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H A Lester

Publications and source records attributed to H A Lester.

At least 19 recordsLinked to original sources
PubMed2002

NRSF causes cAMP-sensitive suppression of sodium current in cultured hippocampal neurons.

The neuron restrictive silencer factor (NRSF/REST) has been shown to bind to the promoters of many neuron-specific genes and is able to suppress transcription of Na(+) channels in PC12 cells, although its functional effect in terminally differentiated neurons is unknown. We constructed lentiviral vectors to express NRSF as a bicistronic message with green fluorescent protein (GFP) and followed infected hippocampal neurons in culture over a period of 1-2 wk. NRSF-expressing neurons showed a time-dependent suppression of Na(+) channel function as measured by whole cell electrophysiology. Suppression was reversed or prevented by the addition of membrane-permeable cAMP analogues and enhanced by cAMP antagonists but not affected by increasing protein expression with a viral enhancer. Secondary effects, including altered sensitivity to glutamate and GABA and reduced outward K(+) currents, were duplicated by culturing GFP-infected control neurons in TTX. The striking similarity of the phenotypes makes NRSF potentially useful as a genetic "silencer" and also suggests avenues of further exploration that may elucidate the transcription factor's in vivo role in neuronal plasticity.

Animals↗
PubMed2001

Kir4.1 potassium channel subunit is crucial for oligodendrocyte development and in vivo myelination.

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 marked motor impairment, and the cellular basis is hypomyelination in the spinal cord, accompanied by severe spongiform vacuolation, axonal swellings, and degeneration. Immunostaining in the spinal cord of wild-type mice up to postnatal day 18 reveals that Kir4.1 is expressed in myelin-synthesizing oligodendrocytes, but probably not in neurons or glial fibrillary acidic protein-positive (GFAP-positive) astrocytes. Cultured oligodendrocytes from developing spinal cord of Kir4.1-/- mice lack most of the wild-type K(+) conductance, have depolarized membrane potentials, and display immature morphology. By contrast, cultured neurons from spinal cord of Kir4.1-/- mice have normal physiological characteristics. We conclude that Kir4.1 forms the major K(+) conductance of oligodendrocytes and is therefore crucial for myelination. The Kir4.1 knock-out mouse is one of the few CNS dysmyelinating or demyelinating phenotypes that does not involve a gene directly involved in the structure, synthesis, degradation, or immune response to myelin. Therefore, this mouse shows how an ion channel mutation could contribute to the polygenic demyelinating diseases.

Animals↗
PubMed2001

Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors.

ATP-gated P2X(2) receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X(2) receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X(2)-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X(2)-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X(2)-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X(2) receptor distribution on the seconds time scale.

Adenosine Triphosphate↗
PubMed2001

Gamma-aminobutyric acid type A receptors modulate cAMP-mediated long-term potentiation and long-term depression at monosynaptic CA3-CA1 synapses.

cAMP induces a protein-synthesis-dependent late phase of long-term potentiation (LTP) at CA3-CA1 synapses in acute hippocampal slices. Herein we report cAMP-mediated LTP and long-term depression (LTD) at monosynaptic CA3-CA1 cell pairs in organotypic hippocampal slice cultures. After bath application of the membrane-permeable cAMP analog adenosine 3',5'-cyclic monophosphorothioate, Sp isomer (Sp-cAMPS), synaptic transmission was enhanced for at least 2 h. Consistent with previous findings, the late phase of LTP requires activation of cAMP-dependent protein kinase A and protein synthesis. There is also an early phase of LTP induced by cAMP; the early phase depends on protein kinase A but, in contrast to the later phase, does not require protein synthesis. In addition, the cAMP-induced LTP is associated with a reduction of paired-pulse facilitation, suggesting that presynaptic modification may be involved. Furthermore, we found that Sp-cAMPS induced LTD in slices pretreated with picrotoxin, a gamma-aminobutyric acid type A (GABA(A)) receptor antagonist. This form of LTD depends on protein synthesis and protein phosphatase(s) and is accompanied by an increased ratio of failed synaptic transmission. These results suggest that GABA(A) receptors can modulate the effect of cAMP on synaptic transmission and thus determine the direction of synaptic plasticity.

Animals↗
PubMed2001

Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety.

Knock-in mice were generated that harbored a leucine-to-serine mutation in the alpha4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.

