Evidence for a Centrally Located Gate in the Pore of a Serotonin-Gated Ion Channel

Panicker, Sandip; Cruz, Hans; Arrabit, Christine; Slesinger, Paul A.

doi:10.1523/jneurosci.22-05-01629.2002

Cited by 76 publications

(90 citation statements)

References 42 publications

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“…Such Cd 2ϩ binding sites have been introduced into other proteins, such as K ATP channels, voltage-gated K ϩ channels, and glutamate transporters (30 -33). Previously, we determined the structure of the M2 and the location of the gate using cysteinesubstituted 5-HT 3A receptors (9). Using these cysteine-substituted receptors, we now demonstrate that the selectivity filter of the 5-HT 3A receptor is sufficiently narrow to form high affinity Cd 2ϩ binding sites.…”

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confidence: 78%

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

Panicker

Cruz

Arrabit

et al. 2004

Journal of Biological Chemistry

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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...

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confidence: 78%

“…Molecular Biology-Cysteine mutants of 5-HT 3A were constructed previously (9). Four mutants (L8ЈC, G10ЈC, Y11ЈC, F14ЈC) were not tested because they yielded little or no currents when expressed alone (see Ref.…”

Section: Methodsmentioning

confidence: 99%

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

Panicker

Cruz

Arrabit

et al. 2004

Journal of Biological Chemistry

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“…Position 15′ of the 5-HT 3A receptors TM2 domain is a non-pore-facing amino acid residue (Reeves et al, 2001;Panicker et al, 2002). In addition to its role in channel gating, the amino acid residue at 15′ also appears to have important roles in channel modulation by alcohol and anesthetics (Mihic et al, 1997;Wick et al, 1998), and binding of these drugs to a hydrophobic pocket involving this residue alters receptor function.…”

Section: Discussionmentioning

confidence: 99%

“…Residue L293 is in the same 15′ position as a residue in the GABA A receptor that has important roles in channel gating (Findlay et al, 2001;Scheller & Forman, 2002) and modulation by alcohols and general anesthetics (Mihic et al, 1997;Wick et al, 1998). This amino acid residue is thought to reside on the non-porefacing side of TM2 domain (Panicker et al, 2002;Reeves et al, 2001). We recently demonstrated that alcohol enhancement of 5-HT 3A receptor-mediated current was abolished with L293C and L293S mutations (Hu et al, 2006).…”

Section: Role Of L293 In the Modulatory Action Of 5-himentioning

confidence: 99%

The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole

Lovinger

2008

Neuropharmacology

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Allosteric modulation of ligand-gated ion channels can play important roles in shaping synaptic transmission. The function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT 3 ) receptor, a member of the Cys-loop ligand-gated ion channel superfamily, is modulated by a variety of compounds such as alcohols, anesthetics and 5-hydroxyindole (5-HI). In the present study, the molecular determinants of allosteric modulation by 5-HI were explored in N1E-115 neuroblastoma cells expressing the native 5-HT 3 receptor and HEK 293 cells transfected with the recombinant 5-HT 3A receptor using molecular biology and whole-cell patch-clamp techniques. 5-HI potentiated 5-HT-activated currents in both N1E-115 cells and HEK 293 cells, and significantly decreased current desensitization and deactivation. Substitution of Leu293 (L293, L15′) in the second transmembrane domain (TM2) with cysteine (L293C) or serine (L293S) abolished 5-HI modulation. Other mutations in the TM2 domain, such as D298A and T284F, failed to alter 5-HI modulation. The L293S mutation enhanced dopamine efficacy and converted 5-HI into a partial agonist at the mutant receptor. These data suggest that 5-HI stabilizes the 5-HT 3A receptor in the open state by decreasing both desensitization and 5-HT unbinding/channel closing; and L293 is a common site for both channel gating and allosteric modulation by 5-HI. Our observations also indicate existence of a second 5-HI recognition site on the 5-HT 3A receptor which may overlap with the 5-HT binding site and is not involved in the positive modulation by 5-HI. These findings support the idea that there are two discrete sites for 5-HI allosteric modulation and direct activation in the 5-HT 3A receptor.

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“…7D). In future studies, it should be feasible to employ the substituted cysteine accessibility method to identify all residues within the MA stretch that impinge upon the permeation pathway in a manner analogous to that adopted to evaluate the channel-lining TM2 residues of, for example, the 5-HT 3A receptor (18,19).…”

Section: Discussionmentioning

confidence: 99%

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

Deeb¹,

Carland

Cooper

et al. 2007

Journal of Biological Chemistry

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Collectively, the data demonstrate that the dynamic modification of the charge of a cytoplasmic residue regulates ␥, consistent with the existence of cytoplasmic portals that impose a rate-limiting barrier to ion conduction in Cys loop receptors.Cysteine loop (Cys loop) receptors encompass five families of gene products. Nicotinic-acetylcholine (nACh) 3 receptors, 5-hydroxytryptamine type-3 (5-HT 3 ) receptors, and Zn 2ϩ -activated channels conduct cations, whereas ␥-aminobutyric acid type A and glycine receptors conduct anions (1). Cys loop receptor subunits combine as pentamers forming a central ion pore that traverses the plasma membrane. Studies examining the role of residues comprising the pore-lining second transmembrane (TM2) helices of the nACh receptor led to the traditional view that this region of Cys loop receptors serves as the rate-limiting barrier to ion flux. Among the cationselective Cys loop receptor subunits, conserved acidic residues influence unitary conductance (␥) by forming concentric rings of negative charge positioned along the vertical axis of the central pore (2).Although the role of residues within the TM2 helix of Cys loop receptors in controlling ␥ is well established, recent studies demonstrate a similar function for residues located in the large cytoplasmic TM3-4 loop (1, 3, 4). Based on a 4.6 Å resolution cryoelectron microscopy image of the Torpedo marmorata nACh receptor, Miyazawa et al. (5) first speculated that amino acids in the TM3-4 loop form "transverse tunnels" that may contribute to the ion conduction pathway. Subsequent electrophysiological studies demonstrated that cytoplasmic residues within membrane-associated (MA) helices of the 5-HT 3 and ␣ 4 ␤ 2 nACh receptors are determinants of ␥ (3, 4). Arg 436 in the 5-HT 3A subunit at a position termed MA 0Ј is critical for maintaining the anomalously low ␥ (ϳ900 femtosiemens) that is a hallmark of the homomeric 5-HT 3A receptor. Furthermore, introduction of MA 0Ј Arg into ␣ 4 ␤ 2 nACh receptors halved ␥ (4).A 4 Å resolution model of the T. marmorata nACh receptor indicates that adjacent MA helices frame portals with apertures only slightly larger than partially hydrated permeant cations (6). Using this structure as a template, homology models of the 5-HT 3A and ␣ 4 ␤ 2 nACh receptors indicate that charged amino acids line their cytoplasmic portals (4).Although it is now clear that specific MA helix residues influence the ␥ of nACh and 5-HT 3 receptors, it remains to be determined whether their charge plays a role. We used substituted cysteine modification to address this question, a method fre-

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Evidence for a Centrally Located Gate in the Pore of a Serotonin-Gated Ion Channel

Cited by 76 publications

References 42 publications

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

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

The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

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