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H A Lester

Publications and source records attributed to H A Lester.

At least 145 records · Page 8Linked to original sources
PubMed1990

A neutral amino acid change in segment IIS4 dramatically alters the gating properties of the voltage-dependent sodium channel.

Sodium channels encoded by the rat IIA cDNA clone [Auld, V. J., Goldin, A. L., Krafte, D. S., Marshall, J., Dunn, J., Catterall, W. A., Lester, H. A., Davidson, N. & Dunn, R. J. (1988) Neuron 1, 449-461] differ at seven amino acid residues from those encoded by the rat II cDNA [Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takeshima, H., Kurasaki, M., Takahashi, H. & Numa, S. (1986) Nature (London) 320, 188-192]. When expressed in Xenopus oocytes, rat IIA channels display a current-voltage relationship that is shifted 20-25 mV in the depolarizing direction relative to channels expressed from rat II cDNA or rat brain poly(A)+ mRNA. By modifying each variant residue in rat IIA to the corresponding residue in rat II, we demonstrate that a single Phe----Leu substitution at position 860 in the S4 segment of domain II is sufficient to shift the current-voltage relationship to that observed for channels expressed from rat brain poly(A)+ RNA or rat II cDNA. Rat genomic DNA encodes leucine but not phenylalanine at position 860, indicating that the phenylalanine at this position in rat IIA cDNA likely results from reverse transcriptase error.

Amino Acid Sequence↗
PubMed1990

Rat brain expresses a heterogeneous family of calcium channels.

We describe the isolation and characterization of several rat brain cDNAs that are homologous to the alpha 1 subunit of heart and skeletal muscle dihydropyridine-sensitive Ca channels. Northern blot analysis of 32 cDNAs shows that they can be grouped into four distinct classes (A, B, C, and D), each corresponding to a distinct hybridization pattern of brain mRNAs. Southern blot and DNA sequencing suggest that each class of cDNA represents a distinct gene or gene family. In the regions sequenced, the rat brain class C and D gene products share approximately 75% amino acid identity with the rabbit skeletal muscle Ca channel. In addition, the class C polypeptide is almost identical to the rabbit cardiac Ca channel (97% identity). In contrast, the rat brain class A and B cDNAs are more distantly related to dihydropyridine-sensitive Ca channels (47-64% amino acid identity) and to the brain class C and D genes (51-55% amino acid identity). To examine the functional significance of the isolated brain cDNAs, hybrid depletion experiments were performed in Xenopus oocytes. Antisense oligonucleotides against class A and B cDNAs each partially inhibited, and a class C oligonucleotide almost fully inhibited, the expression of Ba current in rat brain mRNA injected oocytes; but none of the oligonucleotides affected the expression of voltage-gated Na or K conductances. The clone characterization and sequencing results demonstrate that a number of distinct, yet related, voltage-gated Ca-channel genes are expressed in the brain. The antisense oligonucleotide experiments specifically show that one or several of the Ca-channel classes are related to the Ca channels observed in rat brain mRNA injected oocytes.

Amino Acid Sequence↗
PubMed1990

Inactivation of cloned Na channels expressed in Xenopus oocytes.

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.

Animals↗
PubMed1989

Functional expression of the yeast alpha-factor receptor in Xenopus oocytes.

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a 431-residue polypeptide that has been shown by chemical cross-linking and genetic studies to be a component of the receptor for the peptide mating pheromone, alpha-factor. To demonstrate directly that the ligand binding site of the alpha-factor receptor is comprised solely of the STE2 gene product, the STE2 protein was expressed in Xenopus oocytes. Oocytes microinjected with synthetic STE2 mRNA displayed specific surface binding for 35S-labeled alpha-factor (up to 40 sites/micron2/ng RNA). Oocytes injected with either STE2 antisense RNA or heterologous receptor mRNA (nicotinic acetylcholine receptor alpha, beta, gamma, and delta subunit mRNAs) showed no binding activity (indistinguishable from uninjected control oocytes). The apparent KD (7 nM) of the alpha-factor binding sites expressed on the oocyte surface, determined by competition binding studies, agreed with the values reported for intact yeast cells and yeast plasma membrane fractions. These findings demonstrate that the STE2 gene product is the only yeast polypeptide required for biogenesis of a functional alpha-factor receptor. Electrophysiological measurements indicated that the membrane conductance of oocytes injected with STE2 mRNA, or with both STE2 and GPA1 (encoding a yeast G protein alpha-subunit) mRNAs, did not change and was not affected by pheromone binding. Thus, the alpha-factor receptor, like mammalian G protein-coupled receptors, apparently lacks activity as an intrinsic or ligand-gated ion channel. This report is the first instance in which a membrane-bound receptor from a unicellular eukaryote has been expressed in a vertebrate cell.

