Search PubMed⌕ Search

Biomedical subjects

H A Lester

Publications and source records attributed to H A Lester.

At least 127 records · Page 7Linked to original sources
PubMed1992

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.

Animals↗
PubMed1992

Structure of the gamma-less nicotinic acetylcholine receptor: learning from omission.

The nicotinic acetylcholine receptor can be expressed in Xenopus oocytes by injection of in vitro synthesized RNA for the alpha, beta, gamma, and delta mouse muscle subunits. However, detectable responses can also be obtained by injection of alpha, beta, and delta subunit RNA only. The receptors expressed in this case (gamma-less receptors) share many of the properties of the normal receptor, including relaxation time constants, Hill slope, and relative permeability for Na+, K+, Cs+, and Tris+. The major single-channel conductances of alpha beta gamma delta and alpha beta delta receptors are similar (34.2 +/- 2.9 and 38.5 +/- 0.6 pS, respectively) but clearly different from the major conductances seen after the combined injection of alpha beta delta mouse subunit RNA and Xenopus gamma subunit RNA. Mutations in the second transmembrane segment of the alpha and beta subunits, known to affect open time and blockade by QX-222, are equally effective in the gamma-less receptor. These data strongly suggest that the gamma-less receptor has the same pore diameter as the normal receptor and that alpha, beta, and delta subunits participate in its formation. Injection of alpha beta gamma delta well as alpha beta delta RNA produced additional subconductance states of around 25 pS. The low conductance state was sensitive to mutations introduced in the alpha or beta subunits with or without the gamma subunit, indicating that this channel did not need the gamma subunits but required at least the alpha and beta subunits to be produced. Injection of alpha beta delta and the adult-type epsilon subunit RNA gave rise to channels with conductances of 35 and 55 pS when the stoichiometry of the injection was 2:1:1:1, but only the 55-pS channel was recorded when the epsilon subunit RNA concentration was increased by 10-fold (stoichiometry of 2:1:1:10). The gamma-less receptor can thus be expressed even when the adult epsilon subunit is present. Whether gamma-less receptors are expressed at normal adult neuromuscular junctions remains unknown.

Amino Acid Sequence↗
PubMed1991

Heterologously expressed serotonin 1A receptors couple to muscarinic K+ channels in heart.

In cardiac atrial cells, muscarinic acetylcholine receptors activate a K+ current directly via a guanine nucleotide-binding protein (G protein). Serotonin type 1A receptors may activate a similar pathway in hippocampal neurons. To develop a system in which receptor/G protein/K+ channel coupling can be experimentally manipulated, we have used a highly efficient recombinant vaccinia virus vector system to express human serotonin 1A receptors in primary cultures of rat atrial myocytes. The expressed 1A receptors activated the inwardly rectifying K+ conductance that is normally activated by the endogenous muscarinic acetylcholine receptors. Maximal responses to either agonist occluded further activation by the other agonist. The average activation time constants for serotonin were about 5 times slower than for acetylcholine. The data support suggestions that the intracellular signaling pathway from seven-helix receptors to G proteins and directly to ion channels is widespread in excitable cells. After a fraction of the G proteins are activated irreversibly by guanosine 5'-[gamma-thio]triphosphate, subsequent transduction proceeds more efficiently. One possible interpretation is that multiple G-protein molecules are required to activate each channel. Vaccinia virus expression vectors are thus useful for expressing seven-helix receptors in primary cultures of postmitotic cells and have provided a heterologous expression system for the signaling pathway from seven-helix receptors to G proteins and directly to ion channels.

Acetylcholine↗
PubMed1991

K+ channel expression in primary cell cultures mediated by vaccinia virus.

A recombinant vaccinia virus (VV) was used to express functional Drosophila Shaker H4 K+ channels in primary cell cultures from rat heart (atrial and ventricular myocytes, fibroblasts), autonomic ganglia (SCG neurons) and CNS (hippocampal neurons, cerebral astroglia). In most cells the expressed currents possessed the typical characteristics of the native Drosophila muscle A currents; a few cells showed evidence of hetero-oligomers with new properties. The maximum current density corresponded to a channel density of 2-3/microns 2. Voltage recordings in heart cells showed altered action potential waveforms after successful infection. VV vectors thus are useful for studying altered excitability and cell-specific processing of ion channel proteins.

Action Potentials↗
PubMed1991

Recording of voltage and Ca(2+)-dependent currents in Xenopus oocytes using an intracellular perfusion method.

