Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-07-24
2004-11-16
Shukla, Ram R. (Department: 1632)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S007100, C435S007200, C435S007210, C435S325000
Reexamination Certificate
active
06818391
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of neurobiology. In particular this invention pertains to the identification of a number of novel glutamate transporters.
BACKGROUND OF THE INVENTION
Excitatory neurotransmission involves the exocytotic release of synaptic vesicles filled with glutamate. Glutamate is synthesized in the cytoplasm, and undergoes transport into synaptic vesicles for quantal release. Like the uptake of other classical transmitters, vesicular glutamate transport depends on a proton electrochemical gradient (&Dgr;&mgr;
H+
) generated by the vacuolar H
+
-ATPase (Disbrow et al. (1982)
Biochemical and Biophysical Res. Commun
., 108: 1221-1227; Naito and Ueda (1983)
J. Biol. Chem
. 258: 696-6990). However, unlike the uptake of monoamines and acetylcholine, vesicular glutamate transport relies predominantly on the electrical component of this gradient (&Dgr;&PSgr;) rather than the chemical component (&Dgr;pH) (Carlson et al. (1989)
J. Biol. Chem
. 264: 7369-7376; Maycox et al. (1988)
J. Biol. Chem
. 263: 15423-15428). Consistent with this different mechanism, the two protein families responsible for vesicular uptake of monoamines, ACh and &ggr;-aminobutyric acid (GABA) (Liu and Edwards (1997)
Ann. Rev. Neurosci
. 20: 125-156; Reimer et al. (1998)
Curr. Opin. Neurobiol
. 8: 405-412; Schuldiner et al. (1995)
Physiol. Rev
. 75, 369-392; Varoqui et al., (1994)
FEBS Lett
. 342: 97-102) have not been found to include a glutamate transporter.
SUMMARY OF THE INVENTION
This invention pertains to the identification of a family of novel glutamate transporters. In particular, certain brain-specific Na+-dependent phosphate transporter are shown to be glutamate transporters. Designated herein as VGLUT glutamate transporters, members of this family include, but are not limited to VGLUT1 (formerly BNPI), VGLUT2 (formerly DNPI), and VGLUT3.
The VGLUT transporters of this invention provide good targets to screen for agents that modulate (e.g. upregulate or downregulate) glutamate uptake by a cell (e.g. by a neuron). Thus, in one embodiment, this invention provides a method of screening for an agent that modulates the uptake of glutamate into a cell (e.g. into a synaptic vesicle). The method preferably involves contacting a cell comprising a nucleic acid selected from the group consisting of VGLUT1, VGLUT2, and VGLUT3 with a test agent; and detecting expression or activity of VGLUT1, VGLUT2, or VGLUT3 where an increase or decrease in the expression or activity of VGLUT1, VGLUT2, or VGLUT3 as compared to a control indicates that the test agent modulates the uptake of glutamate into a cell. The control can be a positive or a negative control. In certain embodiments, the control is a negative control comprising contacting a cell at a lower concentration or in the absence concentration of the test agent. Preferred cells include somatic cells or oocytes. Particularly preferred cells include vertebrate cells, more preferably mammalian (e.g. human, rabbit, mouse, goat, equine, porcine, feline, canine, etc.) cells.
In certain preferred embodiments, the detecting comprises detecting a VGLUT (e.g. VGLUT1 and/or VGLUT2, and/or VGLUT3, etc.) nucleic acid and/or a (VGLUT) polyeptide (e.g. VGLUT1 polypeptide and/or VGLUT2 polypeptide, and/or VGLUT3 polypeptide, etc.) VGLUT1 polypeptide, a VGLUT2 polypeptide, or a VGLUT3 polypeptide. In certain embodiments, the VGLUT nucleic acid is detected via a nucleic acid hybridization (e.g., a Northern blot, a Southern blot using DNA derived from the VGLUT1, VGLUT2, or VGLUT3 mRNA, an array hybridization, an affinity chromatography, an in situ hybridization, etc.) and/or a nucleic acid amplification (e.g. PCR, LCR, etc.).
