Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1995-06-07
2002-11-26
Pak, Michael (Department: 1646)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C536S023500
Reexamination Certificate
active
06485967
ABSTRACT:
This invention relates to DNA encoding human neuronal nicotinic acetylcholine receptor protein subunits, as well as the proteins themselves. In particular, human neuronal nicotinic acetylcholine receptor &agr;-subunit-encoding DNA, &agr;-subunit proteins, &bgr;-subunit-encoding DNA, &bgr;-subunit proteins, and combinations thereof are provided.
BACKGROUND OF THE INVENTION
Ligand-gated ion channels provide a means for communication between cells of the central nervous system. These channels convert a signal (e.g., a chemical referred to as a neurotransmitter) that is released by one cell into an electrical signal that propagates along a target cell membrane. A variety of neurotransmitters and neurotransmitter receptors exist in the central and peripheral nervous systems. Five families of ligand-gated receptors, including the nicotinic acetylcholine receptors (nAChRs) of neuromuscular and neuronal origins, have been identified (Stroud et al. (1990) Biochemistry 29:11009-11023). There is, however, little understanding of the manner in which the variety of receptors generates different responses to neurotransmitters or to other modulating ligands in different regions of the nervous system.
The nicotinic acetylcholine receptors (nAChRs) are multisubunit proteins of neuromuscular and neuronal origins. These receptors form ligand-gated ion channels that mediate synaptic transmission between nerve and muscle and between neurons upon interaction with the neurotransmitter acetylcholine (ACh). Since various neuronal nicotinic acetylcholine receptor (nAChR) subunits exist, a variety of nAChR compositions (i.e., combinations of subunits) exist. The different nAChR compositions exhibit different specificities for various ligands and are thereby pharmacologically distinguishable. Thus, the nicotinic acetylcholine receptors expressed at the vertebrate neuromuscular junction, in vertebrate sympathetic ganglia and in the vertebrate central nervous system have been distinguished on the basis of the effects of various ligands that bind to different nAChR compositions. For example, the elapid &agr;-neurotoxins that block activation of nicotinic acetylcholine receptors at the neuromuscular junction do not block activation of some neuronal nicotinic acetylcholine receptors that are expressed on several different neuron-derived cell lines.
Muscle nAChR is a glycoprotein composed of five subunits with the stoichiometry (&agr;)
2
&bgr;(&ggr; or &egr;) &dgr;. Each of the subunits has a mass of about 50-60 kilodaltons (kd) and is encoded by a different gene. The (&agr;)
2
&bgr;(&ggr; or &egr;) &dgr; complex forms functional receptors containing two ligand binding sites and a ligand-gated transmembrane channel. Upon interaction with a cholinergic agonist, muscle nicotinic nAChRs conduct sodium ions. The influx of sodium ions rapidly short-circuits the normal ionic gradient maintained across the plasma membrane, thereby depolarizing the membrane. By reducing the potential difference across the membrane, a chemical signal is transduced into an electrical signal at the neuromuscular junction that induces muscle contraction.
Functional muscle nicotinic acetylcholine receptors have been formed with &agr;&bgr;&dgr;&ggr; subunits, &agr;&bgr;&ggr; subunits, &agr;&bgr;&dgr; add subunits, &agr;&bgr;&ggr; subunits or &agr;&dgr; subunits, but not with only one subunit (see e.g., Kurosaki et al. (1987) FEBS Lett. 214: 253-258; Camacho et al. (1993) J. Neuroscience 13:605-613). In contrast, functional neuronal nAChRs can be formed from a subunits alone or combinations of &agr; and &bgr; subunits. The larger a subunit is generally believed to be a ACh-binding subunit and the lower molecular weight &bgr; subunit is generally believed to be the structural subunit, although it has not been definitively demonstrated that the a subunit does not have the ability to bind ACh or participate in the formation of the ACh binding site. Each of the subunits which participate in the formation of a functional ion channel are, to the extent they contribute to the structure of the resulting channel, “structural” subunits, regardless of their ability (or inability) to bind ACh. Neuronal nAChRs, which are also ligand-gated ion channels, are expressed in ganglia of the autonomic nervous system and in the central nervous system (where they mediate signal transmission), in post-synaptic locations (where they modulate transmission), and in pre- and extra-synaptic locations (where they modulate neurotransmission and may have additional functions; Wonnacott et al.,
In: Progress in Brain Research
(A. Nordberg et al., (Eds) Elsevier, Amsterdam) 157-163 (1990)).
DNA encoding nAChRs has been isolated from several sources. Based on the information available from such work, it has been evident for some time that nAChRs expressed in muscle, in autonomic ganglia, and in the central nervous system are functionally diverse. This functional diversity could be due, at least in part, to the large number of different nAChR subunits which exist. There is an incomplete understanding, however, of how (and which) nAChR subunits combine to generate unique nAChR subtypes, particularly in neuronal cells. Indeed, there is evidence that only certain nAChR subtypes may be involved in diseases such as Alzheimer's disease. Moreover, it is not clear whether nAChRs from analogous tissues or cell types are similar across species.
Accordingly, there is a need for the isolation characterization of DNAs encoding each human neuronal nAChR subunit, recombinant cells containing such subunits and receptors prepared therefrom. In order to study the function of human neuronal nAChRs and to obtain disease-specific pharmacologically active agents, there is also a need to obtain isolated (preferably purified) human neuronal nAChRs, and isolated (preferably purified) human neuronal nAChR subunits. In addition, there is also a need to develop assays to identify such pharmacologically active agents.
The availability of such DNAs, cells, receptor subunits and receptor compositions will eliminate the uncertainty of speculating as to human neuronal nAChR structure and function based on predictions drawn from non-human nAChR data, or human or non-human muscle or ganglia nAChR data.
Therefore, it is an object herein to isolate and characterize DNA encoding subunits of human neuronal nicotinic acetylcholine receptors. It is also an object herein to provide methods for recombinant production of human neuronal nicotinic acetylcholine receptor subunits. It is also an object herein to provide purified receptor subunits and to provide methods for screening compounds to identify compounds that modulate the activity of human neuronal nAChRs.
These and other objects will become apparent to those of skill in the art upon further study of the specification and claims.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, there are provided isolated DNAs encoding novel human alpha and beta subunits of neuronal nAChRs. In particular, isolated DNA encoding human &agr;
6
and &bgr;
3
subunits of neuronal nAChRs are provided. Messenger RNA and polypeptides encoded by the above-described DNA are also provided.
Further in accordance with the present invention, there are provided recombinant human neuronal nicotinic nAChR subunits, including &agr;
6
and &bgr;
3
subunits, as well as methods for the production thereof. In addition, recombinant human neuronal nicotinic acetylcholine receptors containing at least one human neuronal nicotinic nAChR subunit are also provided, as well as methods for the production thereof. Further provided are recombinant neuronal nicotinic nAChRs that contain a mixture of one or more nAChR subunits encoded by a host cell, and one or more nAChR subunits encoded by heterologous DNA or RNA (i.e., DNA or RNA as described herein that has been introduced into the host cell), as well as methods for the production thereof.
Plasmids containing DNA encoding the above-described subunits are also provided. Recombinant cells containing the above-described DNA, mR
Elliott Kathryn J.
Harpold Michael M.
Kohli Vineet
Merck & Co. , Inc.
Pak Michael
Tribble Jack L.
LandOfFree
Human neuronal nicotinic acetylcholine receptor &agr;6 and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Human neuronal nicotinic acetylcholine receptor &agr;6 and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Human neuronal nicotinic acetylcholine receptor &agr;6 and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2925396