Components of the serotonergic system

The wide range of functions mediated by 5-HT results from the existence of several 5-HTRs.

In mammalians, seven distinct 5-HTRs (5-HTR1-7) are described with at least fourteen known subtypes (Figure 9). All 5-HTRs are GPCRs with the exception of 5-HTR3, which is a ligand-gated cation channel (Na⁺ and Ca²⁺ influx, K⁺ efflux). GPCRs are seven transmembrane receptors characterized by the ability to activate heterotrimeric G proteins comprised of the three subunits α, β and γ. The complexity of the serotonergic system is further amplified by the fact that the 5-HTRs couple to different G proteins and may also assemble to receptor homo- and heterodimers.⁸⁵

Figure 9: 5-HTR subtypes and their main signaling pathways.

Overview about the main signaling pathways of the different 5-HTRs. All 5-HTRs are GPCRs with the exception of 5-HTR3, which is a ligand-gated cation channel. 5-HTR1 and 5-HTR5 are associated with coupling to Gαi/o causing a decrease in cAMP, whereas 5-HTR4, 5-HTR6 and 5-HTR7 are known to increase cAMP by activation of Gαs. 5-HTR2, however, couples to Gαq/11 leading to the release of intracellular calcium.⁸⁶ 5-HTR1 and 5-HTR5 couple to Gαi/o, which inhibits adenylyl cyclases (ACs), resulting in downregulation of cyclic adenosine monophosphate (cAMP).⁸⁶ The 5-HTR1 subfamily, which is linked with migraine and anxiety, consists of five subtypes: 5-HTR1a, 5-HTR1b, 5-HTR1d, 5-HTR1e, 5-HTR1f. The subtype 5-HTR1c was reassigned to 5-HTR2c because of its different signaling. Little is known about the 5-HTR5 subfamily, which is comprised of 5-HTR5a and

5-HTR5b, mainly due to the lack of selective agonists.⁸⁷ 5-HTR4, 5-HTR6 and 5-HTR7 are known to activate ACs via Gαs causing an increase in cAMP. The 5-HTR2 subfamily encompasses the three subtypes 5-HTR2a, 5-HTR2b and 5-HTR2c and is associated with coupling to Gαq/11. This signaling pathway leads to the activation of PLC, which in turn hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3), triggering the release of intracellular calcium.⁸⁶ However, in cell culture 5-HTR2a and 5-HTR2c were also shown to exhibit Gαi/o activation.⁸⁷

To define a specific function for each 5-HTR is difficult, at least based on the current literature.

For instance, contradictory reports were published about the role of 5-HTR2a in inflammation.

On the one hand, 5-HT2a stimulation was shown to trigger a pro-inflammatory response by IL6 release in vascular SMCs.⁸⁸ In line with these findings, inhibition of 5-HTR2 blocked the TNFα-induced ICAM1 expression in human umbilical vein endothelial cells via nitric oxide (NO) release.⁸⁹ On the other hand, a different report revealed anti-inflammatory properties of 5-HTR2a in primary aortic SMCs. Specific activation led to the inhibition of TNFα-mediated inflammation including a decrease in the expression of ICAM1 and IL6.⁹⁰ The little consistency in studies arise through the complexity of the serotonergic system. As different cell types express a different 5-HTR expression pattern, 5-HT function is diverse. For instance, 5-HT seems to have two faces in the regulation of the vascular tone. Firstly, 5-HT acts as a vasoconstrictor via the 5-HT2A receptor in VSMC. Secondly, 5-HT induces vasodilation through eNOS production via 5-HTR1b present on endothelial cells.⁹¹ Even within the same organ, different receptors can be stimulatory or inhibitory, leading to the bivalent action of increased or decreased 5-HT.⁷⁶ Conflicting results in cell culture experiments may be explained by contaminations of 5-HT since fetal bovine serum (FBS) contains ~300 nM 5-HT, which is enough to stimulate most 5-HTRs.⁹² Moreover, the absence of sufficiently selective ligands makes it difficult to attribute a specific function to a receptor.⁹³ A few years ago Wang et al.