We describe a method for internal perfusion of Xenopus laevis oocytes that allows control of the composition of intracellular and extracellular solutions, including the possibility of sequential introduction of different substances inside and outside the cell. Using this method, it was possible to record Ca2+ dependent Cl- current and to inhibit it by intracellular perfusion of EGTA-containing solution. With a high BA2+ solution at the external surface of the perfused oocyte, Ba2+ currents through voltage-dependent Ca2+ channels were observed in native and in cardiac RNA-injected oocytes. Finally, a delayed rectifier K+ current was recorded and blocked by internally perfused Cs+ in oocytes injected with mRNA of a cloned (MBK1) K+ channel. The method is expected to be useful for the study of function and modulation of ion channels and transporters in the oocyte, which is an important and widely used model system.

Animals↗
PubMed1991

Characterization of maintained voltage-dependent K(+)-channels induced in Xenopus oocytes by rat brain mRNA.

The voltage-dependent K+ currents encoded by rat brain mRNA were studied in Xenopus oocytes after the voltage-dependent Na+ currents and the Ca(2+)-activated Cl- currents were eliminated pharmacologically. This paper describes the maintained K+ currents (IK), defined primarily by resistance to inactivation for 1 s at a holding potential of -40 mV. IK activates at potentials more positive than -60 to -70 mV and consists of both low-threshold and high-threshold components. IK is partially blocked by both tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which appear to be blocking the same component. Long depolarizing pulses result in incomplete inactivation of IK; the inactivating component is inhibited by TEA. Sucrose density gradient fractionation partially resolves the RNA encoding the several components of IK; most IK arises from size classes between 3.8 and 9.5 kb. The study gives further evidence for the existence of numerous distinct RNA populations that encode brain K+ channels different from previously reported cloned K+ channels that have been expressed in Xenopus oocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Single-channel properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes.

This report analyzes the contribution of individual nicotinic acetylcholine receptor (AChR) subunits to the single-channel properties of the AChR ion channel. By in vitro synthesis of mRNA from cDNA clones encoding each AChR subunit (alpha, beta, gamma, and delta) from mouse BC3H-1 cells and Torpedo electric organ and microinjection of appropriate mRNA combinations into Xenopus oocytes, we studied the single-channel properties of both 'homologous' (all subunits from the same species) and 'hybrid' (subunits from both species) AChRs as they were expressed in the oocyte membrane. AChR expression was determined by surface binding of 125I-labeled alpha-bungarotoxin to intact oocytes, and those with binding sites of 1 fmol/cell or more were chosen for patch-clamp studies. Our results indicate the following: (1) Species difference in single-channel conductance can be explained largely by the charge distribution flanking the M2 transmembrane domain. (2) The alpha and delta subunits from mouse AChR independently lengthen the channel open time, in some cases by 10-fold; the beta subunit from mouse shortens the channel open time; the mouse gamma subunit lengthens open time less dramatically. (3) Voltage sensitivity, as measured by the ratio of channel open times at -60 mV and +60 mV, is influenced by the beta and delta subunits, in agreement with our previous study by two-electrode voltage-clamp recording. We conclude that single-channel properties of the AChR are governed by multiple elements located on different AChR subunits.

Amino Acid Sequence↗
PubMed1991

The mouse 5-HT1C receptor contains eight hydrophobic domains and is X-linked.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) exerts diverse physiological effects in the central and peripheral nervous systems and in smooth muscle by interacting with pharmacologically distinct membrane receptors. We report here the cDNA cloning of the mouse 5-HT1C receptor and its functional expression in Xenopus oocytes. This receptor possesses the unusual feature of containing eight hydrophobic domains capable of forming membrane-spanning alpha-helices, contrary to the usual '7-helix' paradigm for other membrane receptors that function through coupling to GTP-binding proteins. By hybridization analysis of Chinese hamster x mouse somatic cell hybrid lines, the gene for the receptor, designated Htr1c, has been assigned to the mouse X chromosome.

Amino Acid Sequence↗
PubMed1991

Modulation of a cloned mouse brain potassium channel.

The mouse brain K+ channel (MBK), previously cloned by others, has been independently cloned and shown to express in Xenopus oocytes. This K+ current (IK) inactivated over a time course of seconds and was sensitive to the K+ channel-blocking reagent tetraethylammonium. When the K+ channel was coexpressed with a cloned mouse brain serotonin receptor (5HT1c) in oocytes, activation of the 5HT1c receptor by a brief application of serotonin resulted in a suppression of the IK amplitude over the next 20 min. IK could also be suppressed by activation of G proteins. Suppression was also caused by intracellular Ca2+ injections and was blocked by intracellular injection of EGTA. Calmodulin antagonists block the IK suppression, but a known protein kinase inhibitor did not block suppression. The 5HT1c suppression was reversible; recovery from suppression was blocked by the protein kinase inhibitor H-7. These data suggest that the IK suppression occurs through a novel mechanism independent of A- or C-type protein kinases; suppression is best explained as being due to the action of a Ca2+/calmodulin-activated phosphatase; recovery from suppression is due to the action of a protein kinase.

