An Interaction Involving an Arginine Residue in the Cytoplasmic Domain of the 5-HT3A Receptor Contributes to Receptor Desensitization Mechanism

Abstract

A large cytoplasmic domain accounts for approximately onethird of the entire protein of one superfamily of ligand-gated membrane ion channels, which includes nicotinic acetylcholine (nACh), ␥-aminobutyric acid type A (GABA A ), serotonin type 3 (5-HT 3 ), and glycine receptors. Desensitization is one functional feature shared by these receptors. Because most molecular studies of receptor desensitization have focused on the agonist binding and channel pore domains, relatively little is known about the role of the large cytoplasmic domain (LCD) in this process. To address this issue, we sequentially deleted segments of the LCD of the 5-HT 3A receptor and examined the function of the mutant receptors. Deletion of a small segment that contains three amino acid residues (425-427) significantly slowed the desensitization kinetics of the 5-HT 3A receptor. Both deletion and point mutation of arginine 427 altered desensitization kinetics in a manner similar to that of the (425-427) deletion without significantly changing the apparent agonist affinity. The extent of receptor desensitization was positively correlated with the polarity of the amino acid residue at 427: the desensitization accelerates with increasing polarity. Whereas the R427L mutation produced the slowest desensitization, it did not significantly alter single channel conductance of 5-HT 3A receptor. Thus, the arginine 427 residue in the LCD contributes to 5-HT 3A receptor desensitization, possibly through forming an electrostatic interaction with its neighboring residues. Because the polarity of the amino acid residue at 427 is highly conserved, such a desensitization mechanism may occur in other members of the Cys-loop family of ligand-gated ion channels.The Cys-loop pentameric ligand-gated ion channel (LGIC) 2 superfamily includes nicotinic acetylcholine (nACh), ␥-aminobutyric acid type A (GABA A ), glycine, and serotonin type 3 (5-HT 3 ) receptors (1). These receptors play crucial roles in fast synaptic transmission through agonist-induced opening of transmembrane ion channels in the central and peripheral nervous systems. Topologically, these receptors consist of a large extracellular N-terminal domain, a large cytoplasmic domain (LCD), and four transmembrane domains (1). The 5-HT 3 receptor can modulate the release of neurotransmitters such as glutamate, GABA, and dopamine (DA), and plays a role in the reward mechanisms of drug addiction (2, 3). Of all five 5-HT 3 subunits identified by molecular cloning, the 5-HT 3A and 5-HT 3B subunits have been the most thoroughly characterized (4). Although the 5-HT 3B subunit can form a functional heteromer when co-expressed with the 5-HT 3A subunit (5), the 5-HT 3A subunit is capable of forming a functional channel (1), and such a homomer is thought to be the dominant functional form in the central nervous system (6).One common feature shared by members of the LGIC superfamily is that these receptors desensitize at synaptic sites when exposure to agonist is prolonged. Desensitization plays a critical role in the...

Discrete M3-M4 Intracellular Loop Subdomains Control Specific Aspects of γ-Aminobutyric Acid Type A Receptor Function

“…5). A similar effect has been observed in the 5-HT 3 R, where a single intracellular arginine near M4 has also been shown to control receptor desensitization (43). In our sequence alignment, this arginine is homologous to ␣1(Lys-383) (Fig.…”

“…Previous studies of pLGIC ILDs have focused on receptor surface stability, mobility (16,17,22), phosphorylation (26,27), binding interactions with accessory proteins (23), and trafficking machinery (19). Recent studies point to an intracellular component of desensitization (42)(43)(44), and intracellular control of conductance has been linked to the MA stretch (29,34), but this has yet to be confirmed for the GABA A R.…”

Differential Effects of Serotonin and Dopamine on Human 5-HT3AReceptor Kinetics: Interpretation within an Allosteric Kinetic Model

“…This explains why the rate of deactivation is comparable with the rate constant for desensitization and why recovery from deactivation and resensitization occur on similar timescales. The conclusion that desensitization contributes significantly to the deactivation process when 5-HT is the agonist may also explain why mutations in transmembrane-linking domains that alter the rate of 5-HT-induced desensitization produce parallel changes in the rate of deactivation (Hu and Lovinger, 2005;Hu et al, 2006 values for channel activation and desensitization are similar. The most parsimonious explanation for this observation is that agonist-induced desensitization occurs primarily from the open state.…”

“…On removal of agonist, currents decay in a process termed deactivation. Currents also decay during continuous agonist exposure, reflecting receptor isomerization to one or more ion-impermeable desensitized states that cannot be activated even by high concentrations of agonist (Reeves et al, 2005;Hu et al, 2006). Such desensitization is reversible because 5-HT 3 receptors resensitize after the removal of agonist.…”

An Internally Modulated, Thermostable, pH-sensitive Cys Loop Receptor from the Hydrothermal Vent Worm Alvinella pompejana

“…It has been shown earlier that changes in the M3-M4 loop in eukaryotic CLRs alter expression and desensitization (37)(38)(39), modulate interaction with other proteins (40), and affect the single channel conductance in cation-selective CLRs (41). Alva1-pHCl has an intracellular M3-M4 loop that is 25-30 amino acids long.…”