Professor:  Henry A. Lester

Norman Chandler Professor Emeritus of Chemical Biology:  Norman Davidson

Visiting Associates:  Robert Farley, Justine Garvey, Clemens Neusch, Ken Philipson, Johannes Schwarz

Member of the Professional  Staff:  Cesar Labarca

Senior Research Fellow:  Baljit Khakh

Research Fellows: Chi-Sung Chiu, David Dahan1, Hong Dang, Carlos Fonck, Gabriela Greif, Abraham Kovoor, Ming Li, Hendrickje Nadeau, Raad Nashmi, Wenmei Shi, Yanhe Tong, Tzu-Ping Yu

Graduate Students:  Darren Beene1, Gabriel Brandt1, Don Elmore1, Justin Gallivan1, Lintong Li1, Joshua Maurer1, Sarah Monahan1, James Petersson1, George Shapovalov2, Eric Slimko, Steve Spronk1, Niki Zacharias1

Rotating Graduate Students:  Magdalena Bak, Sarah Farivar

Associate Biologist:  Purnima Deshpande

Research and Laboratory Staff:  Shannan Boss, Angela Eickhorst, Pam Fong, Donghong Ju, Kira Kostenko, Hai-Rong Li, Sheri McKinney, Vanna Santoro, Jason Sydes, Michael Walsh, Yuping Wang

1Division of Chemistry and Chemical Engineering, California Institute of Technology

2Division of Physics, Mathematics and Astronomy, California Institute of Technology

Support:  The work described in the following research reports has been supported by:

Alexander von Humboldt Foundation

American Heart Association

Burroughs-Wellcome Fund

California Tobacco-Related Disease Research Program

Deutsche Forschungsgemeinschaft

Huntington Medical Research Institute

National Institute of General Medical Science

National Institute of Neurological Diseases and Stroke

National Institute of Mental Health

National Institute of Drug Abuse

National Parkinson Foundation

Plum Foundation

Wellcome Trust

 

Summary:  Work in our laboratory group emphasizes the molecules of excitability in the nervous system.  We have an active program to construct mouse lines that are biophysically based models for neurological and psychiatric disease.  These mice carry gain of function mutations for ion channels, deleted genes for channels, and deletions for the regulators of G-protein signaling (RGS proteins).  We also wish to construct mice that allow the experimenter to silence selected neurons so that behavioral results can be assessed, in collaboration with Professor David Anderson.  In related work, we are  constructing strains carrying fluorescent markers that will allow quantitation of ion channel and transporter density, in collaboration with Professor Stephen Quake’s group in the Division of Engineering and Applied Physics at Caltech.

            We are also interested in the relationship between the structure and function of ion channels, receptors, and RGS proteins.  Our tools for these experiments include site-directed mutagenesis and heterologous expression.  In collaboration with Professor Dennis Dougherty in the Division of Chemistry and Chemical Engineering, we are conducting fine-grained site-directed mutagenesis with unnatural amino-acid residues.  We also study post-translational modulation with these chemical tools.  With Dougherty and Quake, we are also studying the mechanosensitive ion channels of bacteria.

            The subgroup led by Professor Norman Davidson studies the modification of synaptic transmission by cyclic AMP analogs.  We are applying gene transfer techniques to study the role of individual proteins activation during synaptic modifications.

 

343.     “Knock-in” mice with hypersensitive neuronal a4b2 nAChR

            Cesar Labarca, Purnima Deshpande, Johannes Schwarz, Sigrid Schwarz, B.S. Khakh, P. Kofuji2, Mark Nowak3, Hong Dang, Zhoufeng Chen, J. Boulter4, B.J. Bowers1, J.M. Wehner1

We are examining the physiological role of nicotinic acetylcholine receptors (nAChR) in the nervous system by mutating the corresponding mouse genes to significantly alter the function of the receptors.  We have generated mice with a Leu9’Ser mutation in the M2 region of the a4 subunit of the AChR.  a4b2 receptors carrying this mutation have ACh sensitivity about 30-fold higher than the wild-type receptors when examined in the Xenopus oocyte system.  A 129/SvJ genomic clone containing exon 5 was used to construct a targeting vector, with a neomycin resistance cassette flanked by loxP sites for positive selection.  This cassette was deleted in some of the recombinant ES cells by transfecting them with a CMV-Cre plasmid.  Two lines of mice were generated, one with the neo-cassette still present (neo-intact) and the other with the cassette deleted (neo-deleted).  Heterozygous mice from the neo-deleted line die soon after birth.  Heterozygous animals from the neo-intact line are viable and fertile, but homozygous animals from this line die soon after birth; they show loss of dopaminergic neurons in the substantia nigra after ED14.  Behavioral analyses of the heterozygous mice from the neo-intact line showed differences with the wild type in activity tests, anxiety, pre-pulse inhibition, the ability to perform on the accelerated rotarod, and the effect of very low levels of nicotine. 

1Institute of Behavioral Genetics, University of Colorado, Boulder, CO

2Neuroscience, University of Minnesota School, Minneapolis, MN

3Psychiatry, Medical University of South Carolina, Charleston, SC

4Psychiatry & Biobehavioral Science, UCLA, L.A., CA 90024

 

344.     Locomotor deficits in aging mice with a hypersensitive nicotinic receptor

                C.I. Fonck, C. Labarca, J. Schwarz

            As described in the previous abstract, knock-in mice with hypersensitive a4b2 receptors were generated; and neuroanatomical analysis of homozygous embryos revealed a large deficit in the substantia nigra cell population.  In order to investigate whether heterozygous animals also develop a dopaminergic phenotype, a behavioral longitudinal study was performed comparing wild-type and heterozygous mice.  Locomotor activity in response to amphetamine injection was used as a behavioral measure of nigrostriatal health.  Amphetamine stimulates such activity by increasing extracellular dopamine, primarily due to release from nigrostriatal neurons.  Three month-old wild-type and heterozygous animals were injected intraperitonealy with 5 mg/kg amphetamine and their locomotive behavior was recorded in an activity cage.  There was no significant difference in the amphetamine response between wild-type and heterozygous mice.  The same animals were tested again at 11 months of age.  The amphetamine response for both wild-type and heterozygous animals was lower than at three months of age.  The decrease in response was much larger in the heterozygous mice, and 11-month-old heterozygous animals had a significantly lower response to amphetamine than wild-type animals of the same age.  A decreased locomotor response to amphetamine administration may be due to a dopamine deficit in the nigrostriatal pathway.  Thus, the normal neurodegeneration that occurs with aging in wild-type mice was accelerated in these mutated mice.  These animals will be tested again as they advance in age.  Currently we are analyzing tissue of aged-matched wild-type and heterozygous animals to determine if the differences in behavior are accompanied by neuroanatomical changes as a result of the mutation.  The apparent gradual progression of the neurological deficit in the mutated mice might be relevant for the study of slowly progressing neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases.

