Modular Design of Cys-loop Ligand-gated Ion Channels: Functional 5-HT3 and GABA ρ1 Receptors Lacking the Large Cytoplasmic M3M4 Loop

Abstract

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the α-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT3A) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT3A-glvM3M4 receptors were comparable to 5-HT3A wild type. The channels remained cation-selective but the 5-HT3A-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT3 and acetylcholine receptors, significantly attenuated 5-HT–induced currents with wild-type 5-HT3A but not 5-HT3A-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-ρ1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.

Identifying the elusive link between amino acid sequence and charge selectivity in pentameric ligand-gated ion channels

“…We found that neither the cation selectivity of the 5-HT 3A R (see also ref. 18) nor the anion selectivity of the α1 GlyR is any lower upon removal of their intracellular domains (P K + /P Cl -= 88 and P Cl -/P K + = 271, respectively; Fig. 2 E and F).…”

“…S1; ref. 21) that ions must traverse upon entering or exiting the channel, and their removal was found to increase the 5-HT 3A R's single-channel conductance from <1 to 20-40 pS (9,18). Whereas the 5-HT 3A R has seven basic residues per subunit framing these portals, the muscle AChR has only two (e subunit) or three (α1, β1, and δ subunits), and although these residues exert little to no effect on the charge selectivity of wild-type pLGICs ( Fig.…”

“…In the case of the 5-HT 3A R, we used the 5-HT 3A -glvM3M4R construct described in ref. 18 where most of the M3-M4 linker (115 out of a total of ∼135 residues) was replaced with the SQPARAA heptapeptide (SI Materials and Methods). In the case of the α1 GlyR, we replaced most of the M3-M4 linker (more specifically, the fragment between Lys-312 and Lys-385, that is, 74 out of a total of ∼80-90 residues) with a string of eight alanines (19).…”

“…To address this point, we first mutated the glutamates at position -1′ to alanines in the background of the 5-HT 3A -glvM3M4R construct (that is, the 5-HT 3A R with much shortened M3-M4 linkers; ref. 18) and estimated its charge selectivity. We found that, indeed, neutralizing position -1′ in the background of a channel that lacks the argininelined portals lowers the cation selectivity to a more moderate extent (P K + /P Cl -= 6.6) than does neutralizing these glutamates in the wild-type 5-HT 3A R background (P K + /P Cl -≅ 2.5).…”

Involvement of 5-hydroxytryptamine type 3 receptors in sevoflurane-induced hypnotic and analgesic effects in mice

“…Of these subtypes, the 5-HT 3 receptor is a member of the superfamily of ligand-gated ion channel receptors. This family shares structural similarities with the nicotinic acetylcholine, glycine, and g-aminobutyric acid type A (GABA A ) receptors [3,11,12]. The 5-HT 3 receptors are widely distributed in both the central and peripheral nervous system and are involved in the physiologic and pathologic processes that mediate nausea, vomiting, peripheral nociception, and central antinociception [5,20].…”