Animals↗
PubMed2001

Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with 'knock-in' animals and other expression systems.

Quantitative aspects of synaptic transmission 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 histidine-tagged GFP molecules (His6-GFP) bound to Ni-NTA moieties on transparent beads (80-120 microm diameter) over a density range comprising nearly four orders of magnitude (to 30000 GFP/microm2). The procedures employ commonly available Hg lamps, fluorescent microscopes, 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 root mean square (rms) deviation of 11-21% 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 > approximately 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%.

Animals↗
PubMed2001

The tethered agonist approach to mapping ion channel proteins--toward a structural model for the agonist binding site of the nicotinic acetylcholine receptor.

BACKGROUND: 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 one of the most powerful probes of ion channels and related structures. The nonsense suppression methodology can also be used to incorporate functional side chains designed to deliver novel structural probes to membrane proteins. In this vein, we sought to generalize a potentially powerful tool - the tethered agonist approach - for mapping the agonist binding site of ligand-gated ion channels. RESULTS: 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 alpha149, there is a clear preference for a three-carbon tether, while at position alpha93 tethers of 2-5 carbons are comparably effective. At position gamma55/delta57 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 acetylcholine esterase crystal structure. CONCLUSION: Through the use of nonsense suppression techniques, the tethered agonist approach has been made into a general tool for probing receptor structures. When applied to the nicotinic receptor, the method places new restrictions on developing models for the agonist binding site.

Animals↗
PubMed2001

Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel.

Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used 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 decaging-induced 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 results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor (32)P labeling produced 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 interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems.

Animals↗
PubMed2001

Ion channel diseases of the central nervous system.

In the last decade, advances in molecular genetics and cellular electrophysiology have increased our understanding of ion channel function. A number of diseases termed "channelopathies" have been discovered that are caused by ion channel dysfunction. Channelopathies can be caused by autoimmune, iatrogenic, toxic or genetic mechanisms. Mutations in genes encoding ion channel proteins that disrupt channel function are now the most commonly identified cause of channelopathies, perhaps because gene disruption is readily detected by the methods of molecular genetics. Ion channels are abundant in the central nervous system (CNS), but CNS channelopathies are rare; however, they overlap with some important neurological disorders, such as epilepsy, ataxia, migraine, schizophrenia, Alzheimer's disease and other neurodegenerative diseases. It is possible that more CNS channelopathies will be discovered when additional ion channels are characterized and the complex mechanisms of brain function are better understood. At present, increased knowledge of the identity, structure and function of ion channels is facilitating diagnosis and treatment of many channelopathies.

Animals↗
PubMed2001

Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor.

The nonsense codon suppression technique was used to incorporate o-nitrobenzyl cysteine or o-nitrobenzyl tyrosine (caged Cys or Tyr) into the 9' position of the M2 transmembrane segment of the gamma-subunit of the muscle nicotinic ACh receptor expressed in Xenopus oocytes. The caged amino acids replaced an endogenous Leu residue that has been implicated in channel gating. ACh-induced current increased substantially after ultraviolet (UV) irradiation to remove the caging group. This represents the first successful incorporation of caged Cys into a protein in vivo and the first incorporation of caged amino acids within a transmembrane segment of a membrane protein. The bulky nitrobenzyl group does not prevent the synthesis, assembly, or trafficking of the ACh receptor. When side chains were decaged using 1-ms UV light flashes, the channels with caged Cys or caged Tyr responded with strikingly different kinetics. The increase in current upon photolysis of caged Cys was too rapid for resolution by the voltage-clamp circuit [time constant (tau) <10 ms], whereas the increase in current upon photolysis of caged Tyr was dominated by a phase with tau approximately 500 ms. Apparently, the presence of a bulky o-nitrobenzyl Tyr residue distorts the receptor into an abnormal conformation. Upon release of the caging group, the receptor relaxes, with tau approximately 500 ms, into a conformation that allows the channel to open. Tyr at the 9' position of the gamma-subunit greatly increases the ability of ACh to block the channel by binding within the channel pore. This is manifested in two ways. 1) A "rebound," or increase in current, occurs upon removal of ACh from the bathing medium; and 2) at ACh concentrations >400 microM, inward currents are decreased through the mutated channel. The ability to incorporate caged amino acids into proteins should have widespread utility.