 

345.     Mice with a hypersensitive 5‑HT3 receptor die from obstructive uropathy

            Hong Dang, Purnima Deshpande, Gabriela Greif, Nora Rozengurt2, Nicolas Guy1, David Julius1

            Mouse lines carrying a hypersensitive 5‑HT3Ra receptor gene were generated through targeted exon replacement.  The V13’S mutation in the channel-lining M2 domain renders the 5HT3 receptor ~70-fold more sensitive to serotonin, and produces constitutive activity when combined with the 5‑HT3Rb subunit in Xenopus oocytes.  Male mice homozygous for the mutant allele die at the age of 2-3 months apparently from obstructive uropathy, also termed murine urologic syndrome (MUS).  Heterozygous males and mutant females also die from MUS, but more older and more rarely.  This phenotype of death by MUS has not been reported for 5‑HT3Ra knock-out mice.  RT-PCR suggests that the homozygous mutant mice have decreased levels of 5‑HT3Ra mRNA.  Electro-physiological studies show reduced whole-cell serotonin responses but hypersensitive 5-HT3 receptors.  These findings are consistent with cellular death caused by persistent activation of the mutant receptors.  The 5‑HT3Ra receptor is found in both central and peripheral nervous system, and is thought to be involved in nausea, anxiety, alcohol dependency, and intestinal motility.  However this is the first study showing that disturbances of 5-HT3 receptor function lead to genitourinary disease.

1Department of Cell and Molecular Pharmacology, UCSF, San Francisco, CA 94143

2Department of Pathology and Laboratory Medicine, UCLA, Los Angeles, CA 90095 

 

246.     Lack of RGS9 increases dopamine D2 receptor-mediated locomotion

            Johannes Schwarz, Ching-Kang Chen, Sigrid C. Schwarz, Gabriela J. Greif, Melvin I. Simon

            RGS9 (regulator of G-protein signaling) accelerates deactivation kinetics of Gi, Gq, G12, but not Gs subfamilies of G-proteins.  It is specifically expressed in retina and medium spiny neurons in striatum.  A mouse line was generated that lacks RGS9 (Chen et al., 2000 Nature 403:557-560).  We studied spontaneous and drug-induced locomotion in unlesioned and dopamine depleted mice.  We scored ambulation using activity cages in 6 bins of 5 min duration.  The first 5 min were omitted from the analysis.  One count equaled traveling at least 10 cm.

            RGS9-deficient mice showed a 30% increase in amphetamine (5 mg/kg BW, 71.1 ± 10.9 vs. 106.5 ± 12.1 per 5 min., mean ± SEM, p < 0.05) induced but not baseline or SKF38393 (10 mg / kg)-induced ambulation.  Quinpirole (1 mg/kg) induced a dramatic reduction in locomotion, most likely due to stimulation of presynaptic autoreceptors, that was less pronounced in RGS9 deficient (3.0±1.4) compared to wild-type and heterozygous mice (0.5 ± 0.2, n.s.).  Dopamine-depleted animals (reserpine 1 mg/kg x 5 days) showed a two-fold difference (RGS9 KO: 3.6 ± 0.5 vs. WT/HET: 1.4 ± 0.8, n.s.) in baseline ambulation.  A subsequent injection of apomorphine (5 mg/kg BW) induced a four-fold increase of ambulation in RGS9 deficient (8.8 ± 1.8) compared to wild-type and heterozygous mice (2.0 ± 1.0, p < 0.05).  Following the apomorphine injection, all RGS9 deficient animals showed marked abnormal motor behavior such as dystonia, chorea and myoclonic jerks.  Western blotting showed an increase of RGS9 protein in dopamine-depleted striata of wild-type and heterozygous mice similar to the increase in dopamine D2 receptor density.

            We conclude that loss of RGS9 enhances dopamine D2 receptor-mediated locomotion.  The abnormal motor behavior following apomorphine injection in dopamine-depleted animals indicates that these mice may serve as a model for dyskinesia related to dopamine D2 receptor up-regulation.  Our data may have implications for the pathophysiology of dyskinesia induced by neuroleptics or long-term levodopa treatment of Parkinsonian patients.

 

347.     Mice with a functional, correctly targeted GABA transporter-GFP fusion protein

            Chi-Sung Chiu, Marc Unger1, Emil Kartalov1, Stephen Quake1

            We wish to measure the density of neurotransmitter transporters such as mGAT1 by constructing and studying mice whose mGAT1 has been replaced by mGAT1-GFP fusion.  To test the function of GAT1-GFP fusion in cDNA level, the GFP37 (K.G. Beam lab construct) was fused to the C-terminal of mGAT1 (from N. Nelson, in pcDNAIII) and tested by transfection into HEK 293T cells.  The mGAT1-GFP has EC50 and Vmax values indistinguish-able from those of WT GAT1 indicating that this fusion protein shows no functional difference with wild type.

            To study the localization of mGAT1-GFP in hippocampal neurons, lentivirus and Sindbis virus expression constructs were made.  The expressed mGAT1-GFP fusion protein is targeted to neuronal processes and accumulates at synaptic sites, for instance by colocalization with synapsin I. 

            We constructed a mouse gene targeting vector containing the linker-GFP in the final coding exon of mGAT1.  This construct successfully replaced the wild-type mGAT1 genome in 129 embryonic stem cell and successfully generated chimeras.  These chimeras have been mated and germline transmission verified.  GFP fluorescence is localized to GABAergic neurons.

1Division of Engineering and Applied Science, Caltech

 

348.     Single molecules calibrate GFP surface densities on transparent beads

            Chi-Sung Chiu, Marc Unger1, Emil Kartalov1, Stephen Quake1

            Quantitative aspects of synaptic trans-mission can be studied by inserting green fluorescent protein (GFP) moieties into the genes encoding membrane proteins.  To provide calibrations for measurements on synapses expressing such proteins, we developed methods to quantify His6-tagged GFP molecules bound to Ni-NTA moieties on transparent beads (80 – 120 mm dia) over a density range comprising nearly four orders of magnitude (to 30,000 GFP/mm2).  The procedures employ commonly available Hg lamps, fluorescent micro-scopes, and CCD cameras.  Two independent routes are employed:  (1) Single-molecule fluorescence measurements are made at the lowest GFP densities, providing an absolute calibration for macroscopic signals at higher GFP densities.  (2) Known numbers of His6-GFP molecules are coupled quantitatively to the beads.  Each of the two independent routes provides linear data over the measured density range, and the two independent methods agree with an rms deviation of 11 to 21% over this range.  These satisfactory results are obtained on two separate microscope systems.  The data can be corrected for bleaching rates, which are linear with light intensity and become appreciable at intensities > ~ 1 W/cm2.  If a suitable GFP-tagged protein can be chosen and incorporated into a “knock-in” animal, the density of the protein can be measured with an absolute accuracy on the order of 20%.

