Regulation and Trafficking of 5-HT3A Receptors Open Access
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Serotonin or 5-Hydroxytryptamine (5-HT) is a major neurotransmitter in the central and peripheral nervous systems that regulates diverse functions such as mood and gut motility. The 5-HT type 3 receptor is unique among the 5-HT receptors because it is the only ligand-gated ion channel for this neurotransmitter. 5-HT3 receptors are expressed throughout the body and specifically in multiple areas of the brain that have been implicated in drug addiction and mental states. In neurons, ion channels rapidly depolarize or hyperpolarize the membrane, which results in the induction or suppression of an action potential, respectively. Therefore, the regulation of ligand gated ion channel expression at the cell surface is essential for modulating neuronal responses to specific transmitters. However, the targeting and regulation of 5-HT3 receptors at the cell membrane are poorly understood. Therefore, in these studies, I set out to investigate both the targeting and regulation of functional 5-HT3A receptors using HEK-293 cells and the neuroblastoma cell line N1E-115. I demonstrate that 5-HT3A receptors tagged with the alpha-bungarotoxin binding sequence (BBS) are constitutively internalized in the absence of agonist. 5-HT3A/BBS receptors are also constitutively internalized in HEK-293 cells and in N1E-115 cells, which endogenously express 5-HT3 receptors. During internalization, 5-HT3A/BBS receptors co-localize with transferrin in clathrin-coated vesicles. However, 30 minutes after internalization, 5-HT3A/BBS receptors are not co-localized with transferrin in recycling endosomes, nor are they trafficked to lysosomes after 60 minutes. These data suggest that after internalization 5-HT3A/BBS receptors are targeted to early and/or late endosomes. I have employed the use of fluorophore assisted light inactivation (FALI) to manipulate 5-HT3A/BBS receptors with bound alpha-bungarotoxin conjugated to Alexa fluorophores. FALI was capable of inhibiting the constitutive internalization and the function of 5-HT3A/BBS receptors. Inhibition of 5-HT3A/BBS receptors by FALI was dependent on the irradiation energy, which is the product of time and intensity of light. FALI has been suggested to be limited to the protein tagged, however, my studies as well as others show that there is collateral damage to surrounding proteins and/or lipids. The inactivation of 5-HT3A/BBS receptor internalization and function was mediated by reactive oxygen species implicating the FALI process. FALI of 5-HT3A/BBS receptors gives us the ability to inhibit 5-HT3 receptors in a spatially and temporally defined manner that could be used to help elucidate receptor turnover or the role of 5-HT3 receptors in the central nervous system. Selective serotonin re-uptake inhibitors increase extracellular concentrations of 5-HT and are commonly used to treat depression. The effects of increased 5-HT on the surface expression of 5-HT3 receptors remains unclear. My data show that prolonged exposure to agonist increases the surface expression of 5-HT3A/BBS receptors. The increase in surface 5-HT3A/BBS receptors results in increased 5-HT induced current peak amplitudes. The agonist-induced up-regulation of 5-HT3A/BBS receptors is concentration and time dependent. Competitive antagonists can also induce the up-regulation of 5-HT3A/BBS receptors suggesting that the occupancy of the agonist-binding site is sufficient to induce the up-regulation. Agonist- or antagonist-induced up-regulation of 5-HT3A/BBS receptors is not mediated by the translation of new receptors. These studies suggest that 5-HT3 mediated signaling could be affected with the use of SSRIs. Next, I examined the regulation of the 5-HT3A receptors by evaluating whether the second intracellular loop is required for the proper trafficking to the cell surface. My data suggest that lower temperatures aid in the trafficking of 5-HT3A receptors lacking the second intracellular loop, however, it is unclear how the decrease in temperature affects trafficking. Furthermore, mutations within the second intracellular loop eliminating the serine and threonine phosphorylation residues did not significantly inhibit the trafficking of 5-HT3A receptors to the cell surface. However, mutating the single tyrosine in addition to the serines and threonines significantly inhibited the surface expression of 5-HT3A receptors. These data suggest that manipulations to the second intracellular loop affect the trafficking of 5-HT3A receptors and in combination significantly inhibit the trafficking of 5-HT3A receptors to the cell surface. Taken together my data suggest that the trafficking of 5-HT3A receptors can be modulated by FALI, or prolonged exposure to agonist, and proper trafficking is mediated by phosphorylation within the second intracellular loop.