5-HT3 receptors are membrane ion channels

The neurohormone 5-hydroxytryptamine (5HT or serotonin) exerts its effects by binding to several distinct receptors. One of these is the M-receptor of Gaddum and Picarelli, now called the 5-HT3 receptor, through which 5-HT acts to excite enteric neurons. Ligand-binding and functional studies have shown that the 5-HT3 receptor is widely distributed in peripheral and central nervous tissue and evidence suggests that the receptor might incorporate an ion channel permeable to cations. We now report the first recordings of currents through single ion channels activated by 5-HT3 receptors, in excised (outside-out) membrane patches from neurons of the guinea pig submucous plexus. Whereas application of acetylcholine activated predominantly a 40-pS channel, 5-HT caused unitary currents apparently through two channels of conductances of 15 and 9 pS, which were reversibly blocked by antagonists of the 5-HT3 receptor. Receptors for amine neurotransmitters, including 5-HT1 and 5-HT2, have previously been thought to transduce their effects through GTP-binding proteins: the direct demonstration that 5-HT3 receptors are ligand-gated ion channels implies a role for 5-HT, and perhaps other amines, as a 'fast' synaptic transmitter.     

A biological role for prokaryotic ClC chloride channels

An unexpected finding emerging from large-scale genome analyses is that prokaryotes express ion channels belonging to molecular families long studied in neurons. Bacteria and archaea are now known to carry genes for potassium channels of the voltage-gated, inward rectifier and calcium-activated classes, ClC-type chloride channels, an ionotropic glutamate receptor and a sodium channel. For two potassium channels and a chloride channel, these homologues have provided a means to direct structure determination. And yet the purposes of these ion channels in bacteria are unknown. Strong conservation of functionally important sequences from bacteria to vertebrates, and of structure itself, suggests that prokaryotes use ion channels in roles more adaptive than providing high-quality protein to structural biologists. Here we show that Escherichia coli uses chloride channels of the widespread ClC family in the extreme acid resistance response. We propose that the channels function as an electrical shunt for an outwardly directed virtual proton pump that is linked to amino acid decarboxylation.     

A cytoplasmic region determines single-channel conductance in 5-HT3 receptors

5-hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the 'HA-stretch') within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.     

New mammalian chloride channel identified by expression cloning.

Ion channels selectively permeable to chloride ions regulate cell functions as diverse as excitability and control of cell volume. Using expression cloning techniques, a complementary DNA from an epithelial cell line has been isolated, sequenced and its putative structure examined by site-directed mutagenesis. This cDNA, encoding a 235-amino-acid protein, gave rise to a chloride-selective outward current when expressed in Xenopus oocytes. The expressed, outwardly rectifying chloride current was calcium-insensitive and was blocked by nucleotides applied to the cell surface. Mutation of a putative nucleotide-binding site resulted in loss of nucleotide block but incurred dependence on extracellular calcium concentration. The unusual sequence of this putative channel protein suggests a new class of ion channels not related to other previously cloned chloride channels.     

Cloning of cDNA for the glutamate-binding subunit of an NMDA receptor complex.

The amino acids L-glutamic and L-aspartic acids form the most widespread excitatory transmitter network in mammalian brain. The excitation produced by L-glutamic acid is important in the early development of the nervous system, synaptic plasticity and memory formation, seizures and neuronal degeneration. The receptors activated by L-glutamic acid are a target for therapeutic intervention in neurodegenerative diseases, brain ischaemia and epilepsy. There are two types of receptors for the excitatory amino acids, those that lead to the opening of cation-selective channels and those that activate phospholipase C (ref. 11). The receptors activating ion channels are NMDA (N-methyl-D-aspartate) and kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive receptors. The complementary DNAs for the kainate/AMPA receptor and for the metabotropic receptor have been cloned. We report here on the isolation and characterization of a protein complex of four major proteins that represents an intact complex of the NMDA receptor ion channel and on the cloning of the cDNA for one of the subunits of this receptor complex, the glutamate-binding protein.     