1Division of Engineering and Applied Science, Caltech

 

349.     Selective silencing of mammalian neurons:  strategies using chloride channels

            Eric Slimko, David Anderson*

Selectively reducing the excitability of glutamatergic neurons will (1) allow for the creation of animal models of human neurological disorders and (2) provide insight into possible treatments for conditions such as epilepsy, stroke, and excitotoxin poisoning.  Because there are no pharmacological agents that target subsets of glutamatergic cells, we are focusing on genetic approaches.  Our strategy is to express invertebrate ivermectin-sensitive chloride channels (C. elegans GluCl a and b) under tissue-specific promoters and activate them with the drug to produce the silencing effect through a Cl- conductance.  Ivermectin is an anthelmintic used to suppress a variety of parasitic infections in both humans and animals.  Our goal is to construct in vitro models that mimic conditions that can be obtained in transgenic mice and in human gene therapy.  We have constructed a three-cistron Sindbis virus that expresses the a and b subunits of GluCl along with EGFP.  Using this construct, we have shown that the C. elegans channel can express well in HEK293 cells, delivering robust (1 nA at –60 mV) Cl- currents in response to glutamate and ivermectin.  This virus also infects cultured rat sympathetic cervical ganglion and hippocampal neurons.  When activated by ivermectin at concentrations as low as 50 nM, the Cl- current effectively reduces the excitability of the infected cell.  Although high concentrations of the drug in brain can be lethal, such concentrations may be well below toxic levels.

*Professor, Division of Biology, Caltech

 

350.     Dominant negative Rab3a:  Effects on synaptic transmission

            Hendrickje Nadeau

Lentiviral vectors were constructed to express the synaptic vesicle protein Rab3a, either wild-type or a GTPase-deficient mutant (81 Q®L), as a bicistronic message with green fluorescent protein (GFP).  The vector was used to transduce cultured E18 or P1 hippocampal neurons at 0-14 days in vitro (div).  Cultures from the entire hippocampus as well as mossy-fiber-enriched cultures from CA3 and dentate gyrus were used.  Neurons infected with the wild type survived for 4-6 wk, and with the mutant, 2-3 wk; cells in dense culture and those >7 div were less sensitive to toxic effects of the mutant.  Paired electrophysiological recordings and FM-143 imaging revealed a suppression of vesicle release from neurons expressing the mutant protein, but not in those expressing the wild type.  Response of infected neurons to stimuli from uninfected neurons was unaffected.

 

351.     ROMK1 (Kir1.1) causes apoptosis and chronic silencing of hippocampal neurons

                H. Nadeau, S. McKinney, D.J. Anderson

            Lentiviral vectors were constructed to express the weakly-rectifying kidney K+ channel ROMK1 (Kir1.1), either fused to enhanced green fluorescent protein (EGFP) or as a bicistronic message  (ROMK1-CITE-EGFP).  The channel was stably expressed in cultured rat hippocampal neurons.  Infected cells were maintained for 2-4 weeks without decrease in expression level or evidence of viral toxicity, although 15.4 mM external KCl was required to prevent apoptosis of neurons ex-pressing functional ROMK1.  No other trophic agents tested could prevent cell death, which was probably caused by K+ loss.  This cell death did not occur in glia, which were able to support ROMK1 expression indefinitely.  Functional ROMK1, quantified as the non-native inward current at –130 mV in 5.4 mM external K+ blockable by 500 mM Ba2+, ranged from 1 to 40 pA/pF.  Infected neurons exhibited a Ba2+-induced depolarization of 7 ± 2 mV relative to matched EGFP-infected controls, as well as a 30% decrease in input resistance and a shift in action potential threshold of 2.6 ± 0.5 mV.  This led to a shift in the relation between injected current and firing frequency, without changes in spike shape, size, or timing.  This shift, which quantifies silencing as a function of ROMK1 expression, was predicted from Hodgkin‑Huxley models.  No cellular compensatory mechanisms in response to expression of ROMK1 were identified, making ROMK1 potentially useful for transgenic studies of silencing and neurodegeneration, although its lethality in normal K+ has implications for the use of K+ channels in gene therapy.

 

352.     Two-compartment model for whole-cell data analysis and transient compensation

                Hendrickje Nadeau

            Recording and analysis of neuronal patch-clamp data involve many assumptions about membrane properties and cell morphology.  Some of these assumptions introduce large errors or oversimplifications into the results.  In particular, dendritic branching with high intracellular resistance leads to difficulty with capacitance calculation and transient subtraction, and may significantly distort measured currents.  A two-compartment model, presented in detail here, provides a simple method of reducing many of these problems for the relatively simple case of cultured neurons studied with whole-cell patch electrodes.  Some passive membrane properties may be accurately calculated, and the results may be used to correct recorded currents for resulting series resistance, intracellular resistance, and capacitive transient errors.  The model may be tailored to particular cell types or experimental conditions.

 

353.     Kir4.1 knockout mice: phenotypic impact in retina

            Paulo Kofuji1, Paul Ceelen1, Kathleen R. Zahs2, Leslie W. Surbeck1, Henry A. Lester, Eric A. Newman1

            The inwardly rectifying potassium channel Kir4.1 has been suggested to underlie the principal K+ conductance of mammalian Müller cells and to participate in the generation of field potentials and regulation of extracellular K+ in the retina.  To further assess the role of Kir4.1 in the retina, we generated a mouse line with targeted disruption of the Kir4.1 gene (Kir4.1 -/-).  Müller cells from Kir4.1 -/- mice were not labeled with an anti-Kir4.1 antibody although they appeared morphologically normal when stained with an anti-glutamine synthetase antibody.  By contrast, in Müller cells from wild-type littermate (Kir4.1 +/+) mice, Kir4.1 was present and localized to the proximal endfeet and perivascular processes.  In situ whole-cell patch clamp recordings showed a ten-fold increase in the input resistance and a large depolarization of Kir4.1 -/- Müller cells in comparison to Kir4.1 +/+  cells.  The slow PIII response of the light-evoked electroretinogram (ERG), which is generated by K+ fluxes through Müller cells, was totally absent in retinas from Kir4.1 -/- mice.  The b-wave of the ERG, in contrast, was spared in the null mice.  Overall, these results indicate that Kir4.1 is the principal K+ channel subunit expressed in mouse Müller glial cells.  The highly regulated localization and the functional properties of Kir4.1 in Müller cells suggest the involvement of this channel in the regulation of extracellular K+ in the mouse retina.

