Tatsuya Haga

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist.

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

Choline uptake systems of rat brain synaptosomes

The uptake of [3H]choline into synaptosomes and the subsequent synthesis of [3H]acetylcholine were examined as functions of choline, Na+ and hemicholinium-3 concentrations in the incubation medium. The results indicate the presence of two carrier-mediated uptake systems for choline. One system requires Na+ for the uptake and the other does not. About 60% of the choline taken up into synaptosomes by the Na+-dependent system were converted to acetylcholine, whereas only a few percent of the choline taken up into synaptosomes by the Na+-independent system were converted to acetylcholine.Km, the choline concentration giving half-maximum transport was 4–8 μM for the Na+-dependent system and about 40 μM for the Na+-independent system.Ki values for hemicholinium-3 were 0.05–0.1 μM for the Na+-dependent system and 40–50 μM for the Na+-independent system.

Identification of G protein-coupled receptor genes from the human genome sequence

We have identified novel G protein‐coupled receptors (GPCRs) with no introns in the coding region from the human genome sequence: 322 olfactory receptors; 22 taste receptors; 128 registered GPCRs for endogenous ligands; 50 novel GPCR candidates homologous to registered GPCRs for endogenous ligands; and 59 novel GPCR candidates not homologous to registered GPCRs. The total number of GPCRs with and without introns in the human genome was estimated to be approximately 950, of which 500 are odorant or taste receptors and 450 are receptors for endogenous ligands.

Identification and characterization of the high-affinity choline transporter

In cholinergic neurons, high-affinity choline uptake in presynaptic terminals is the rate-limiting step in acetylcholine synthesis. Using information provided by the Caenorhabditis elegans Genome Project, we cloned a cDNA encoding the high-affinity choline transporter from C. elegans (cho-1). We subsequently used this clone to isolate the corresponding cDNA from rat (CHT1). CHT1 is not homologous to neurotransmitter transporters, but is homologous to members of the Na+-dependent glucose transporter family. Expression of CHT1 mRNA is restricted to cholinergic neurons. The characteristics of CHT1-mediated choline uptake essentially match those of high-affinity choline uptake in rat brain synaptosomes.

Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence

The complete amino acid sequence of the porcine cardiac muscarinic acetylcholine receptor has been deduced by cloning and sequencing the cDNA. The tissue location of the RNA hybridizing with the cDNA suggests that this muscarinic receptor species represents the M2 subtype.

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [14C]choline, about half or more of which was converted to [I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μM‐[14C] choline and neostigmine. The amount of [14C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na+ concentration in the incubation medium. The effect of Na+ on the uptake of [I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.

Functional characterization of the human high-affinity choline transporter1

Na+‐dependent, high‐affinity choline uptake in cholinergic neurons is the rate‐limiting step in acetylcholine synthesis. Here we report the molecular cloning and functional characterization of the human high‐affinity choline transporter (hCHT1). The hCHT1 exhibits significant homology with known members of the Na+‐dependent glucose transporter family, but not with members of the neurotransmitter transporter family. The human CHT1 gene is 25 kb in length with 9 exons and was assigned to chromosome II at position IIq11–12. Northern blot analysis showed that a 5.4 kb hCHT1 transcript was expressed exclusively in tissues containing cholinergic neurons. When expressed in Xenopus oocytes, the human clone induced Na+‐ and Cl−‐dependent, high‐affinity choline uptake, which was sensitive to the specific inhibitor hemicholinium‐3, with a K i of 1.3 nM. The hCHT1‐mediated choline uptake increased with increasing concentrations of choline, Na+ and Cl−, with EC50 values of 2.0 μM, 76 mM, and 48 mM, and with apparent Hill coefficients of 1, 2.5 and 2.3, respectively.

Distribution of the high-affinity choline transporter in the central nervous system of the rat

In cholinergic nerve terminals, Na(+)- and Cl(-)-dependent, hemicholinium-3-sensitive, high-affinity choline uptake is thought to be the rate-limiting step in acetylcholine synthesis. The high-affinity choline transporter cDNA responsible for the activity was recently cloned. Here we report production of a highly specific antibody to the high-affinity choline transporter and distribution of the protein in the CNS of the rat. The antibody stained almost all known cholinergic neurons and their terminal fields. High-affinity choline transporter-immunoreactive cell bodies were demonstrated in the olfactory tubercle, basal forebrain complex, striatum, mesopontine complex, medial habenula, cranial nerve motor nuclei, and ventral horn and intermediate zone of the spinal cord. Noticeably, high densities of high-affinity choline transporter-positive axonal fibers and puncta were encountered in many brain regions such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem. Transection of the hypoglossal nerve resulted in a loss of high-affinity choline transporter immunoreactivity in neurons within the ipsilateral hypoglossal motor nucleus, which paralleled a loss of immunoreactivity to choline acetyltransferase. The antibody also stained brain sections from human and mouse, suggesting cross-reactivity. These results confirm that the high-affinity choline transporter is uniquely expressed in cholinergic neurons and is efficiently transported to axon terminals. The antibody will be useful to investigate possible changes in cholinergic cell bodies and axon terminals in human and rodents under various pathological conditions.

G protein--coupled receptor kinases.

Rhodopsin, P-adrenergic receptors (PARS), and muscarinic acetylcholine receptors (mAChRs) are prototypes of receptors that are linked to GTP-binding regulatory proteins (G proteins) and that interact with transducin (G), G5 , and Gi/Go/Gq , respectively . These receptors are known to be phosphorylated in a lightor agonist-dependent manner by G protein-coupled receptor kinases (GR kinases) such as rhodopsin kinase and PAR kinase (PARK) . This phosphorylation is thought to be involved in the desensitization of these receptors . Several reviews have been published on the phosphorylation and desensitization of rhodopsin (Kiihn, 1987 ; Palczewski and Benovic, 1991), OARS (Benovic et al ., 1988x ; Lefkowitz et al., 1990 ; Palczewski and Benovic, 1991 ; Lefkowitz, 1993), and mAChRs (Hosey, 1992; Haga et al ., 1993) . In the present review, we will deal with the properties of GR kinases, particularly the regulation of GR kinase activity by G proteins .

High-affinity choline transporter

The cholinergic neurons have long been a model for biochemical studies of neurotransmission. The components responsible for cholinergic neurotransmission, such as choline acetyltransferase, vesicular acetylcholine transporter, nicotinic and muscarinic acetylcholine receptors, and acetylcholine esterase, have long been defined as functional units and then identified as molecular entities. Another essential component in the cholinergic synapses is the one responsible for choline uptake from the synaptic cleft, which is thought to be the rate-limiting step in acetylcholine synthesis. A choline uptake system with a high affinity for choline has long been assumed to be present in cholinergic neurons. Very recently, the molecular entity for the high-affinity choline transporter was identified and is designated CHT1. CHT1 mediates Na+- and Cl−-dependent choline uptake with high sensitivity to hemicholinium-3. CHT1 has been characterized both at the molecular and functional levels and was confirmed to be specifically expressed in cholinergic neurons.