Ion permeation mechanism of the potassium channel

Ion-selective channels enable the specific permeation of ions through cell membranes and provide the basis of several important biological functions; for example, electric signalling in the nervous system. Although a large amount of electrophysiological data is available, the molecular mechanisms by which these channels can mediate ion transport remain a significant unsolved problem. With the recently determined crystal structure of the representative K+ channel (KcsA) from Streptomyces lividans, it becomes possible to examine ion conduction pathways on a microscopic level. K+ channels utilize multi-ion conduction mechanisms, and the three-dimensional structure also shows several ions present in the channel. Here we report results from molecular dynamics free energy perturbation calculations that both establish the nature of the multiple ion conduction mechanism and yield the correct ion selectivity of the channel. By evaluating the energetics of all relevant occupancy states of the selectivity filter, we find that the favoured conduction pathway involves transitions only between two main states with a free difference of about 5 kcal mol(-1). Other putative permeation pathways can be excluded because they would involve states that are too high in energy.     

Mechanism of ion permeation through calcium channels

Calcium channels carry out vital functions in a wide variety of excitable cells but they also face special challenges. In the medium outside the channel, Ca2+ ions are vastly outnumbered by other ions. Thus, the calcium channel must be extremely selective if it is to allow Ca2+ influx rather than a general cation influx. In fact, calcium channels show a much greater selectivity for Ca2+ than sodium channels do for Na+ despite the high flux that open Ca channels can support. Relatively little is known about the mechanism of ion permeation through Ca channels. Earlier models assumed ion independence or single-ion occupancy. Here we present evidence for a novel hypothesis of ion movement through Ca channels, based on measurements of Ca channel activity at the level of single cells or single channels. Our results indicate that under physiological conditions, the channel is occupied almost continually by one or more Ca2+ ions which, by electrostatic repulsion, guard the channel against permeation by other ions. On the other hand, repulsion between Ca2+ ions allows high throughput rates and tends to prevent saturation with calcium.     

Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel

5-hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2-M3), can link binding to gating through a cis-trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis-trans energy gap of the proline analogue and the activation of the channel, suggesting that cis-trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2-M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2-M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.     

Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels

From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. Here we show that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca(2+) concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling.     

The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors

We have cloned and sequenced cDNAs of the strychnine-binding subunit of the rat glycine receptor, a neurotransmitter-gated chloride channel protein of the CNS. The deduced polypeptide shows significant structural and amino-acid sequence homology with nicotinic acetylcholine receptor proteins, indicating that there is a family of genes encoding neurotransmitter-gated ion channels.     

Identification and distribution of 5-HT3 receptors in rat brain using radioligand binding

Functional serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into three subtypes: 5-HT1-like, 5-HT2 and 5-HT3 (ref. 1). Brain binding sites have been identified for both the 5-HT1 and 5-HT2 subtypes. Receptors of the 5-HT3 type have been characterized on isolated peripheral tissue models such as the rat vagus nerve, guinea-pig ileum and isolated rabbit heart. Using these models, selective 5-HT3 receptor antagonists such as MDL 72222 (ref. 5), ICS 205-930 (ref. 6), GR38032F (ref. 7) and BRL 43694 (ref. 8) have been developed. Recently, GR38032F, MDL 72222 and ICS 205-930 have been shown to have behavioural effects in rodents and primates that undoubtedly reflect an action in the central nervous system (refs 9-11 and unpublished observations), suggesting the existence of 5-HT3 receptors in the brain. Here we report direct evidence for the existence of 5-HT3 receptors in rat brain tissue and their distribution, based on high affinity binding of the potent 5-HT3 receptor antagonist 3H-GR65630 to homogenates of rat entorhinal cortex. Selective 5-HT3 receptor antagonists and agonists inhibited binding of 3H-GR65630 with high affinities which correlated well with their actions on the rat isolated vagus nerve. Binding was differentially distributed throughout the brain with high concentrations in cortical and limbic areas.     

The protein kinase A-regulated cardiac Cl- channel resembles the cystic fibrosis transmembrane conductance regulator.

Stimulation of beta-adrenoceptors in cardiac ventricular myocytes activates a strong chloride ion conductance as a result of phosphorylation by cyclic AMP-dependent protein kinase (PKA). This Cl- conductance, which is time- and voltage-independent, counters the tendency of the simultaneously enhanced Ca2+ channel current to prolong the ventricular action potential. Using inside-out giant patches excised from guinea-pig myocytes, we show here that phosphorylation by the PKA catalytic subunit plus Mg-ATP elicits discrete Cl- channel currents. In almost symmetrical Cl- solutions (approximately 150 mM), unitary current amplitude scales with membrane potential, and reverses sign near 0 mV, to yield a single channel conductance of approximately 12 pS. Opening of the phosphorylated channels requires hydrolysable nucleoside triphosphate, indicating that phosphorylation by PKA is necessary, but not sufficient, for channel activation. The properties of these PKA-regulated cardiac Cl- channels are very similar, if not identical, to those of the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial cell Cl- channel whose regulation is defective in patients with cystic fibrosis. The full cardiological impact of these Cl- channels and of their possible malfunction in patients with cystic fibrosis remains to be determined.     