Animals↗
PubMed2000

Modulation of early growth response (EGR) transcription factor-dependent gene expression by using recombinant adenovirus.

Early growth response (EGR) transcription factors link initial cytoplasmic events to long-term alterations of cellular gene expression and are induced by various stimuli. To test their roles in cell physiology, we constructed adenoviral recombinants encoding NGFI-A binding protein 2 (NAB2, a repressor of EGR1, EGR2, and EGR3), EGR1, NAB-insensitive EGR1(I293F) (EGR1*), EGR2, and the NAB-binding, repressive domain 1 (R1) of EGR1. These viruses regulated EGR-dependent expression of GFP and luciferase reporter genes in heterologous expression assays. Infection of a myoblast cell line with EGR1 and EGR1* adenovirus induced the endogenous gene for platelet-derived growth factor A chain (PDGF-A). In addition, in neuroblastoma cells, the two novel EGR1 target genes EGR3 and NAB2 were identified by using adenoviral transfer of EGR1 and EGR1*. Our results demonstrate that recombinant adenovirus is useful to regulate heterologous and endogenous EGR target gene expression, and suggest that EGR transcription factors can autoregulate themselves.

Adenoviridae↗
PubMed2000

Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: phenotypic impact in retina.

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. In 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 10-fold increase in the input resistance and a large depolarization of Kir4.1 -/- Müller cells compared with 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.

Amino Acid Sequence↗
PubMed2000

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

Transmitter-gated cation channels are detectors of excitatory chemical signals at synapses in the nervous system. Here we show that structurally distinct alpha3beta4 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 owing 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 are occluded during activation of endogenously co-expressed P2X channels. Our data provide a molecular basis and a synaptic context for cross-inhibition between transmitter-gated channels.

Acetylcholine↗
PubMed2000

Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL). New gain of function mutations in the loop region.

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that 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; for instance, normal phenotypes were noted for mutations at Ala(20), the analogue of the highly sensitive Gly(22) 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. Cross-linkable residues were substituted for the residues involved in the hydrogen bond, and cross-linking 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.

Amino Acid Sequence↗
PubMed2000

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

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. Programs to implement the algorithms are available from http://www.its.caltech.edu/ approximately nadeau/Rscomp.html.

Animals↗
PubMed2000

Co-expression of Gbeta5 enhances the function of two Ggamma subunit-like domain-containing regulators of G protein signaling proteins.

Regulators of G protein signaling (RGS) stimulate the GTPase activity of G protein Galpha subunits and probably play additional roles. Some RGS proteins contain a Ggamma subunit-like (GGL) domain, which mediates a specific interaction with Gbeta5. The role of such interactions in RGS function is unclear. RGS proteins can accelerate the kinetics of coupling of G protein-coupled receptors to G-protein-gated inwardly rectifying K(+) (GIRK) channels. Therefore, we coupled m2-muscarinic acetylcholine receptors to GIRK channels in Xenopus oocytes to evaluate the effect of Gbeta5 on RGS function. Co-expression of either RGS7 or RGS9 modestly accelerated GIRK channel kinetics. When Gbeta5 was co-expressed with either RGS7 or RGS9, the acceleration of GIRK channel kinetics was strongly increased over that produced by RGS7 or RGS9 alone. RGS function was not enhanced by co-expression of Gbeta1, and co-expression of Gbeta5 alone had no effect on GIRK channel kinetics. Gbeta5 did not modulate the function either of RGS4, an RGS protein that lacks a GGL domain, or of a functional RGS7 construct in which the GGL domain was omitted. Enhancement of RGS7 function by Gbeta5 was not a consequence of an increase in the amount of plasma membrane or cytosolic RGS7 protein.

Animals↗
PubMed2000

Expression of GIRK (Kir3.1/Kir3.4) channels in mouse fibroblast cells with and without beta1 integrins.

G protein-activated K+ channel (GIRK) subunits possess a conserved extracellular integrin-binding motif (RGD) and bind directly to beta1 integrins. We expressed GIRK1/GIRK4 channels labeled with green fluorescent protein in fibroblast cell lines expressing or lacking beta1 integrins. Neither plasma membrane localization nor agonist-evoked GIRK currents were affected by the absence of beta1 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 beta1 integrins remained intact. Thus, beta1 integrins are not essential for functional GIRK expression; and the GIRK-integrin interactions involve structural elements other than the RGD motif.

Animals↗