-factor called coelenterazine.52 The second class of probes, which includes Pericams and G-CaMPs, has a Ca2+-binding sequence, for example, from calmodulin (CaM), inserted into a single reporter protein, so that Ca2+ binding will change the spectral properties of the reporter protein.53 The third class of probes, which includes cameleon, has a Ca2+-binding motif inserted between two different reporter proteins so that, upon Ca2+ binding, the efficiency of fluorescence resonance energy transfer (FRET) changes.54

One major advantage of GECIs over fluorescent dyes is that GECIs can be targeted to desired organelles by fusing the construct to organelle-specific targeting motifs.55 Thus, GECIs can be tuned to measure Ca2+ levels in different intracellular compartments, such as the ER or LELs. In addition, GECIs can detect Ca2+ levels in microdomains in living cells. Furthermore, low-affinity (micromolar range) GECIs allow luminal Ca2+ measurements if they are engineered to localize on the luminal side of target proteins or TRPs. In contrast, high-affinity (several hundred nanomolar range) GECIs allow juxta-organellar Ca2+ measurement if they are engineered to accumulate on the cytoplasmic side of target proteins or TRPs. For example, aequorin fused to the luminal side of vesicle-associated membrane protein-2 (VAMP-2), a single transmembrane SNARE protein expressed on the surface of secretory vesicles/ granules, was used to monitor the Ca2+ levels of dense core vesicles in neuroendocrine cells.56 Similar approaches could be applied to monitor Ca2+ levels in other organelles by generating chimeric proteins between aequorin and organelle-specific proteins, including intracellular TRPs. For example, a chimera between VAMP-7, a LEL-specific SNARE protein, and aequorin could be used to monitor the channel activities of TRPMLs.

19.4. CONCLUSION

Several intracellular TRPs have been well characterized with regard to their subcellular localization and channel properties. Further, the functions of intracellular TRPs can be revealed by correlating their subcellular localization and channel properties. Considering the Ca2+ permeability of most TRPs and the Ca2+ dependence of membrane traffic, intracellular TRPs are likely involved in regulating vesicular trafficking. Indeed, the accumulation of lipids and other macromolecules in LELs observed in MLIV patients suggests a role for TRPMLs in lipid trafficking and lysosome biogenesis.57,58 Lysosomal biogenesis is a Ca2+-dependent process involving membrane fusion/fission of vacuoles containing different materials along the endosomal–lysosomal pathway.59 It is possible that unidentified trafficking cues activate TRPMLs to release Ca2+, which then bind to Ca2+ sensors, for example, CaM and synaptotagmins, in the LEL and induce membrane fusion/fission events.7 In the near future, with the TRP-resident compartments identified, more intracellular functions of TRPs will be revealed.

ACKNOWLEDGMENTS

The work in the authors’ laboratory is supported by start-up funds to H. X. from the Department of MCDB and Biological Science Scholar Program, the University of Michigan, and an NIH RO1 grant (NS062792 to H. X). We appreciate the encouragement and helpful comments from other members of the Xu laboratory.

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