Fluorophore assisted light inactivation (FALI) of recombinant 5-HT3A receptor constitutive internalization and function

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Abstract

Fluorescent proteins and molecules are now widely used to tag and visualize proteins resulting in an improved understanding of protein trafficking, localization, and function. In addition, fluorescent tags have also been used to inactivate protein function in a spatially and temporally-defined manner, using a technique known as fluorophore-assisted light inactivation (FALI) or chromophore-assisted light inactivation (CALI). In this study we tagged the serotonin3 A subunit with the α-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HT3A/BBS receptors with fluorescently conjugated α-bungarotoxin in live cells. We show that 5-HT3A/BBS receptors are constitutively internalized in the absence of an agonist and internalization as well as receptor function are inhibited by fluorescence. The fluorescence-induced disruption of function and internalization was reduced with oxygen radical scavengers suggesting the involvement of reactive oxygen species, implicating the FALI process. Furthermore, these data suggest that intense illumination during live-cell microscopy may result in inadvertent FALI and inhibition of protein trafficking.

Introduction

The 5-hydroxytryptamine type 3 (5-HT3) receptor mediates the rapid excitatory currents evoked by serotonin both in the peripheral and central nervous systems (Maricq et al., 1991, Tecott et al., 1993). Currently 5-HT3 antagonists are used clinically to treat irritable bowel syndrome and nausea and emesis during chemotherapy (Hesketh, 2008). Furthermore, 5-HT3 receptor polymorphisms are associated with schizophrenia and bipolar disorder (Niesler et al., 2001). In addition, 5-HT3A receptors may have roles in addiction to alcohol and other drugs of abuse as well as anxiety (Hodge et al., 1993, Olivier et al., 2000 reviewed in Grant, 1995, McKinzie et al., 2000). Therefore, regulation of 5-HT3A receptors at the cell membrane may play important roles in a number of neural functions, including nausea, anxiety and drug addiction.

The 5-HT3 receptor is a member of the cysteine-loop pentameric ligand gated ion channel (pLGIC) family, which includes the nicotinic-acetylcholine receptor, γ-aminobutyric acid type A (GABAA), and glycine receptors. Nicotinic-acetylcholine and 5-HT3 receptors form cation channels, whereas GABA and glycine receptors form anion channels. Five 5-HT3 isoforms have been described and have been termed 5-HT3A–E (Maricq et al., 1991, Davies et al., 1999, Niesler et al., 2003, Karnovsky et al., 2003). The A subtype can combine to form functional homo-pentameric structures whereas B–E subunits must combine with A subunits to form functional hetero-pentameric receptors (Maricq et al., 1991, Davies et al., 1999, Niesler et al., 2007). Heteromeric 5-HT3AB receptors have larger single channel conductance and are less permeable to Ca2+ compared to 5-HT3A homomeric receptors (Davies et al., 1999). Despite our extensive knowledge of 5-HT3 receptor structure and function, little is known about the cell surface stability and trafficking of this receptor.

The development of fluorescent proteins and strategies for tagging proteins with other fluorescent molecules has made it possible to visualize specific proteins and investigate their localization and trafficking. Furthermore, fluorescent proteins and molecules have been used to specifically inhibit proteins upon the excitation of the fluorophore by light and this technique is known as fluorophore assisted light inactivation (FALI) or chromophore assisted light inactivation (CALI) (Jay, 1988; reviewed in Jacobson et al., 2008). Fluorescent proteins such as enhanced Green Fluorescent Protein (eGFP) (McLean et al., 2009, Rajfur et al., 2002, Tanabe et al., 2005, Vitriol et al., 2007) and synthetic fluorophores like fluorescein or red biarsenical dye (ReAsH) (McLean et al., 2009, Tour et al., 2003, Yan et al., 2006, Guo et al., 2006, Marek and Davis, 2002, Lee et al., 2008) have been used to produce FALI.

FALI is mediated by the actions of singlet oxygen and is dependent on the irradiation energy (Horstkotte et al., 2005, McLean et al., 2009). Singlet oxygen is a reactive oxygen species (ROS) that can cause oxidation of tryptophan, tyrosine, methionine, histidine, and cysteine residues that may result in the cross-linking of residues (reviewed in Davies, 2003). The generation of singlet oxygen is a byproduct of all fluorescence in the presence of oxygen. Due to the close proximity of the fluorophore and the target protein, the singlet oxygen most likely reacts with the target protein (McLean et al., 2009). It has been suggested that FALI is specific to the protein labeled (Jay, 1988, Rajfur et al., 2002, Tanabe et al., 2005, Surrey et al., 1998, Yan et al., 2006, Marek and Davis, 2002, Tour et al., 2003). However, it has also been shown that FALI can lead to collateral damage to non-targeted proteins (Guo et al., 2006, Rahmanzadeh et al., 2007).

In this study, we identified robust constitutive internalization of recombinant 5-HT3A receptors expressed in HEK-293 cells and N1E-115 neuroblastoma cells in the absence of an agonist. Furthermore, we show that receptor internalization and function were attenuated by fluorophore assisted light inactivation.

Section snippets

α-bungarotoxin tagged 5-HT3A receptor construct

5-HT3A receptors were tagged with the binding sequence for α-bungarotoxin allowing us to label surface receptors with fluorescently conjugated α-bungarotoxin in live cells described previously (Sanders and Hawrot, 2004) and used by other groups (Wilkins et al., 2008, Sekine-Aizawa and Huganir, 2004, McCann et al., 2005, Guo et al., 2006). The bungarotoxin binding sequence (BBS) WRYYESSLEPYPD was added to the short extracellular c-terminus of the 5-HT3A receptor by primer addition PCR. The

Discussion

In the present study we were able to tag the 5-HT3A subunit with the α-bungarotoxin pharmatope tag (Sanders and Hawrot, 2004, McCann et al., 2005, Sekine-Aizawa and Huganir, 2004, Wilkins et al., 2008, Guo et al., 2006, Watschinger et al., 2008) on the extracellular carboxyl terminal. Using this method we were able to specifically label surface receptors in live cells with α-bungarotoxin conjugated to Alexa fluorophores. We show that 5-HT3A/BBS receptors are constitutively internalized in the

5-HT3A/BBS construct

The α-bungarotoxin binding sequence was added to the carboxyl terminal of the mouse 5-HT3A by primer addition PCR using the QuickChange system (Stratagene, USA). The stop codon was removed and the first bungarotoxin binding sequence were added using the following forward primer:

  • 5′tggtccatttggcattatacttggagatactacgagagctccctggagccctaccctgactaatctagagggcccgtttaaacc3′

and the following reverse primer:
  • 5′ggtttaaacgggccctctagattagtcagggtagggctccagggagctctcgtagtatctccaagtataatgccaaatggacca3′.

The second

Acknowledgments

We thank Dr. Henry H. Puhl III for the 5-HT3A/HA and the mGluR8/BBS constructs. We thank Dr. Stephen Vogel for use of the light power meter and comments on the manuscript. Thank you to Steve Ikeda for comments on the manuscript. And lastly, we thank Dr. Jennifer Gillette and Dr. Jennifer Lippincott-Schwartz for the use of the microscopes.

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