and Wacker et al. deciphered the GPCR crystal structures of the 5-HT subtypes 5-HTR1b and 5-HTR2b.^94,95 While both receptors show high similarity in the structure of the 5-HT binding site, there is a subtle difference in the width of the binding pocket, which is already enough to cause differences in signaling. This paved the way for the development of novel more selective compounds for these two receptors. Further research on 5-HTR crystal structures will provide a better understanding of the serotonergic receptor function and will enable the design of more selective compounds and thereby more specific drugs.⁹⁶

3.3.2.2 Serotonin transporter

The 5-HT transporter (SERT or 5-HTT) is a Na⁺/Cl^--dependent twelve transmembrane domain spanning monoamine transporter, which is encoded in humans by the gene solute carrier family 6, member 4 (SLC6A4).⁸¹ It is a key regulator for 5-HT signaling, which terminates signaling by removing extracellular 5-HT through transportation across the plasma membrane

into the cell. In this way, the 5-HT reuptake can mediate the duration as well as the strength of the autocrine and paracrine signaling of 5-HT. After reuptake, 5-HT is either recycled and packed into vesicles by VMAT or degraded by MAO. Although SERT is mainly studied and therapeutically targeted for controlling the 5-HT concentration in the synaptic cleft, it is also crucial for platelet function including the regulation of plasma 5-HT levels.^76,79,81 Because of its important function, SERT is regulated in several ways. Like many receptors, SERT is not constitutively located on the plasma membrane but dynamically traffics due to post-translational modifications such as phosphorylation. Apart from trafficking, phosphorylation was also postulated to modulate SERT activity. Thus, p38 mitogen activated protein kinase (MAPK)-dependent phosphorylation, induced by the pro-inflammatory cytokines IL1β and TNFα, enhances SERT activity.⁹⁷ Moreover, 5-HT itself regulates the density of SERT on the plasma membrane. Increased 5-HT concentrations display a bivalent effect on SERT. In neurons, high extracellular 5-HT levels decrease the density of SERT on the membrane, whereas SERT on the surface of platelets is initially upregulated with increasing plasma 5-HT levels.⁹⁸ However, with continuously rising plasma levels, the SERT density on platelets falls below normal levels, suggesting that 5-HT limits its own uptake into platelets by down-regulating SERT.⁷⁹ This is further supported by the observation that high intracellular 5-HT levels in platelets result in SERT internalization, which is dependent on GTPase serotonylation.⁸¹

It is well established that SERT is implicated in mental disorders such as depression, although the precise mechanism is still debated.⁹³ This is further supported by the observation that genetic variations and altered SERT expression are associated with behavioral phenotypes and disorders. Clinical evidence arises from polymorphisms in the promoter region of SERT, the so-called serotonin-transporter-gene-linked polymorphic region (5-HTTLRP), which has been associated with neuropsychiatric disorders.⁹⁹ In accordance, mice deficient for SERT exhibit behavioral abnormalities linked to anxiety and depression. As a result, SERT is the primary target of many antidepressant medications. The most common antidepressants are the selective serotonin reuptake inhibitors (SSRIs). The effectiveness of SSRIs is assumed to be mediated by enhanced serotonergic neurotransmission. More precisely, SSRIs block SERT and thereby the 5-HT reuptake, resulting in an increased 5-HT concentration in the synaptic cleft and subsequently to an amplified signaling. Just recently, Coleman and colleagues were the first who described a high resolution structure of the human SERT bound to two different SSRIs.^100,101 While others suggest that SERT functions by forming oligomers, Coleman et al.

postulate that SERT-dimers observed in crystal form are unlikely to occur under physiological conditions in the cell membrane because the predicted membrane-spanning regions of each protomer are not properly aligned.¹⁰⁰