1Dept. of Neuroscience and, 2Dept. of Physiology, University of Minnesota, Minneapolis, MN

 

354.     Myelination is controlled by the Kir4.1 potassium channel

            Clemens Neusch, Nora Rozengurt1, Russell E. Jacobs, Paulo Kofuji2

            To understand the cellular and in vivo functions of specific K+ channels in glia, we have studied mice with a null mutation in the weakly inwardly rectifying K+ channel subunit Kir4.1.  Kir4.1-/- mice display a profound ataxic phenotype, and the cellular basis is severe spongiform vacuolation in the spinal cord, accompanied by axonal swellings and degeneration.  Immunostaining of wild-type mice reveals that Kir4.1 is expressed in myelin-synthesizing oligodendrocytes.  Oligodendrocytes from Kir4.1-/- mice lack most of the wild-type K+ conductance, have depolarized membrane potentials, and display immature morphology.  We conclude that Kir4.1 is crucial for myelination of axons.  These studies show how a defect in an ion channel of oligodendrocytes can lead to hypomyelination resembling neurological diseases.

1Department of Pathology, UCLA School of Medicine, Los Angeles, CA  90095

2Department of Neuroscience, University of Minnesota, Minneapolis, MN  55455

 

355.     Expression of GIRK channels in fibroblasts with and without b1 integrins

            Tatiana Ivanina, Clemens Neusch, Yong-Xin Li, Yanhe Tong, Cesar Labarca, Deane F. Mosher1

            G protein-activated K+ channel (GIRK) subunits possess a conserved extracellular integrin-binding motif (RGD) and bind directly to b1 integrins.  We expressed GIRK1/GIRK4 channels labeled with green fluorescent protein in fibroblast cell lines expressing or lacking b1 integrins.  Neither plasma membrane localization nor agonist-evoked GIRK currents were affected by the absence of b1 integrins or by incubation with externally applied RGD-containing peptide.  Mutation of the aspartate (D) of RGD impaired currents, GIRK glycosylation, and membrane localization, but the interaction with b1 integrins remained intact.  Thus, b1 integrins are not essential for functional GIRK expression; and the GIRK-integrin interactions involve structural elements other than the RGD motif.

1Section of Hematology, Department of Medicine, University of Wisconsin, Madison, WI

 

356.     Hearing and inner ear function in a Kir4.1 knock-out mouse

            C. Neusch, N. Rozengurt1, P. Kofuji2

            The inwardly rectifying potassium channel Kir4.1 is expressed in the rat cochlea.  In particular, the stria vascularis and satellite cells of the spiral ganglia show considerable expression of Kir4.1.  Thus, an important role in regulating the positive endocochlear potential in the scala media has been suggested for Kir4.1.  Additionally, its expression in satellite cells of the spiral ganglia suggests a role in regulating the concentration of K+ extruded from ganglion neurons during excitation.  We constructed a knock-out mouse lacking the Kir4.1 channel by gene targeting inactivation.  Homozygous mice for the inactivated Kir4.1 (Kir4.1-/-) displayed a strong phenotype: they had limb paralysis, ataxia and died prematurely 2-3 weeks after birth.  In startle response testings, Kir4.1-/- mice lacked a response to various audible stimuli (80, 100, 120 db).  Histological examination of the ear revealed marked swelling of the tectorial membrane with partial disruption of membrane structures and misarrangement of membrane fibers.  The Reissner membrane was found to adhere to the tectorial membrane.  The maculae of the utricle and saccule and the crista ampullaris of the vestibular system, however, appeared unremarkable.  Immunohistochemical staining confirmed earlier results showing prominent signals in satellite cells of the spiral ganglia and in the stria vascularis.  This knock-out mouse allows us to investigate the function of this particular channel for the development of the cochlea and in particular for its role in regulating the endocochlear potential and the K+ concentration around spiral ganglia cells in the mouse cochlea.

1Dept. of Pathology, UCLA Medical Center

2Dept. of Physiology, Univ. of Minnesota, Minneapolis MN

 

357.     Co-expression of Gb5 enhances function of the GGL domain-containing RGS7

            Abraham Kovoor, Ching-Kang Chen, Wei He1, Theodore G. Wensel1, Melvin I. Simon

            Regulators of G-protein signaling (RGS proteins) stimulate the GTPase activity of G‑protein Ga subunits.  A subset of RGS proteins, RGSs 6, 7, 9 and 11, contain a Gg subunit-like (GGL) domain, which mediates a specific interaction between these RGS proteins and Gb5.  The role of such interactions in RGS function is unclear.  It was previously established in Xenopus oocytes that the time-course of GIRK channel deactivation following agonist removal is determined by the rate at which the G proteins gating the GIRK channels hydrolyze their bound GTP.  Therefore we coupled GIRK channels (Kir3.1/Kir3.2) to m2-muscarinic acetylcholine receptors (m2-mAChR) by heterologous expression in Xenopus oocytes to evaluate the role of Gb5 in RGS7 function.  We found that the co-expression of RGS7 with GIRK channels and m2-mAChR modestly accelerated GIRK channel deactivation.  In addition, when we co-expressed Gb5 and RGS7 with GIRK channels and m2-mAChR, the acceleration of GIRK channel kinetics was strongly increased over that produced by RGS7 alone.  This enhancement of RGS7 function by Gb5 was not achieved by co-expression of Gb1.  Gb5 did not modulate the function of RGS4, an RGS protein that does not contain a GGL domain.  We showed that the enhancement of RGS7 function by Gb5 was not a consequence of an increase in the amount of plasma membrane or cytosolic RGS7 protein.

1Verna and Marrs McLean Dept. of Biochemistry, Baylor College of Medicine, Houston, TX 77030

 

358.     A P2X2 channel EGFP fusion protein in oocytes and hippocampal neurons

            Baljit S. Khakh, W. Bryan Smith2, Erin M. Schuman1, Norman Davidson

It is an important goal to determine P2X2 channel cellular and subcellular distribution, density and regulation in live cells.  To this end we report the engineering and functional evaluation of a fusion protein between rat P2X2 channels and a thermostable mutant of EGFP.  P2X2-f-EGFP and wtP2X2 channels were expressed in oocytes, and ATP evoked currents with EC50s of 14 ± 2, and 5 ± 1 mM, and Hill coefficients of 1.7 ± 0.1 and 1.8 ± 0.1, respectively.  P2X2-f-EGFP and P2X2 channels showed little desensitization, and suramin (30 mM) blocked ATP‑evoked currents at P2X2-f-EGFP and P2X2 channels similarly (k+1 1.2 ± 0.2 and 1.2 ± 0.4 x104 M-1.s-1).  P2X2-f-EGFP channels formed functional heteromers with wtP2X3 channels as assessed by a diagnostically slowly desensitizing response to abmeATP.  We next generated Sindbis viruses for P2X2-f-EGFP channels and infected E18 hippocampal neurons in primary culture.  P2X2-f-EGFP channels were functional in neuronal somata, dendrites and presynaptic terminals where activation by ATP enhanced mini EPSC frequency, but not amplitude.  20 hours post-infection we observed green fluorescence that localized to the plasma membrane, dendritic shafts, and punctuate bulbous protrusions along the dendrites.  The functional significance of this distribution pattern is under investigation.

