Inependent projects

Rig1

Hi,

Here's a tentative proposal for our project...right now it'd be largely a
replication of a study last year, but we're thinking of more original
things based around

Modulation of N-methyl-D-aspartate receptor function by
glycine transport

Richard Bergeron, Torsten M. Meyer, Joseph T. Coyle*, and Robert W. Greene
Proc Natl Acad Sci USA 1998 Dec 22; 95(26):15730-4
 

Control of NMDA receptor activation by a glycine transporter co-expressed
in Xenopus oocytes.
Supplisson S, Bergman C; J Neurosci 1997 Jun 15;17(12):4580-90
These papers report that glycine excites NMDA receptor/channel so if
you block glycine transporter, the NMDA current will be larger because
there is more glycine around. And these glycine transport antagonists
are/will be used as drugs for schizophrenia (it's similar to serotonin
transporter antagonist as anti-depressants). The earlier paper reports the
modulation effect in Xenopus oocytes, while the later one used whole-cell
patch-clamping in hippocampal slices. The paper that looked at Xenopus
oocytes didn't look at antagonist activity, however. We'd want to
look at NMDAR / GLYT1 co-expression, and look at both modulation and
antagonist activity. We'd probably want to do voltage-clamping rather than
patch-clamping.

What we'd need is:  1 healthy oocyte
                      NMDA receptors
                      GLY1 transporters
                      2-amino-5-phosphonopentanoic acid (NMDA
    antagonist)
                      7-chlorokynurenate (GLY1 antagonist)
                      glycine

Experiments: 1. measure current (control)
             1a. add glycine and measure current (control2)
             2. add NMDA antagonist and measure current to see
                how much current is due to NMDA
             2a. maybe add different concantrations of glycine to find
                 saturation level for NMDA current
             3. add GLY1 antagonist
             4. measure current - it should increase
             5. repeat step 2

-------------------------------------

That's the proposal so far. Let us know what you think, and we'll revise
it.

-Rory, Svjetlana, Kayla, Steven

Rig2

Josh Maurer
Eugene Pivovarov
Luis E. Vazquez

Title:
Effects of DTT on the ACh nicotinic receptor

Introduction:
It has been suggested, that upon binding, acetylcholine (ACh) blocks a
readily reducible disulfide bond between Cys 192 and Cys 193 in the
a-subunit of the nicotinic acetocholine receptor.   The binding of ACh to
the nicotinic receptor causes ion conductance through the channel followed
by its desensitization.

Previous work has shown that the reducing agent dithiothreitol (DTT) can
inactivate the Torpedo nAChR, by reduction of the disulfide bonds between
C192 and C193 in the a-subunits.  We are interested in studying this
effect more carefully by testing whether simultaneous application of DTT
and various agonists decreases the number of receptors inactivated by DTT
reduction.  It is expected that if agonist occupies the nAChR's binding
site, DTT will be unable to reduce the disulfide bond between Cys 192-Cys
193.  The extent to which DTT reduction of the 192-193 disulfide bond is
inhibited by agonist, will depend on the number of receptors to which the
agonist is bound, the length of time which DTT is applied, and on the
binding strength of the agonist.  This can be quantitated by first
determining the amount of DTT needed to inactivate 50% of the ACh
receptors and then redetermining this when agonist and DTT are applied
simultaneously.

Experimental overview:

a. Current will be measured from nAChR mRNA injected oocytes held under
voltage-clamp.
b. The concentration of DTT needed to inactivate 50% of the ACh receptors
will be determined by applying various DTT concentrations and measuring
the current induce by ACh, at its EC50 concentration, before and after DTT
application.
c. To examine agonist protection of the disulfide bond a solution
containing DTT  plus agonist will be applied in place of the DTT solution
and the experiment will be repeated as above.  It is expected that higher
concentrations of DTT will be necessary in order to achieve inactivation
of 50% of the ACh receptors.   Acetocholine and nicotine will be examined
as agonists.
 