Animals↗
PubMed1989

Further evidence demonstrating that N-methyl-D-aspartate and kainate activate distinct ion channels.

Several excitatory amino acid receptors encoded by rat brain mRNA were expressed in Xenopus oocytes. Experimental protocols using an open channel blocker (MK-801) were designed to test the common receptor-channel hypothesis in which N-methyl-D-aspartate (NMDA) and kainate activate the same ion channel but induce different open channel conformations with different ionic permeabilities. The present data demonstrate that NMDA exposes previously trapped MK-801 molecules to the transmembrane field and accelerates their dissociation from the channel at positive potentials, while kainate lacks this effect. Therefore, kainate does not activate the same ion channel as NMDA does. Furthermore, differential inhibition of the NMDA response or the kainate response by the competitive antagonists D-2-amino-5-phosphonopentanoic acid (D-AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) indicates that NMDA and kainate do not share the same binding site. Thus, these several lines of evidence demonstrate that two distinct receptor-channels are activated by NMDA and kainate, respectively.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1989

Expression of functional sodium channels in stage II-III Xenopus oocytes.

We have injected mRNA from rabbit brain into stage II-III Xenopus oocytes to determine whether they will translate exogenous RNA and incorporate functional ion channels into the membrane. Our results show that 48 h after RNA injection, functional voltage-dependent Na channels are present at sufficient densities to allow quantitative electrophysiological recording. The smaller oocytes have at least 2 experimental advantages over the stage V-VI oocytes normally used for electrophysiological experiments: (1) the smaller membrane capacitance (approximately 5-fold) allows a faster settling time following a voltage step and a more detailed kinetic analysis of membrane currents than was previously possible with the 2-microelectrode technique, and (2) roughly 8-fold less RNA is needed for each injection. Thus, the stage II-III Xenopus oocyte is a suitable preparation for the study of ion channels.

Animals↗
PubMed1989

Expression of Drosophila Shaker potassium channels in mammalian cells infected with recombinant vaccinia virus.

A recombinant vaccinia virus containing a Drosophila potassium channel (Shaker H4) cDNA was constructed by homologous recombination between wild-type vaccinia virus DNA and a transfer plasmid. The new virus was used to infect four types of mammalian cells in culture. Electrophysiological recording 24-72 hr after infection revealed the expression of voltage-gated transient potassium channels in all four cell types. The properties of the induced currents were identical to those previously observed following injection of the Shaker H4 transcript into oocytes. Vaccinia promises to be an effective vehicle for the heterologous expression of transmembrane ion channels in a variety of cell types.

4-Aminopyridine↗
PubMed1988

Evidence that the M2 membrane-spanning region lines the ion channel pore of the nicotinic receptor.

Site-directed mutagenesis and expression in Xenopus oocytes were used to study acetylcholine receptors in which serine residues (i) were replaced by alanines (alpha, delta subunits) or (ii) replaced a phenylalanine (beta subunit) at a postulated polar site within the M2 transmembrane helix. As the number of serines decreased, there were decreases in the residence time and consequently the equilibrium binding affinity of QX-222, a quaternary ammonium anesthetic derivative thought to bind within the open channel. Receptors with three serine-to-alanine mutations also displayed a selective decrease in outward single-channel currents. Both the direction of this rectification and the voltage dependence of QX-222 blockade suggest that the residues mutated are within the aqueous pore of the receptor and near its cytoplasmic (inner) surface.

Animals↗
PubMed1988

Heterologous expression of excitability proteins: route to more specific drugs?

Many clinically important drugs act on the intrinsic membrane proteins (ion channels, receptors, and ion pumps) that control cell excitability. A major goal of pharmacology has been to develop drugs that are more specific for a particular subtype of excitability molecule. DNA cloning has revealed that many excitability proteins are encoded by multigene families and that the diversity of previously recognized pharmacological subtypes is matched, and probably surpassed, by the diversity of messenger RNAs that encode excitability molecules. In general, the diverse subtypes retain their properties when the excitability proteins are expressed in foreign cells such as oocytes and mammalian cell lines. Such heterologous expression may therefore become a tool for testing drugs against specific subtypes. In a systematic research program to exploit this possibility, major considerations include alternative processing of messenger RNA for excitability proteins, coupling to second-messenger systems, and expression of enough protein to provide material for structural studies.