Animals↗
PubMed1991

Low molecular weight mRNA encodes a protein that controls serotonin 5-HT1c and acetylcholine M1 receptor sensitivity in Xenopus oocytes.

Serotonin 5-HT1c and acetylcholine M1 receptors activate phosphoinositidase, resulting in an increased formation of IP3 and 1,2 diacylglycerol. In Xenopus oocytes injected with mRNA encoding either of these receptors, Ca2+ released from intracellular stores in response to IP3 then opens Ca(2+)-gated Cl-channels. In the present experiments, oocytes expressing a transcript from a cloned mouse serotonin 5-HT1c receptor were exposed to identical 15-s pulses of agonist, administered 2 min apart; the second current response was two to three times that of the first. However, in those oocytes coinjected with the 5-HT1c receptor transcript and a low molecular weight fraction (0.3-1.5 kb) of rat brain mRNA, the second current response was approximately 50% of the first. Thus, the low molecular weight RNA encodes a protein (or proteins) that causes desensitization. Experiments using fura-2 or a Ca(2+)-free superfusate indicated that desensitization of the 5-HT1c receptor response does not result from a sustained elevation of intracellular Ca2+ level or require the entry of extracellular Ca2+. Photolysis of caged IP3 demonstrated that an increase in IP3 and a subsequent rise in Ca2+ do not produce desensitization of either the IP3 or 5-HT1c peak current responses. Furthermore, in oocytes coinjected with the low molecular weight RNA and a transcript from the rat M1 acetylcholine receptor, the M1 current response was greatly attenuated. Our data suggest that the proteins involved in attenuation of the M1 current response and desensitization of the 5-HT1c current response may be the same.

Animals↗
PubMed1991

Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus.

The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors. We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV). 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes. 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors. The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor. After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes. However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes. Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method. Potential applications to structure-function studies and expression cloning are discussed.

Animals↗
PubMed1991

Reverse pharmacology of the nicotinic acetylcholine receptor. Mapping the local anesthetic binding site.

We have been examining the interaction of a local anesthetic derivative, QX-222, with the ion channel pore of the muscle AChR, using a combination of mutagenesis, oocyte expression, and electrophysiology. Single channel recording, together with macroscopic voltage-jump relaxations, provides a measure of the residence time of the open channel blocker within the pore. We have found systematic changes in the apparent affinity of the open channel for QX-222 following amino acid substitutions in the proposed M2 transmembrane helix of each of the four subunits of the AChR. Assigning the number 1' to the residue at the cytoplasmic end of the M2 helix, positions 2',6',10',14', and 18' are modeled as forming the lining of the pore. Polar to nonpolar substitutions at 6' decrease QX-222 residence time, while the opposite effect is seen at position 10'. Nonpolar to polar substitutions have the converse effect. The distance between the aromatic and quaternary amine moieties of QX-222 corresponds almost exactly to the repeat distance of an alpha helix. This structural feature is common to many local anesthetic drugs. We propose a model for the binding of QX-222 within the ion channel of the AChR that is consistent with these observations.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of a rat brain GABA transporter.

A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

Amino Acid Sequence↗
PubMed1990

An open-channel blocker interacts with adjacent turns of alpha-helices in the nicotinic acetylcholine receptor.

The binding site for an open-channel blocker, QX-222, at mouse muscle nicotinic acetylcholine receptors was probed using site-directed mutagenesis, oocyte expression, and electrophysiological analysis. The proposed cytoplasmic end of the M2 transmembrane helix is termed position 1'. At position 10' (alpha S252, beta T263, gamma A261, delta A266), Ala residues yield stronger and longer binding of QX-222 than Ser or Thr residues. These effects are opposite and roughly equal (30%-50% per mutation) to previously reported effects at position 6'. The polar end of an anesthetic molecule seems to bind to the position 6' OH groups, which provide a water-like region; the nonpolar moiety is near position 10' and binds more strongly in a nonpolar environment. Interactions with adjacent OH-rich turns of an amphiphilic helix may explain the widespread blocking effects of local anesthetics at the conduction pore of ion channels.

Amino Acid Sequence↗