1Professor, Division of Biology, Caltech

2Grad Student, Prof. Erin Schuman’s Lab, Division of Biology, Caltech

 

359.     A link between gating and permeation for a transmitter-gated cation channel

            Angela Eickhorst, Baljit S. Khakh

In standard models, ion channel conductance, gating and permeation are independent.  In previous work we have shown that P2X2 channels change their ionic permeability in seconds, becoming more permeable to organic cations such as NMDG+.  But many quantitative questions still remain.  In an extension of these studies we have now found that ionic permeability is directly linked to gating.  The P2X2 channel opens to distinct sizes in a manner dependent on gating.  Low doses of ATP open a pore with a diameter of ~0.9nm, whereas doses of ATP higher than the EC50 open a pore ~1.5nm diameter.  The rate constant for dilation is also directly linked to gating.  By using ions of different size we conclude that the P2X2 pore dilates progressively, and not in a stepwise manner:  the rate of dilation is ~0.15 nm per second.  Generally, one thinks that channel activation in-creases only open probability, but here we have provided further quantitative evidence, to show that for P2X2 channels gating and permeation are absolutely linked.  Further mechanistic studies are underway.

 

360.     Determining the disulfide connectivity of the P2X2 receptor

            Gabriel S. Brandt1, Pamela M. England1, Baljit S. Khakh, Dennis A. Dougherty1

            The disulfide linkage is an important structural element in many proteins such as ion channels.  In this work, we attempt to determine the disulfide connectivity of the P2X2 receptor. 

The 472 amino acid P2X2 receptor from rat has ten Cys residues in its approximately 274 aa extracellular domain.  The ability to site-specifically cleave the protein would answer the question of whether or not any two cysteine residues are connected.  Ordinarily, cleavage of a protein at a single position gives rise to two fragments.  However, cleavage within a disulfide loop does not generate two fragments, since the two pieces are held together by the covalent disulfide bond.  Under conditions where the disulfide is reduced, two fragments will, of course, be seen.  As a result, cleavage of the protein under both native and reducing conditions is an effective assay for disulfide connectivity.  Through the use of nonsense suppression methodology, we can introduce an ester linkage into the otherwise all-peptide backbone of a protein at any given position.  The ester linkage is susceptible to alkaline hydrolysis, satisfying the conditions that we require for being able to address the question of disulfide connectivity.

To date, an epitope tag has been introduced into the rat P2X2 receptor to permit its visualization in Western blots.  The hemagglutinin epitope at the C-terminus has no detectable effect on expression in oocytes, and the protein is easily detectable by blotting with an anti-HA antibody.  In addition, the amber codon has been introduced in between each of the ten cysteines which make up the five potential disulfide pairs in the P2X2 receptor.  Suppression with tRNA charged with the hydroxy acid derivative of valine (Vah, for valine hydroxy acid) has been successful at five of the eight sites examined thus far.  In each of these cases, electrophysiological measurements have been carried out to establish that the Vah-containing receptors respond to ATP.  In addition, membranes isolated from these oocytes and probed with the anti-HA antibody in Western blots clearly show that full-length protein is being produced.  Experiments are under way to determine disulfide connectivity.

1Division of Chemistry, Chemical Engineering, Caltech

 

361.     Identifying the nicotine binding site in the neuronal a4b2 nAChR

            Gabriel S. Brandt1, Lisa J. Turner1, Dennis A. Dougherty1

The a4b2 nicotinic acetylcholine receptor (nAChR) is estimated to account for 90% of nicotine binding in the brain.  Previous work in our lab has shown a potentially interesting distinction in the way nicotine and the natural agonist acetylcholine bind to the muscle-type nAChR.  The work described here represents an attempt to understand how nicotine binds to the a4b2 receptor.

The cationic nature of nAChR agonists and the prevalence of aromatic residues in the putative binding site of the receptor led our lab to identify, in previous work, an interaction between acetylcholine and Trp149 of the alpha subunit of the muscle nAChR.  This interaction is only possible at extremely short range and led to the conclusion that Trp149 is intimately involved in ACh binding.  At the same time, this interaction appeared to be absent in nicotine binding.  The technique which allowed this cation-π interaction to be identified involves replacing Trp149 with analogs of tryptophan which are essentially isosteric, but which differ in their electrostatic potential surfaces.  Those which are more electropositive than Trp show decreased dose response to the agonist. 

To date, the a4b2 subunits have been expressed in oocytes and shown to produce receptors which give measurable responses to both nicotine and ACh.  The subunits have recently been sub-cloned into a vector optimized for oocyte expression.  Codons for the relevant Trp residues have been mutated to the amber codon for subsequent suppression experiments.  In addition, the HA epitope has been introduced into the alpha4 subunit in order to detect protein synthesis by the oocyte.  Another line of experimentation has sought to understand in greater detail nicotine binding to the muscle receptor, which has proven to be a more experimentally tractable system.  Several Trp residues which are conserved in the putative binding site have been examined for evidence of a cation-π interaction with nicotine. 

1Division of Chemistry and Chemical Engineering, Caltech

 

362.     State-dependent cross-inhibition between transmitter-gated cation channels

            Baljit S. Khakh, Xiaoping Zhou1, Jason Sydes, James J. Galligan1

Transmitter-gated cation channels are detectors of excitatory chemical signals at synapses in the nervous system.  Here we show that structurally distinct a3b4 nicotinic and P2X2 channels influence each other when co-activated.  The activation of one channel type affects distinct kinetic and conductance states of the other, and co-activation results in non-additive responses due to inhibition of both channel types.  State-dependent inhibition of nicotinic channels is revealed most clearly with mutant P2X2 channels, and inhibition is decreased at lower densities of channel expression.  In synaptically-coupled myenteric neurons, nicotinic fast excitatory postsynaptic currents (EPSC’s) are occluded during activation of endogenously co-expressed P2X channels.  These data provide a molecular basis and a synaptic context for cross-inhibition between transmitter-gated channels.

1Department of Pharmacology and Toxicology and the Neuroscience Program, Michigan State University

 

363.     The tethered agonist approach to mapping the agonist binding site of the nAChR

            Lintong Li1, Wenge Zhong1, Niki Zacharias1, Caroline Gibbs1, Dennis A. Dougherty1

The integral membrane proteins of neurons and other excitable cells are generally resistant to high-resolution structural tools.  Structure-function studies, especially those enhanced by the nonsense suppression methodology for unnatural amino acid incorporation, constitute powerful probes of ion channels and related structures.  The nonsense suppression methodology can also be used to incorporate functional sidechains designed to deliver novel structural probes to membrane proteins.  We have developed a new approach - the tethered agonist approach - to mapping the agonist binding site of  ligand-gated ion-channels.