Animals↗
PubMed1988

Properties of two classes of rat brain acidic amino acid receptors induced by distinct mRNA populations in Xenopus oocytes.

The Xenopus laevis oocyte expression system was used to study the molecular composition of mRNAs encoding acidic amino acid (AA) receptors from rat brain. Xenopus oocytes injected with poly(A) mRNA express two general classes of AA receptors. One class consists of AA-gated cation channels. Responses are evoked by N-methyl-D-aspartate (NMDA), by kainate, and to a lesser extent by L-glutamate or quisqualate. The second class of receptor is coupled to an intracellular second messenger pathway activating an oocyte-encoded Ca2+-activated Cl- conductance. This second messenger-coupled AA receptor can be activated by L-glutamate or quisqualate. DL-2-amino-5-phosphonopentanoic acid and D-alpha-aminohexanedioic acid inhibit the AA-gated cation conductances activated by NMDA or kainate with different potencies but do not inhibit the second messenger-coupled AA receptor. Responses to NMDA are enhanced by micromolar level of glycine and are inhibited by Mg2+, Zn2+, or MK-801. Dose-response analysis reveals that the AA-gated cation conductance activated by kainate requires the binding of two agonist molecules. To study the molecular composition, the mRNAs were size-fractionated by denaturing agarose gel electrophoresis. About 20-fold purification in specific activity (nA/ng of mRNA injected) of mRNAs encoding the second messenger-coupled AA receptor was achieved. In contrast, only a slight enrichment of the mRNAs encoding the AA-gated channel was observed. This suggests that the second messenger-coupled AA receptor is encoded by a single size class of mRNA, whereas the AA-gated cation channel(s) is encoded by multiple species of mRNAs or by mRNAs whose size distribution is heterogeneous.

Amino Acids↗
PubMed1988

Tetrodotoxin-sensitive voltage-dependent Na currents recorded from Xenopus oocytes injected with mammalian cardiac muscle RNA.

Voltage-sensitive sodium (Na) channel currents recorded from mammalian cardiac muscle are blocked by tetrodotoxin (TTX) with a Kd of 1-3 microM. We have observed a Kd for TTX of 4-10 nM for Na currents recorded from Xenopus oocytes injected with RNA extracted from rabbit cardiac muscle. This result suggests that the degree of TTX sensitivity of Na channels encoded by cardiac muscle mRNA is in part determined by post-translational modification(s) or of associations with accessory proteins in the membrane.

Animals↗
PubMed1988

At least two mRNA species contribute to the properties of rat brain A-type potassium channels expressed in Xenopus oocytes.

Fast transient K+ channels (A channels) of the type operating in the subthreshold region for Na+ action potential generation were expressed in Xenopus oocytes injected with rat brain poly(A) RNA. Sucrose gradient fractionation of the RNA separates mRNAs encoding A-currents (6-7 kb) from mRNAs encoding other voltage-dependent K+ channels. A-currents expressed with fractionated mRNA differ in kinetics and pharmacology from A-currents expressed with total mRNA. The original properties of the A-currents can be reconstituted when small mRNAs (2-4 kb) are added to the large mRNA fraction. Thus the properties of the A-currents expressed with total poly(A) RNA depend on the presence of more than one mRNA species. mRNA(s) present in the large RNA fraction must encode channel subunits since they express an A-current by themselves. The small mRNA(s) may encode a second subunit(s) or a factor, such as an enzymatic activity that modulates the properties of the channels, which could play a role in generating A-channel functional diversity.

Animals↗
PubMed1988

A rat brain Na+ channel alpha subunit with novel gating properties.

We have constructed a full-length rat brain Na+ channel alpha subunit cDNA that differs from the previously reported alpha subunit of Noda et al. at 6 amino acid positions. Transcription of the cDNA in vitro and injection into Xenopus oocytes resulted in the synthesis of functional Na+ channels. Although the single-channel conductance of the channels resulting from cloned cDNA was the same as that of channels resulting from injection of rat brain RNA, we observed two significant differences in the gating properties of the channels. The Na+ currents from cloned cDNA displayed much slower macroscopic inactivation compared with those from rat brain mRNA. In addition, the current-voltage relationship for currents from cloned cDNA was shifted 20-25 mV in the depolarizing direction compared with currents from rat brain RNA. Coinjection of low MW rat brain RNA restored normal inactivation of the channels indicating the presence of a component, either a structural subunit of the channel complex or a modifying enzyme, necessary for normal gating of the channel.

Amino Acid Sequence↗
PubMed1988

A-type potassium channels expressed from Shaker locus cDNA.