Rig3
Bi 161 Independent Project Proposal:
Students: Ryan Simkovsky, Melinda Turner, Brent Kious (Rig 3)

Cation-? bonding interactions in the nicotinic acetylcholine receptor’s nicotine binding site

 Recent research in the acetylcholine binding properties of  the nicotinic AchR has suggested that specificity of the channel’s Ach binding site may be mediated, in part, by cation-? bonding interactions between positively charged residues in the Ach molecule and aromatic ? bonds of the six member ring of  tryptophan 149 in the ? subunit.  Through EC50 measurements on nAchR 9’ mutant channels (which have a more sensitive Ach response) containing artificially incorporated tryptophan derivatives with varying abilities to contribute to cation-? bond interactions, researchers have provided evidence that such interactions are in fact vital to the channel’s affinity for Ach. (Zhong et al., 1998)
 Structural studies of the nAch receptor have also revealed the presence of  aromatic residues (tryptophan and tyrosine) in the channel’s nicotine binding site.  Utilizing in vivo nonsense-suppression, we hope to incorporate the unnatural tryptophan derivative F4 Trp (which contains flourine atoms at four positions in tryptophan’s six-member ring, decreasing its ?-bonding ability) into position ?Trp 55, to study whether cation-? bond interactions at this residue serve to mediate nicotine binding in the wild type receptor. It is our hope that Xenopus oocytes expressing mutant nAchR (? F4Trp 55) can be subjected to voltage clamp experiments to measure the response of the mutant channel to varying concentrations of nicotine. Comparison of the mutant channel’s EC50 for nicotine to that for the wild type nAchR should demonstrate whether or not incorporation of F4 Trp has any appreciable effect on the channel’s  nicotine binding ability.   It is our expectation that incorporation of an amino acid with reduced ? bonding ability should increase the channel’s EC50, if cation-? bonding at this residue is involved in nicotine binding.

Rig4
Subject: bi161 final project for Rig 4
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Bjorn Christianson
Ofer Mazor
Ania Mitros
Javier Perez-Orive

We would like to examine the properties of the NMDAR1 channel.  First, we
would like to spend a day characterizing the basic properties of the
channel.  The remaining two days we would like to focus on more
interesting properties of this channel.

Day 1, Basic properties:  To characterize the behavior of the NMDAR1
channel, we would like to vary the concentrations of some of the ions
associated with the function of this channel.  We will begin by observing
the effect of Mg++ concentration on the voltage-dependent behavior of the
channels.  It would also be interesting to vary the extracellular
concentrations of Ca++, Na+, and K+.  The resulting change in current flow
through the membrane should allow us to compare the relative
conductivities of these three ions through the NMDAR1 channel.

Day 2, Late current effect:  We would need an oocyte expressing NMDAR1
channels and another non-NMDA channel that is not blocked by Mg++ (such as
AMPA).  We will subject the oocyte to a brief pulse of glutamate.   With
Mg++ in solution, the NMDAR1 channels will be blocked and we should see a
peak in current that decays fairly quickly.  Without Mg++ or in a
depolarized cell, the peak should decay more slowly since the NMDA
channels open and close more slowly than other channels.  To create a
brief glutamate pulse, we have considered two options:  (1) in the rigs we
are currently using, use a syringe to inject a small amount of solution
containing a high concentration of glutamate and hope it gets flushed out
fairly quickly; or (2) use the computer-controlled rigs in the Lester lab
for a more controlled glutamate peak.

Day 3, The effects of PCP (phencyclidine) and Zn++:  We would like to
compare the effects of these two channel blockers.  We know that binding
sites for both are within the channel, and the PCP requires the channel to
be open before binding.  We would like to see whether this is also true of
Zn++ and compare the results for the two.  We should determine how
difficult it is to rinse the PCP (or Zn++) from the channel once it has
bound, and perhaps how the removal of the blockers is dependent on how
many times the cell is depolarized to open the NMDA channels.
Furthermore, it would be interesting to see how the effectiveness of PCP
or Zn++ varies with the number of open channels, perhaps by depolarizing
the cell in the presence of PCP, examining its new conductance, and
repeating the procedure with a different voltage (and thus a different
number of open channels).  If time permits, we would also like to observe
the effects of varying glycine concentration on channel activity.

Rig5
Minoree Kohwi
Matt Paul
g. Paul Vigil
 

Purpose:
1. To express TRP in Xenopus oocytes and show that the addition of
diacylglycerol (DAG)  can activate the opening of the TRP channel.
2. To test whether DAG is involved in the serotonin-mediated
signal-transduction pathway.