In preferred embodiments, the VGLUT polypeptide is detected via a method such as capillary electrophoresis, Western blot, mass spectroscopy, ELISA, immunochromatography, immunohistochemistry, thin layer chromatography (TLC), and the like. In preferred embodiments, the VGLUT polypeptide activity involves detecting glutamate transport in a cell expressing an endogenous or a heterologous VGLUT polypeptide (e.g., VGLUT1, VGLUT2, VGLUT3, etc.). In certain embodiments, the test agent is not one or more of the following: an antibody, a nucleic acid, a protein, and an agent that alters &Dgr;pH or &Dgr;&PSgr;. In particularly embodiments the test agent is a small organic molecule. In certain embodiments, the methods further comprise comparing the level of expression or activity of VGLUT1 with the level of expression or activity of VGLUT2 and/or VGLUT3.
In another embodiment, this invention provides a method of prescreening for a potential modulator of glutamate transporter activity (e.g. glutamate uptake into a synaptic vesicle). The method preferably involves contacting a VGLUT glutamate transporter polypeptide (e.g. VGLUT1, VGLUT2, VGLUT3, etc.) or a nucleic acid encoding a VGLUT glutamate transporter polypeptide with a test agent; and detecting binding (e.g. specific binding) of the test agent to the VGLUT glutamate transporter polypeptide or to the nucleic acid encoding a VGLUT glutamate transporter polypeptide where specific binding of said test agent to the VGLUT glutamate transporter polypeptide or VGLUT nucleic acid indicates that the test agent is a potential modulator of glutamate transporter activity. The method can, optionally, further involve recording test agents that specifically bind to the VGLUT glutamate transporter polypeptide or to the nucleic acid encoding a VGLUT glutamate transporter polypeptide in a database of candidate modulators of glutamate transporter activity. In certain embodiments, the test agent is not one or more of the following: an antibody, a nucleic acid, a protein, and an agent that alters &Dgr;pH or &Dgr;&PSgr;. In particularly embodiments the test agent is a small organic molecule. The detecting can involve detecting specific binding of the test agent to the VGLUT nucleic acid (e.g. via Northern blot, a Southern blot using DNA derived from the VGLUT mRNA, array hybridization, affinity chromatography, in situ hybridization, etc.). The detecting can also involve detecting specific binding of the test agent to the VGLUT glutamate transporter polypeptide (e.g. via capillary electrophoresis, Western blot, mass spectroscopy, ELISA, immunochromatography, thin layer chromatography, and immunohistochemistry). In certain embodiments, the test agent is contacted directly to the VGLUT glutamate transporter polypeptide or to the nucleic acid encoding a VGLUT glutamate transporter polypeptide. in certain embodiments, the test agent is contacted to a cell containing the VGLUT glutamate transporter polypeptide or to said nucleic acid encoding a VGLUT glutamate transporter polypeptide. The cell can be a cell cultured ex vivo.
In still another embodiment, this invention provides a cell comprising a heterologous nucleic acid encoding a glutamate transporter wherein said glutamate transporter is selected from the group consisting of VGLUT1, VGLUT2, and VGLUT3. Preferred cells include somatic cells (e.g. nerve cells), or oocytes. Particularly preferred cells include vertebrate cells, more preferably mammalian (e.g. human, rabbit, mouse, goat, equine, porcine, feline, canine, etc.) cells. In a particularly preferred embodiment, the cell transports glutamate via the heterologous VGLUT glutamate transporter. In one embodiment, the cell is a pheochromocytoma PC12 cell.
In yet another embodiment, this invention provides a method of increasing glutamate transport by a mammalian cell. The method can involve transfecting the cell with a nucleic acid encoding a VGLUT polypeptide selected from the group consisting of VGLUT1, VGLUT2, and VGLUT3. The VGLUT nucleic acid is preferably operably linked to a constitutive, tissue-specific or inducible promoter.
This invention also provides a method of decreasing glutamate uptake into a cell. The method involves downregulating expression or activity of a VGLUT polypeptide in the cell. In certain embodiments, the inhibiting comprises a method selected from
Bellocchio Elizabeth E.
Edwards Robert H.
Fremeau, Jr. Robert T.
Reimer Richard J.
Quine Intellectual Property Law Group P.C.
Shukla Ram R.
The Regents of the University of California
LandOfFree
Glutamate transporters does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Glutamate transporters, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Glutamate transporters will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3343228