A-type K+ currents are expressed in Xenopus oocytes injected with in vitro-synthesized transcripts from cDNAs for the Drosophila Shaker (Sh) locus. A single Sh gene product, possibly as a multimer, is sufficient for formation of functional A channels. Various Sh RNAs express A currents with distinct kinetic properties. An analysis of structure-function relationships shows that the conserved central region of Sh polypeptides determines ionic selectivity and overall channel behavior, whereas the divergent amino and carboxyl termini can modify channel kinetics. Alternative splicing of Sh gene transcripts may provide one mechanism for the generation of K+ channel diversity.

Animals↗
PubMed1988

Evidence for the involvement of more than one mRNA species in controlling the inactivation process of rat and rabbit brain Na channels expressed in Xenopus oocytes.

The properties of rat and rabbit brain sodium (Na) channels expressed in Xenopus oocytes following either unfractionated or high-molecular-weight mRNA injections were compared to assess the relative contribution of different size messages to channel function. RNA was size-fractionated on a sucrose gradient and a high-molecular-weight fraction (7-10 kilobase) encoding the alpha-subunit gave rise to functional voltage-dependent Na channels in the oocyte membrane. Single-channel conductance, mean open time, and time to first opening were all similar to the values for channels following injection of unfractionated RNA. In contrast, inactivation properties were markedly different; Na currents from high-molecular-weight RNA inactivated with a several-fold smaller macroscopic inactivation rate and showed a steady-state voltage dependence that was shifted in the depolarizing direction by at least 10 mV relative to that for unfractionated RNA. Single-channel recording revealed that the kinetic difference arose from a greater probability for high-molecular-weight RNA induced channels to reopen during a depolarizing voltage step. Pooling all gradient fractions and injecting this RNA into oocytes led to the appearance of Na channels with inactivation properties indistinguishable from those following injection of unfractionated RNA. These results suggest that mRNA species not present in the high-molecular-weight fraction can influence the inactivation process of rat brain Na channels expressed in Xenopus oocytes. This mRNA may encode beta-subunits or other proteins that are involved in posttranslational processing of voltage-dependent Na channels.

Animals↗
PubMed1987

Expression of mouse-Torpedo acetylcholine receptor subunit chimeras and hybrids in Xenopus oocytes.

In this study, in vitro synthesized mRNA encoding mouse and Torpedo nicotinic acetylcholine receptor subunits was injected into Xenopus oocytes, followed by assays for assembly onto the oocyte surface (using [125I]alpha-bungarotoxin binding) and for acetylcholine-induced conductances (using voltage clamp). We constructed hybrid acetylcholine receptors in Xenopus oocytes by injecting all 8 possible combinations of 4 subunit-specific mRNAs in which a single subunit is derived from the other species. For each hybrid combination, there is detectable assembly and conductance. We also constructed cDNA clones that encode chimeric acetylcholine receptor subunits in which part of the gamma subunit from Torpedo was replaced by the homologous region of the delta subunit from mouse. None of the chimeric subunits was able to replace the Torpedo gamma, mouse delta, or Torpedo delta subunit with regard to assembly or function. We therefore conclude that widely spaced (and unknown) parts of the protein chain are required for the intersubunit interactions that eventually lead to functional assembly of the receptor.

Animals↗
PubMed1987

Activation of a potassium current by rapid photochemically generated step increases of intracellular calcium in rat sympathetic neurons.

Although Ca2+ is a well-established intracellular messenger, there are many questions concerning the kinetics and spatial localization of its effects. Such problems may now be approached with the photosensitive Ca2+ chelator nitr-5. The Ca2+ affinity of this molecule decreases by a factor of 40 after absorption of near-UV light; Ca2+ is liberated with a time constant of approximately equal to 300 microseconds. Nitr-5 or the related compounds nitr-2 and nitr-7, complexed with Ca2+, were introduced into rat sympathetic ganglion cells by dialysis from a patch pipette electrode operating in the whole-cell, voltage-clamp mode. Light flashes released Ca2+ and activated a K+ current. Flash-induced current relaxations followed a simple exponential time course with time constants as brief as 5 ms. Comparison of the kinetics among the chelators, which photolyze at different rates, suggests that release of Ca2+ from nitr-5 is too fast to limit the relaxation. Thus we confirm directly that Ca2+ can modulate membrane properties within a few milliseconds after entering a cell. A preliminary kinetic description of K+ current activation by Ca2+ in rat sympathetic neurons is presented; Ca2+ appears to bind to the channel with a rate constant of at least 2 X 10(7) M-1 X s-1.

Animals↗