Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Abstract

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT 3A ) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd 2؉ ) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2 C and, to a lesser extent, G-2 C, showed high affinity inhibition with Cd 2؉ Ligand-gated ion channel (LGIC) 1 receptors are responsible for rapid chemical transmission between neurons in the nervous system (1). Alterations in the response of ligand-gated receptors have profound effects on neuronal activity in the brain. For example, mutations in nicotinic acetylcholine-gated receptors (nAChRs) and glycine-gated receptors have been linked to certain forms of epilepsy and startle disease, respectively (2-4).LGIC receptors that respond to acetylcholine, ␥-aminobutyric acid, glycine, or serotonin (5-HT) possess a conserved pair of extracellular cysteines in the N-terminal domain ("Cys loop" receptors) and are composed of five subunits, each containing four putative transmembrane domains (5). Of these four domains, the second transmembrane domain (M2) lines the majority of the water-filled pore (Fig. 1A).The rapid response of LGIC receptors is achieved by the energetic coupling of agonist binding in the extracellular Nterminal domain with a "gate" located 60 Å away in the receptor pore (M2 domain) (6). Several studies with LGIC receptors suggest that the gate is situated in the middle of the M2 (6 -12) (but see Ref. 13). The primary determinants of selectivity appear to be located below the gate, near the cytoplasmic membrane surface. Mutations in the cytoplasmic end of the M2 alter ion selectivity (14 -17), and several recent studies show that by mutating sites in this region, cation-conducting LGIC can be converted to anion-conducting channels and vice versa (18 -23). Because permeability of like-charged molecules in LGIC appears to be largely dependent on size, it is believed that the narrowest region of the open LGIC pore coincides with the channel selectivity filter, which is formed by a portion of the M1-M2 loop and the cytoplasmic side of the M2 helix (16,24). Consistent with this region being narrow, site-specific mutations in the filter alter conductivity in a manner that depends upon side-chain volume (8,24). Thus, the pore of the open receptor can be envisaged as a funnel, with a wide extracellular vestibule that tapers to a constriction at the cytoplasmic side of the membrane, where ion selectivity is determined (25).What is known about the extent of movement in the cytoplasmic selectivity filter? The 9-Å three-dimensional structures of open and closed Torpedo nAChRs derived fro...

Pore conformations and gating mechanism of a Cys-loop receptor

“…5A Right) is wider at its bottom and narrower at its upper part. Yet, the cytoplasmic vestibule remains the narrowest part of the pore in both the closed and the open states, in accord with Panicker et al (17). Our open-pore structure is compatible with the inability of Zn 2ϩ to block chimera Hϩ16Ј and with the capacity of Zn 2ϩ to block the open channel and to produce off responses by interacting specifically with histidines substituted at position Ϫ5Ј, Ϫ2Ј, Ϫ1Ј, 2Ј, or 9Ј (Table 1 and Fig.…”

A Role for the Val291 Residue within the Transmembrane Domain 2 in Diltiazem- and TMB-8 [3,4,5-Trimethoxybenzoic Acid 8-(Diethylamino)octyl Ester]-Mediated 5-Hydroxytryptamine Type 3A Receptor Regulations

“…1C, for the conserved Val291 residue of the 5-HT 3A receptor, the present and previous studies have also shown that mutations of Val291 to other amino acids affect the inhibition of I 5-HT in response to anesthetics, cadmium, ginsenoside Rg 3 and TMB-8. 3,4,7,8,18) Thus, Val291 of the 5-HT 3A receptor could be useful targets for studying the receptor-related pharmacology. This residue could also be used for drug developments such as anti-emetic agent if diltiazem or TMB-8 derivatives are selective for 5-HT 3A receptor.…”

“…Based on this, we hypothesized that there are common interaction site(s) with diltiazem and TMB-8. Since TM2 forms the pore region of 5-HT 3A receptors, and mutations in this region affect the inhibition of I 5-HT in response to anesthetics, cadmium and the open channel blocker, 3,4,7) we investigated the effect of diltiazem and TMB-8 on I 5-HT in oocytes expressing 5-HT 3A receptors that contain site- Table 1 (meanϮS.E.M. ; nϭ9-12 oocytes for each point).…”

The structural basis of function in Cys-loop receptors

“…Evidence from SCAM has indicated that the intracellular end of M1 and the M1-M2 linker lie along the path of the permeating ions, and these regions contain residues responsible for anion/ cation selectivity (Filippova et al 2004 ; also see section 4.4). Coordination of cadmium ions in 5-HT 3 R Cys mutants and the use of negatively and positively charged thiol reactive MTS reagents have demonstrated that residues in the M1-M2 loop are accessible (Panicker et al 2002(Panicker et al , 2004.…”