Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine. We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation. The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast. The gene was called CTL1 for its choline transporter-like properties. The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3.5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon. In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain. High levels of rCTL1 were also identified in the mucosal cell layer of the colon. Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2-4). These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons. C ells contain large amounts of choline incorporated in their membranes, and all plant and animal cells, including unicellular organisms, absorb free choline as a nutrient. At cholinergic nerve terminals, the sodium-dependent high affinity choline uptake mechanism that is coupled to acetylcholine synthesis has been particularly well characterized at the functional level (1-3) but has thus far eluded diverse attempts at identification based on protein purification (4), even in conjunction with the use of a selective and irreversible ligand (5).The yeast choline transporter has been isolated by homologous complementation by using a choline transport deficient yeast strain (6), and heterologous complementation of yeast mutants with a yeast expression library made with Arabidopsis thaliana cDNA has been used to identify the potassium transporter of plants (7). We decided to adapt the strategy of complementation cloning to the problem of neuronal choline transport by using choline transport deficient yeast with the highly cholinergic electric lobes of Torpedo as a source of cDNA. However, the single clone isolated, capable of partially restoring choline uptake in the mutant yeast, did not resemble the yeast choline transporter (8) and is thus considered to be a heterologous suppressor for the choline transport mutation. We report the sequences of genes both homologous and orthologous to the Torpedo choline transporter-like protein, tCTL1, and we begin the characterization of this new family of transmembrane proteins by studying ...
We show here that the choline transporter-like (CTL) family is more extensive than initially described with five genes in humans and complex alternative splicing. In adult rat tissues, CTL2-4 mRNAs are mainly detected in peripheral tissues, while CTL1 is widely expressed throughout the nervous system. During rat post-natal development, CTL1 is expressed in several subpopulations of neurones and in the white matter, where its spatio-temporal distribution profile recalls that of myelin basic protein, an oligodendrocyte marker. We identified two major rat splice variants of CTL1 (CTL1a and CTL1b) differing in their carboxy-terminal tails with both able to increase choline transport after transfection in neuroblastoma cells. In the developing brain, CTL1a is expressed in both neurones and oligodendroglial cells, whereas CTL1b is restricted to oligodendroglial cells. These findings suggest specific roles for CTL1 splice variants in both neuronal and oligodendrocyte physiology.
Presynaptic injection of cyclic ADP‐ribose (cADPR), a modulator of the ryanodine receptor, increased the postsynaptic response evoked by a presynaptic spike at an identified cholinergic synapse in the buccal ganglion of Aplysia californica. The statistical analysis of long duration postsynaptic responses evoked by square depolarizations of the voltage‐clamped presynaptic neurone showed that the number of evoked acetylcholine (ACh) quanta released was increased following cADPR injection. Overloading the presynaptic neurone with cADPR led to a transient increase of ACh release followed by a depression. cADPR injections did not modify the presynaptic Ca2+ current triggering ACh release. Ca2+ imaging with the fluorescent dye rhod‐2 showed that cADPR injection rapidly increased the free intracellular Ca2+ concentration indicating that the effects of cADPR on ACh release might be related to Ca2+ release from intracellular stores. Ryanodine and 8‐amino‐cADPR, a specific antagonist of cADPR, decreased ACh release. ADP‐ribosyl cyclase, which cyclizes NAD+ into cADPR, was present in the presynaptic neurone as shown by reverse transcriptase‐polymerase chain reaction experiments. Application of NAD+, the substrate of ADP‐ribosyl cyclase, increased ACh release and this effect was prevented by both ryanodine and 8‐amino‐cADPR. These results support the view that Ca2+‐induced Ca2+ release might be involved in the build‐up of the Ca2+ concentration which triggers ACh release, and thus that cADPR might have a role in transmitter release modulation.
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