Using the in vivo nonsense suppression method for unnatural amino acid incorporation, a series of tethered quaternary ammonium derivatives of tyrosine have been incorporated into the nicotinic acetylcholine receptor.  At three sites a constitutively active receptor results, but the pattern of activation as a function of chain length is different.  At position a149, there is a clear preference for a three-carbon tether, while at position a93 tethers of two to five carbons are comparably effective.  At position g55/d57 all tethers except the shortest one can activate the receptor.  Based on these and other data, a model for the receptor binding site can be developed by analogy to the acetylcholinesterase crystal structure.

Thus a potentially generalizable strategy for probing receptors - the tethered agonist approach - has been developed.  When applied to the nicotinic receptor, the method places new restrictions on developing models for the agonist binding site.

 

 

Structure of Tyr-OnQ

 

 

1Division of Chemistry and Chemical Engineering, Caltech

 

364.     Spectroscopic mapping of agonist-induced conformational changes in the nAChR

            David S. Dahan1, Edward Hawrot2, Arthur Karlin3, Dennis A. Dougherty1

            The nicotinic acetylcholine receptor (nAChR) forms a cation-selective channel that opens transiently when the receptor binds ACh.  Although the nAChR is the most thoroughly studied neurotransmitter-gated ion channel, the detailed structural mechanisms underlying the opening of the channel remain unknown.  Since fluorophores can serve as versatile probes of protein structure and dynamics, our objective is to combine site-specific fluorescent labeling with voltage clamping to monitor the conformational changes that accompany channel gating.  Using fluorescently labeled a-bungarotoxin, a nAChR antagonist that binds with high affinity, we showed the feasibility of detecting nAChR expressed at the surface of Xenopus oocytes.  By site-directed mutagenesis we introduced cysteine residues close to the ACh binding site and at sites within the M1 and M2 transmembrane domains that are exposed in the channel lumen.  These cysteine substitution mutants will be expressed in Xenopus oocytes and reacted with extrinsically applied, membrane impermeable, sulfhydryl-reactive fluorescent probes.  Conformational changes in the protein will be monitored via changes in the fluorescence intensity.  The relative distances between the M3-M4 loop and various cysteine-substituted residues will be determined for the nAChR in the closed versus the open state.  We anticipate that these studies will lead to the development of more detailed conformational models of nAChR gating.

1Division of Chemistry and Chemical Engineering, Caltech

2Division of Biology and Medicine, Brown University, Providence, Rhode Island

3Center for Molecular Recognition, Columbia University, New York, NY

 

365.     Cellular and molecular mechanisms of neuronal nicotinic receptor desensitization

            Raad Nashmi, Sarah Monahan1, Cesar Labarca, Dennis A. Dougherty1

            Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in cholinergic transmission in the PNS and CNS.  nAChRs have been implicated in a variety of functions, including neuronal development, neuritic pathfinding, learning and memory, and enhancing synaptic transmission.  The heteromeric a4b2 and the homomeric a7 subunits form the two major subtypes of functional nAChRs in the brain.  In particular, a4b2 subunits have a high affinity to nicotine.  However, chronic exposure to nicotine could lead to long-lasting desensitization to nAChRs.  Calcium, protein kinase C (PKC) and protein tyrosine kinases are known to modulate nAChR activity.  The over-all aim of this study is to examine the mechanisms of a4b2 nAChR desensitization and recovery from desensitization.  The Lester/Dougherty laboratory is developing methods to incorporate artificial amino acids (AAA) in mammalian cell lines.  This novel technique will allow the study of mechanisms of modulation of nicotinic receptor activity with unprecedented accuracy.

            The following specific hypotheses will be examined:  1) Phosphorylation of nAChRs by PKC can improve the recovery from desensitization.  2) Desensitization and recovery from desensitization are due to the internalization and subsequent reinsertion of nAChRs into the cell membrane.

            There are predicted phosphorylation sites in both a4 and b2 subunits.  Artificial amino acids (AAA) will be employed in site directed mutagenesis experiments to replace serine, with either caged or caged phospho-serine.  The decaging of these AAA with UV illumination will allow us to assess the phosphorylation of each residue in the desensitization and recovery from desensitization of the a4b2 nAChR.  Activators of PKC such as phorbol myristate acetate will also be used.  The electrophysiology data will be correlated with real time fluorescence imaging data to examine cellular trafficking of nAChRs during desensitization and its recovery.  Fluorescent fusion protein constructs will be produced in order to examine with fluorescent microscopy the spatial intracellular trafficking of nAChRs during desensitization and recovery from desensitization.

            This study represents a step in clarifying the mechanisms of nAChR function.  Moreover, imaging studies in conjunction with electrophysiology will provide a fundamental understanding of the role of intracellular receptor trafficking during modulation of nAChRs by signal transduction pathways.  In addition, dysfunction of nAChR activity due to mutations to the a4 subunit has been linked to Alzheimer’s disease and epilepsy.  There-fore, it is anticipated that the knowledge gained from this study would have implications in finding therapies to a variety of diseases of the CNS.

1Division of Chemical and Chemical Engineering, Caltech

 

366.     Unnatural amino acid incorporation by nonsense suppression in mammalian cells

            Sarah L. Monahan1, Dennis A. Dougherty1

            The incorporation of unnatural amino acids into proteins in vivo by nonsense suppression has now been well established in Xenopus oocytes, and studies to understand the biochemistry of neuronal ion channels have been done using this technique (1).  However, these studies would greatly benefit by developing techniques to allow nonsense suppression and unnatural amino acid incorporation using mammalian cell lines, and specifically neurons.  Efforts towards this goal are currently in progress.  Electroporation is being used to deliver an amber mutant EGFP reporter gene, and amber suppressor tRNA derived from Tetrahymena (2), to mammalian cells.  A microporator (previously designed specifically for neuronal transfection) (3) is being used to deliver these materials to a controllable number of cells with a low cell death rate and high transfection efficiency.  As a model cell line, CHO cells have been chosen for these preliminary developmental studies.

1Division of Chemical and Chemical Engineering, Caltech

 

References

Nowak, M.W., Gallivan, J.P., Silverman, S.K., Labarca, C.F., Dougherty, D.A. and Lester, H.A. (1998) Meth. Enzymol. 293:504-529.

Saks, M.E., Sampson, J.R., Nowak, M.W., Kearney, P.C., Du, R., Abelson, J.N., Lester, H.A. and Dougherty, D.A.(1996) J. Biol. Chem. 271.

