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Human Choline Transporter cDNA
Vanderbilt researchers report the isolation and characterization of a human cDNA encoding the high affinity, hemicholinium-3 sensitive choline transporter. This transporter is expressed in cholinergic terminals, and it provides for transport into cholinergic terminals of choline, the precursor for acetylcholine biosynthesis. The cDNA, through HC-3 radio ligand binding assays or choline transport assays, allows for high-throughput screening of choline transporter directed agents or as a negative screen to insure specificity for nicotinic and muscarinic acetylcholine receptor-directed agents (as well as other pharmaceutics). The choline transporter in vivo is highly regulated, and the human choline transporter's regulation is poorly understood. Use of the cDNA may allow for the development of novel cholinergic therapeutics targeted at choline transporter modulation. Antibodies directed against the human choline transporter should be useful probes of human cholinergic neurons. Sequences in the human choline transporter cDNA should allow for the generation of transporter specific gene probes that can be queried by in situ hybridization, PCR analyses of transporter gene expression or "gene chip" approaches evaluating alterations in presynaptic cholinergic function.
This transporter is responsible for choline transport into cholinergic nerve terminals to provide for acetylcholine biosynthesis. Acetylcholine is a major neurotransmitter in the brain and periphery. Acetylcholine signaling is involved in motor and cognitive function, and altered cholinergic signaling is evident in neuromuscular disorders, as well as disruptions of higher cognitive function occurring in Alzheimer's disease. This invention provides for a new tool to develop cholinergic focused therapeutics. This cDNA is of human origin, and thus reagents developed to interfere or modulate the transporter should be more clinically relevant.
Agents that modulate the choline transporter represent a novel opportunity to influence cholinergic signaling therapeutically for neuromuscular, autonomic or CNS disorders. Antibodies derived against specific sequences of the human choline transporter cDNA should be useful in identifying cholinergic neurons in human brain sections postmortem. The availability of the human choline transporter cDNA should also provide an additional test for specificity in the development of acetylcholine receptor therapeutics, as low affinity against the human choline transporter is desired and could be readily detected in binding assays using recombinantly expressed cDNA.
As no cell lines exist that express the human choline transporter, researchers must validate lack of cross-reactivity with the transporter of their medications using postmortem brain tissue. Stable cell lines expressing the transporter will allow for routine membrane preparations enriched in the human choline transporter for testing for such cross-reactivity or for a direct product development of choline transporter modulators. Thus, the human choline transporter cDNA allows for the development of a novel radio ligand binding assay using recombinant transporters expressed in mammalian cell lines. The transporter's high affinity for the radio ligand [3H] hemicholinium-3 can be readily detected in membrane binding assays. The cDNA could also be used to develop novel viral vectors to enhance cholinergic signaling in model systems or disease states. The cDNA or sequences therein could be used for quantitative evaluation of cholinergic gene expression via PCR or used in arrayed approaches termed "gene chips" to evaluate the integrity and regulation of cholinergic signaling. The cDNA may be useful in mapping mutations to the human choline transporter gene that may contribute to autonomic, motor or cognitive disturbances.
The cDNA or sequences therein could be used for quantitative evaluation of cholinergic gene expression via PCR, or they could be used in arrayed approaches termed "gene chips" to evaluate the integrity and regulation of cholinergic signaling. The cDNA may also be useful in mapping mutations to the human choline transporter gene that may contribute to autonomic, motor or cognitive disturbances.
To date, there are no agents in clinical use directed at the choline transporter. Thus, there is no current precedent for utility of drugs targeted at the transporter, though this could also be viewed as an opportunity. Additionally, antagonists targeted to the transporter would diminish the choline transporter function and might not be the first priority for companies seeking to augment cholinergic tone. Drugs that specifically up-regulate the choline transporter are not known, and thus companies considering this approach will need to invest additional resources in studies to identify functional modulators. For the latter case, radio ligand binding assays, which many companies are most familiar with, may be of little value. Companies already committed to high-throughput functional screens of transporter or receptor function should already be poised to develop such agents, but this may narrow the list.
US Patent 7,338,799 was filed December 1, 2003 and issued March 4, 2008.!doctype>
Inventors:Randy BlakelySubbu ApparsundarumShawn Ferguson