A chloride channel widely expressed in epithelial and non-epithelial cells.

Chloride channels have several functions, including the regulation of cell volume, stabilizing membrane potential, signal transduction and transepithelial transport. The plasma membrane Cl- channels already cloned belong to different structural classes: ligand-gated channels, voltage-gated channels, and possibly transporters of the ATP-binding-cassette type (if the cystic fibrosis transmembrane regulator is a Cl- channel). The importance of chloride channels is illustrated by the phenotypes that can result from their malfunction: cystic fibrosis, in which transepithelial transport is impaired, and myotonia, in which ClC-1, the principal skeletal muscle Cl- channel, is defective. Here we report the properties of ClC-2, a new member of the voltage-gated Cl- channel family. Its sequence is approximately 50% identical to either the Torpedo electroplax Cl- channel, ClC-0 (ref. 8), or the rat muscle Cl- channel, ClC-1 (ref. 9). Isolated initially from rat heart and brain, it is also expressed in pancreas, lung and liver, for example, and in pure cell lines of fibroblastic, neuronal, and epithelial origin, including tissues and cells affected by cystic fibrosis. Expression in Xenopus oocytes induces Cl- currents that activate slowly upon hyperpolarization and display a linear instantaneous current-voltage relationship. The conductivity sequence is Cl- greater than or equal to Br- greater than I-. The presence of ClC-2 in such different cell types contrasts with the highly specialized expression of ClC-1 (ref. 9) and also with the cloned cation channels, and suggests that its function is important for most cells.     

Identification of presynaptic serotonin autoreceptors using a new ligand: 3H-PAT

Binding studies with appropriate labelled ligands have revealed the existence of two types of serotonin (5-HT) receptor, 5-HT1 and 5-HT2, in the central nervous system of mammals. The 5-HT1 type is characterized by a higher affinity for agonists than for antagonists, whereas the 5-HT2 type binds preferentially to antagonists. However, neither of these receptor types apparently corresponds to the presynaptic autoreceptor controlling 5-HT release. In an attempt to identify the presynaptic autoreceptor directly, we synthesized the tritiated derivative of 8-hydroxy-2-(di-n-propylamino) tetralin (PAT), a new tetralin derivative with potent 5-HT agonist properties and carried out binding studies with rat brain membranes. As we report here, in the hippocampus, the properties of 3H-PAT binding sites correspond closely to those of 5-HT1 sites. In contrast, in the striatum, 3H-PAT binding sites exhibit a subcellular distribution and pharmacological characteristics usually associated with presynaptic autoreceptors. Furthermore, a marked loss of 3H-PAT binding sites occurs in the striatum (but not in the hippocampus) after the selective degeneration of serotoninergic fibres in 5,7-hydroxytryptamine (5,7-HT)-treated rats. Conversely, the sprouting of additional 5-HT terminals in the brain stem of adult rats treated at birth with 5,7-HT is associated with an increased density of 3H-PAT binding sites in this region. 3H-PAT thus seems to be a useful ligand for studying the biochemical and pharmacological characteristics of presynaptic autoreceptors in selected regions of rat brain.     

An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 muM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists but was blocked by nanomolar concentrations of Cu2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.     

A single amino-acid difference confers major pharmacological variation between human and rodent 5-HT1B receptors.

Neuropsychiatric disorders such as anxiety, depression, migraine, vasospasm and epilepsy may involve different subtypes of the 5-hydroxytryptamine (5-HT) receptor. The 1B subtype, which has a unique pharmacology, was first identified in rodent brain. But a similar receptor could not be detected in human brain, suggesting the absence in man of a receptor with equivalent function. Recently a human receptor gene was isolated (designated 5-HT1B receptor, 5-HT1D beta receptor, or S12 receptor) which shares 93% identity of the deduced protein sequence with rodent 5-HT1B receptors. Although this receptor is identical to rodent 5-HT1B receptors in binding to 5-HT, it differs profoundly in binding to many drugs. Here we show that replacement of a single amino acid in the human receptor (threonine at residue 355) with a corresponding asparagine found in rodent 5-HT1B receptors renders the pharmacology of the receptors essentially identical. This demonstrates that the human gene does indeed encode a 1B receptor, which is likely to have the same biological functions as the rodent 5-HT1B receptor. In addition, these findings show that minute sequence differences between homologues of the same receptor from different species can cause large pharmacological variation. Thus, drug-receptor interactions should not be extrapolated from animal to human species without verification.     

Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs.

The glutamate receptor (GluR) channel plays a key part in brain function. Among GluR channel subtypes, the NMDA (N-methyl-D-aspartate) receptor channel which is highly permeable to Ca2+ is essential for the synaptic plasticity underlying memory, learning and development. Furthermore, abnormal activation of the NMDA receptor channel may trigger the neuronal cell death observed in various brain disorders. A complementary DNA encoding a subunit of the rodent NMDA receptor channel (NMDAR1 or zeta 1) has been cloned and its functional properties investigated. Here we report the identification and primary structure of a novel mouse NMDA receptor channel subunit, designated as epsilon 1, after cloning and sequencing the cDNA. The epsilon 1 subunit shows 11-18% amino-acid sequence identity with rodent GluR channel subunits that have been characterized so far and has structural features common to neurotransmitter-gated ion channels. Expression from cloned cDNAs of the epsilon 1 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional GluR channels with high activity and characteristics of the NMDA receptor channel. Furthermore, the heteromeric NMDA receptor channel can be activated by glycine alone.     

A voltage-gated proton-selective channel lacking the pore domain

Voltage changes across the cell membrane control the gating of many cation-selective ion channels. Conserved from bacteria to humans, the voltage-gated-ligand superfamily of ion channels are encoded as polypeptide chains of six transmembrane-spanning segments (S1-S6). S1-S4 functions as a self-contained voltage-sensing domain (VSD), in essence a positively charged lever that moves in response to voltage changes. The VSD 'ligand' transmits force via a linker to the S5-S6 pore domain 'receptor', thereby opening or closing the channel. The ascidian VSD protein Ci-VSP gates a phosphatase activity rather than a channel pore, indicating that VSDs function independently of ion channels. Here we describe a mammalian VSD protein (H(V)1) that lacks a discernible pore domain but is sufficient for expression of a voltage-sensitive proton-selective ion channel activity. H(v)1 currents are activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H+-selective, and Zn2+-sensitive. Mutagenesis of H(v)1 identified three arginine residues in S4 that regulate channel gating and two histidine residues that are required for extracellular inhibition of H(v)1 by Zn2+. H(v)1 is expressed in immune tissues and manifests the characteristic properties of native proton conductances (G(vH+)). In phagocytic leukocytes, G(vH+) are required to support the oxidative burst that underlies microbial killing by the innate immune system. The data presented here identify H(v)1 as a long-sought voltage-gated H+ channel and establish H(v)1 as the founding member of a family of mammalian VSD proteins.     

Potassium conductances in hippocampal neurons blocked by excitatory amino-acid transmitters

Excitatory amino acids mediate fast synaptic transmission in the central nervous system through the activation of at least three distinct ionotropic receptors: N-methyl-D-aspartate (NMDA), the alpha-amino-3-hydroxy-5-methyl-isoxasole-4-propionate (AMPA)/quisqualate (QUIS) and the kainate subtypes (for reviews, see refs 1, 2). They also activate the additional QUIS 'metabotropic' receptor (sensitive to trans-1-amino-cyclopentyl-1,3-dicarboxylate, ACPD) linked to inositol phospholipid metabolism. We have used hippocampal slice cultures to study the electrophysiological consequences of the metabotropic response. We find that activation of an ACPD-sensitive QUIS receptor produces a 'slow' excitation of CA3 pyramidal cells, resulting from depression of a Ca2(+)-dependent K+ current and a voltage-gated K+ current. Combined voltage-clamp and microfluorometric recordings show that, although these receptors can trigger an increase in intracellular Ca2+ concentration, suppression of K+ currents is independent of changes in intracellular Ca2+. These effects closely resemble those induced by activating muscarinic acetylcholine receptors in the same neurons and suggest that excitatory amino acids not only act as fast ionotropic transmitters but also as slow neuromodulatory transmitters.