Teruel, M.N., Blanpied, T.A., Shen, K., Augustine, G.J. and Meyer, G. (1999) J. Neurosci. Meth. 93:37‑48.

 

367.     Role of conserved proline in transmembrane domain M1 of ligand-gated ion channels

            Hong Dang, Pam England1, S. Sarah Farivar Dennis A. Dougherty1

            A conserved proline residue is found in the first transmembrane domain (M1) of every subunit in the ligand-gated ion channel superfamily.  The position of this proline between the N-terminal extracellular agonist binding and the second transmembrane (M2) channel lining domains in the primary sequence suggests its possible involvement in the gating of the receptor.  Replacing this proline with alanine, glycine, or leucine in the 5HT3A homomeric receptors expressed in Xenopus oocytes resulted in the absence of 5‑HT induced whole cell currents, although there were normal levels of specific surface [3H]granisetron ([3H]-BRL-43694) binding sites.  To identify properties of the conserved proline that are critical for the function of the channel, two amino acids and an a-hydroxy acid were incorporated at the proline position using the nonsense suppression method.  Trans-3-methyl-proline, pipecolic acid, and leucic acid were able to replace the conserved proline to produce active channels with EC50 values similar to those for the wild type receptor.  These trends are preserved in the heteromeric receptors consisting of 5HT3A and 5HT3B subunits in oocytes.  The prominent common feature among these residues and proline is the lack of hydrogen bond donor activity, potentially resulting in a flexible secondary structure in the M1 region.  Thus lack of hydrogen bond donor activity may be a key element in channel gating and may explain the high degree of conservation of this M1 proline.

1Division of Chemistry and Chemical Engineering, Caltech

 

368.     Analysis of the agonist binding site in the 5-HT3 receptor

            Darren L. Beene1, Dennis A. Dougherty1

            The 5-hydroxytryptamine receptor (5‑HT3R) belongs to the superfamily of transmembrane ligand-gated ion channels, which includes the nicotinic acetylcholine (nACh), glycine, and g-aminobutyric acid A receptors.  Like all members of the family, the 5-HT3R exhibits a high degree of sequence and structural homology to the nACh receptor.  Previous studies from our group on the nACh receptor have shown that Trp 149 in the agonist binding-site participates in a cation-p interaction with acetylcholine.  This finding indicates that upon binding the quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side-chain of Trp 149.  5-Hydroxytryptamine (5-HT) contains a primary amino group, which at physiological pH is largely protonated.  In addition Trp 183 in the 5-HT3R aligns with Trp 149 of the nACh receptor.  The aim of the present study is to investigate whether a similar cation-p binding interaction exists in the 5-HT3R.

            The basic method for the study involves the site-specific introduction of a series of fluorinated Trp’s at position 183 via nonsense suppression.  The cation-p interaction is a noncovalent electrostatic binding force, where the negative potential on the face of an aromatic ring binds a cation.  It has been shown that the electron-withdrawing ability of fluorine substantially decreases the cation-p binding of Trp, while presenting minimal steric perturbation.  Preliminary results suggest that Trp 183 of the 5-HT3R does participate in a cation-p interaction with 5-HT.  EC50 values for Trp, 5-F-Trp, and 5,7-F2-Trp are 0.96, 6.71, and 29.2 µM, respectively.

1Division of Chemistry and Chemical Engineering, Caltech

 

369.     Synthesis of caged phosphoamino acids and their analogs

            Gabriel S. Brandt1, Dennis A. Dougherty1

Nonsense suppression presents a unique opportunity to introduce a particular residue in a phosphorylated form.  However, as a regulatory mechanism, phosphorylation is necessarily dynamic.  The static introduction of a phosphoamino acid, then, is insufficient to understand the role of phosphorylation in a cell.  The work described here proposes an approach for experimentally controlling the phosphorylation state of functional ion channel in the membrane of a living cell.

Prior work in the lab has demonstrated the utility of caged tyrosine in studying phosphorylation.  The side-chain hydroxyl group of the introduced tyrosine residue is 'masked' by a nitrobenzyl protecting group.  Upon irradiation of the cell, the photolabile protecting group leaves and reveals the wild-type residue, which can then be phosphorylated by tyrosine kinases.  Two other compounds which would complement the caged tyrosine residue are caged phosphotyrosine, and a caged non-hydrolyzable phosphotyrosine analog.  In this way, the rate of phosphorylation by kinases, the rate of de-phosphorylation by phosphatases, and the effects of constitutive phosphorylation may all be examined.

To date, an appropriately protected caged phosphotyrosine has been synthesized.  Further work is needed to improve the yield and to charge a tRNA with the amino acid to test whether or not it can be incorporated by the ribosomal machinery of the oocyte.  Also, a synthetic scheme for the preparation of difluorophosphonomethyl tyrosine has been devised.  Additionally, schemes have been developed for the synthesis of similarly caged phosphoserine and phosphothreonine residues.

1Division of Chemistry and Chemical Engineering, Caltech

 

370.     Tyrosine decaging leads to membrane trafficking during modulation of Kir2.1

            Yanhe Tong, Gabriel S. Brandt1, Ming Li, George Shapovalov2, Eric Slimko, Andreas Karschin3, Dennis A. Dougherty1

            Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking.  A nonsense suppression procedure was employed to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes.  When tyrosine kinases were active, flash decaging led both to decreased K+ currents and also to substantial (15-26%) decreases in capacitance, implying net membrane endocytosis.  A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the K+ channel inhibition.  Thus decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited.  Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis.  In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation primarily results in endocytosis of Kir2.1 channels.  Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, we found no evidence for direct tyrosine phosphorylation of Kir2.1.  Therefore it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain.  These data establish that sidechain decaging can provide valuable time-resolved data about intracellular signaling systems.

1Division of Chemistry and Chemical Engineering, Caltech

2Division of Physics, Mathematics and Astronomy, Caltech

3Department of Physiology, University of Würzburg, Würzburg, Germany

 

371.     Single-molecule measurements to correlate channel kinetics and structure

            George Shapovalov1, Ido Braslavsky2, Stephen Quake2

            Mechanosensitive (MS) channels comprise an important class of ion channels.  It is believed that the action of MS channels represents a first step in an animal sensation of touch and even in sensing such basic forces as membrane stretch by osmotic pressure.  The Rees group at Caltech has determined the structure of a bacterial mechanosensitive channel, Tb-MscL.  The known structure allows us to address the fundamental question of what happens when the channel opens, especially since the structure is known only for its closed state.  Moreover the channel was crystallized in a high concentration of detergent, a highly unnatural environment for the protein.  Thus there are ongoing debates about the open state structure and even possible orientation of the cytoplasmic alpha helix in the closed state.