Introduction:

The 5HT2C receptor is a G-protein-coupled receptor (Gq/11).
Phospholipase C acts on phosphatidylinositol bis-phosphate (PIP 2),
producing DiAcylGlycerol (DAG) and inositol tris-phosphate (IP3).  IP3
goes on to open intracellular calcium stores, and the calcium released
opens Ca++-activated chlorine channels.  The role of DAG in the
5HT2C-mediated signal-transduction pathway is not clear.

Two articles appeared in Nature in January 1999 discussing the DAG
activation of Transient Receptor Potential (TRP) protein channels in
Drosophila and mammalian homologues (TRPC proteins).  We plan to express
the TRP, or TRP-like (TRPL), channels and the 5HT2C receptor in Xenopus
oocytes, block calcium activity by injecting calcium chelators, and then
determine whether the DAG produced in the normal serotonin pathway can
open the TRP channels.

Materials:

1. cDNA for TRP/TRPL channels to try to express TRP in Xenopus oocytes.
2. EDTA to chelate calcium
3. IP3 blocker to block release of Ca from intracellular stores
4. cDNA for 5HT2C receptor
5. serotonin
6. DAG

Methods:

I. Verify Expression
A. TRP only
B. 5HT2C only
C. TRP and 5HT2C

II. Confirm “normal” activity of DAG (control)
A. DAG -> TRP only cell
B. DAG -> 5HT2C only cell
C. DAG -> 5HT2C + TRP cell

III. Demonstrate blocking of Ca++ dependent pathways
A. in the oocyte expressing 5HT2C
1. Add 5HT
2. Add 5HT + chelators
3. Add 5HT + IP3 blocker

IV. Test for existence of 5HT2C --> DAG-> TRP  pathway
A.  in a 5HT2C + TRP expressed cell
1. Add 5HT
2. Add 5HT + chelators
3. Add 5HT + IP3 blocker

Expected Results:

We hypothesize that the addition of calcium chelators will greatly
reduce if not eliminate the current flow resulting from the activation
of the 5HT2C receptor.  We further hypothesize that in the 5HT2C + TRP
oocytes the production of DAG will open the TRP channels and demonstrate
the possibility of a second, DAG-mediated pathway.

Rig6

Subject: Bi 161 proposal
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For our project in Bi 161, we intend to investigate the effects of the
noncompetative antagonist dextromethorphan on the NMDA glutamate receptor.
Dextromethorphan has been shown to bind to the NMDA glutamate receptor
and the sigma receptor.  While the functionality of the sigma receptor
have not been well characterized, the NMDA receptor is a relatively well
understood glutamate-activated calcium channel.  Thus, NMDA appears to be
a good point to begin investigating the molecular activity of DXM.
Furthermore, there are two different subunits that comprise the NMDA
receptor, NR1 and NR2.  In vivo experiments have demonstrated that
different expression ratios of NR1 and NR2 lead to different reactions to
DXM.  However, the molecular differences in these interactions have not
been characterized.

For our actual project, we will need mRNAs for both the NR1 and NR2
subunits of the NMDA receptor.  We will inject different ratios of these
two mRNAs into a number of groups of Xenepus oocytes.  Assuming that the
expression of these proteins is proportional to the amount of the
respective mRNA injected, we will produce groups of Xenepus oocytes with
known ratios of NR1 and NR2 subunits.  We will then perform a variety of
voltage-clamp experiments on each group to characterize the normal
behavior of the NMDA receptor in the Xenepus oocyte.  After that, we will
apply different concentrations of DXM to the oocytes to characterize the
binding properties of DXM.  We will use voltage-clamp experiments to
investigate the macroscopic effects of DXM and patch clamp techniques to
investigate microscopic effects of DXM on single NMDA channels.

If there is time available after we have completed all the voltge clamp
experiments we are interested in, we would like to use calcium imaging to
get more information on NMDA function in Xenepus oocytes.  In neurons,
when NMDA receptors allow calcium in the cell, it can lead to the release
of intracellular stores that activate a number of pathways.  By using
calcium imaging, we can determine if the intracellular stores of calcium
are activated in the Xenepus oocyte system as well.  This is useful in
determining how closely the NMDA data in Xenepus oocytes models their
actual activity in neurons.

Rig #6:
  Ethan Snyder-Frey
  Patrick Drew
  Gabriel Miller
  Uri Eden