            We intend to use single-channel patch clamping and micromechanical simulation with laser tweezers to test hypotheses about the motions involved in gating of Tb and possibly E. coli MscL channels.  Streptavidin beads connected with the help of a tether to specific residues of the channel will be used to exert the force.  l-phage DNA has the appropriate length and can be connected to the beads and biotinylated residues on the channel and therefore is a likely candidate to be used as a tether.  The beads can be driven with the help of laser tweezers.  The force that can be applied in such a way varies from sub-pN to around 100 pN, which seems to be an appropriate range for opening the channel protein.

            A necessary step is to test the accessibility of different residues by the biotin-streptavidin complex.  Mutants of Tb-Mscl containing cysteines in selected positions will be prepared (a few of these mutants are already available from the Rees group) and reacted with a biotin-maleimide compound for the channel incorporated in an artificial membrane.  Binding of biotin-maleimide will be assayed by coupling the resultant product with the fluorescent streptavidin.  This process will provide important information about accessibility of different residues of properly folded channel in its natural environment (in the membrane).  Combined with the measurements of electrophysiological activity of mutants this information will give important insights into the function of the channel.

1Division of Physics, Mathematics, and Astronomy, Caltech

2Div. of Engineering and Applied Science, Caltech

 

372.     M. tuberculosis MscL channels.  New gain of function mutations in the loop region

            Joshua A. Maurer1, Donald E. Elmore1, Dennis A. Dougherty1

            Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for E. coli and M. tuberculosis MscL and to place these homologues into sequence families.  Using this alignment, previously identified E. coli MscL mutants which displayed severe and very severe gain of function phenotypes were mapped onto the M. tuberculosis MscL sequence.  Not all of the resulting M. tuberculosis mutants displayed a gain of function phenotype, including mutations at A20, the analogue of the highly sensitive G22 site in E. coli.  A previously unnoticed intersubunit hydrogen bond in the extracellular loop region of the M. tuberculosis MscL crystal structure has been analyzed.  Crosslinkable residues were substituted for the residues involved in the hydrogen bond, and crosslinking studies indicated that these sites are spatially close under physiological conditions.  In general, mutation at these positions results in a gain of function phenotype, which provides strong evidence for the importance of the loop region in MscL channel function.  No analogue to this interesting interaction could be found in E. coli MscL by sequence alignment.  Taken together, these results indicate that caution should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies of E. coli MscL.

1Division of Chemistry and Chemical Engineering, Caltech


373.     GABA receptors modulate CA3-CA1 synapses during cAMP-mediated long-term plasticity

            Tzu-ping Yu, Sheri McKinney, Norman Davidson

            cAMP has been reported to selectively induce a protein-synthesis-dependent late phase of long-term potentiation (L-LTP) at CA3-CA1 synapses in acute hippocampal slices.  Here we report long-term synaptic potentiation and depression induced by cAMP at monosynaptic pairs of CA3 and CA1 cells of organotypic hippocampal slice culture.

            Excitatory postsynaptic potentials (EPSPs) of a single CA1 cell were evoked by single action potentials of a synaptically connected CA3 cell.  Following bath application of the membrane permeable cAMP analog, Sp-cAMPS, enhancement of synaptic transmission occurred and could last at least 2-3 hours.  The early phase of potentiation (E-LTP) was associated with a reduction of paired-pulse facilitation and was blocked by the GABA receptor antagonist, picrotoxin.  This E-LTP was mediated through PKA since it could be blocked by the PKA inhibitor, Rp-cAMPS.  L-LTP was accompanied by increases in both frequency and amplitude of spontaneous EPSPs (sEPSPs) of postsynaptic CA1 cells.  Pre-incubation of slice culture with a protein synthesis inhibitor, anisomycin, blocked L-LTP without affecting E-LTP.  Furthermore, we found that instead of producing LTP, Sp-cAMPS induced long-term depression in two slices pretreated with 50mM picrotoxin.  This form of LTD was associated with a reduction in mean amplitude of sEPSPs.  These results show that GABAergic neurons can modulate the effect of Sp-cAMPS on synaptic strength, and thus subsequently determine the direction of synaptic plasticity.

 

Publications

Dang, H., England, P.M., Farivar, S.S., Dougherty, D.A. and Lester, H.A. (2000) Probing the role of a conserved M1 proline residue in 5-Hydroxytryptamine(3) receptor gating.  Mol. Pharmacol. 57:1114-1122.

England, P.M., Lester, H.A. and Dougherty, D.A. (1999) Mapping disulfide connectivity using backbone ester hydrolysis.  Biochemistry 38:14409‑14415.

Ivanina, T., Neusch, C., Li, Y-X., Tong, Y., Labarca, C., Mosher, D.F., and Lester, H.A. (2000) Expression of GIRK (Kir3.1/Kir3.4) channels in mouse fibroblast cells with and without b1 Integrins.  FEBS Lett. 466:327-332.

Khakh, B.S. and Lester H.A. (1999) Dynamic selectivity filters in ion channels.  Neuron 23:653‑658.

Khakh, B.S., Zhou, X., Sydes, J., Galligan, J.J., and Lester, H.A. (2000) State-dependent cross inhibition between transmitter-gated cation channels.  Nature 406:405-410.

Kofuji, P., Ceelen, P., Zahs, K.R., Surbeck, L.W., Lester, H.A. and Newman, E.A. (2000) Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 Subunit) in mice:  Phenotypic impact in retina.  J. Neurosci. 20:5733-5740.

Kovoor, A., Chen, C-K., He, W., Wensel, T.G., Simon, M.I. and Lester, H.A. (2000) Co-expression of Gb5 enhances the function of two Gg subunit-like domain-containing regulators of G protein signaling proteins.  J. Biol. Chem. 275:3397-3402.

Li, M., Farley, R.A. and Lester, H.A. (2000) An intermediate state of the g-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence.  J. Gen. Physiol. 115:491-508.

Maurer, J.A., Elmore, D.E., Lester, H.A. and Dougherty, D.A. (2000) Comparing and contrasting E.coli and M. tuberculosis mechanosensitive channels (MscL).  New gain of function mutations in the loop regions.  J. Biol. Chem. 275:22238-22244.

Nadeau, H. and Lester, H.A. (2000) Two-compartment model for whole-cell data analysis and transient compensation.  J. Neurosci. Meth. 99:25-35.

Nadeau, H., McKinney, S., Anderson, D.J. and Lester, H.A. (2000) ROMK1 (Kir1.1) Causes apoptosis and chronic silencing of hippocampal neurons.   J. Neurophysiol. 84:1062-1075.

Tong, Y., Brandt, G., Li, M., Shapovalov, G., Slimko, E., Karschin, A., Dougherty, D.A. and Lester, H.A.  Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel.  Submitted for publication.