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H A Lester

Publications and source records attributed to H A Lester.

At least 163 records · Page 9Linked to original sources
PubMed1987

cDNA cloning of a serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes.

We describe a strategy for the cloning of neurotransmitter-receptor and ion-channel cDNAs that is based on electrophysiological assays of mRNA-injected Xenopus oocytes. This procedure circumvents the purification of these membrane proteins, which is hindered by their low abundance and their hydrophobic nature. It involves methods for RNA fractionation by high-resolution gel electrophoresis, directional cDNA cloning in a single-stranded vector, and screening of the cDNA library by voltage-clamp measurements of currents induced by serotonin in mRNA-injected oocytes. The applicability of our approach is demonstrated by the isolation of a serotonin receptor cDNA clone from a mouse choroid plexus papilloma. The clone was identified by hybrid-depletion and hybrid-selection procedures. The receptor expressed in oocytes injected with hybrid-selected RNA is fully functional, indicating that it is composed of a single subunit encoded by a 5-kilobase RNA. The pharmacology of the hybrid-selected receptor confirms that we have successfully cloned a serotonin 5-HT1C receptor cDNA.

Animals↗
PubMed1987

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

This study used messenger RNA encoding each subunit (alpha, beta, gamma and delta) of the nicotinic acetylcholine (ACh) receptor from mouse BC3H-1 cells and from Torpedo electric organ. The mRNA was synthesized in vitro by transcription with SP6 polymerase from cDNA clones. All 16 possible combinations that include one mRNA for each of alpha, beta, gamma, and delta were injected into oocytes. After allowing 2-3 d for translation and assembly, we assayed each oocyte for (a) receptor assembly, measured by the binding of [125I]alpha-bungarotoxin to the oocyte surface, and (b) ACh-induced conductance, measured under voltage clamp at various membrane potentials. All combinations yielded detectable assembly (30-fold range among different combinations) and ACh-induced conductances (greater than 1,000-fold range at 1 microM). On double-logarithmic coordinates, the dose-response relations all had a slope near 2 for low concentrations of ACh. Data were corrected for variations in efficiency of translation among identically injected oocytes by expressing ACh-induced conductance per femtomole of alpha-bungarotoxin-binding sites. Five combinations were tested for d-tubocurarine inhibition by the dose-ratio method; the apparent dissociation constant ranged from 0.08 to 0.27 microM. Matched responses and geometric means are used for describing the effects of changing a particular subunit (mouse vs. Torpedo) while maintaining the identity of the other subunits. A dramatic subunit-specific effect is that of the beta subunit on voltage sensitivity of the response: gACh(-90 mV)/gACh(+30 mV) is always at least 1, but this ratio increases by an average of 3.5-fold if beta M replaces beta T. Also, combinations including gamma T or delta M usually produce greater receptor assembly than combinations including the homologous subunit from the other species. Finally, EACh is defined as the concentration of ACh inducing 1 microS/fmol at -60 mV; EACh is consistently lower for alpha M. We conclude that receptor assembly, voltage sensitivity, and EACh are governed by different properties.

Animals↗
PubMed1987

Ca channels induced in Xenopus oocytes by rat brain mRNA.

RNA was isolated from brains of 16-d-old rats and poly(A) samples were injected into stage V and VI oocytes. After allowing 2-5 d for expression, most oocytes were exposed to medium in which the K had been replaced by Cs for 24 hr prior to recording. Ba currents were usually measured in Cl-free Ba-methanesulfonate saline. IBa in noninjected oocytes was often undetectable, but ranged up to 50 nA (22 +/- 4 nA, n = 21). In contrast, injected oocytes showed a peak IBa of 339 +/- 42 nA (n = 33). The threshold for activation of IBa was -40 mV, with peak currents at +10 to +20 mV. After a peak, currents decayed to a nearly steady level along a single-exponential time course (tau = 650 +/- 50 msec at +20 mV). The maintained current was 67 +/- 6% (n = 9) of the early peak amplitude. A prepulse duration of 5 sec was needed to examine the inactivation of barium currents in injected oocytes. The inward IBa could be observed in BaCl2 solutions at potentials positive to ECl and also in Na-free salines, indicating that neither Cl- nor Na+ was carrying the inward current. Although IBa displayed voltage-independent blockade by Cd (50% inhibition at 6 microM), the peptide Ca channel antagonist, omega-CgTX (1 microM), and the organic Ca channel-blocking agents (verapamil, compound W-7, and nifedipine) were uniformly ineffective. No effects were observed with the dihydropyridine antagonist nifedipine (even at 10 microM, or when cells were held at -40 mV) or agonist Bay K-8644. However, IBa was enhanced via activation of protein kinase C with 4-beta-phorbol dibutyrate (PBT2). In contrast, use of forskolin to activate protein kinase A did not alter IBa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Rat brain 5-HT1C receptors are encoded by a 5-6 kbase mRNA size class and are functionally expressed in injected Xenopus oocytes.

Injection of rat brain RNA into Xenopus laevis oocytes induces synthesis of receptors that show an electrophysiological response to bath application of serotonin. While there are at least 4 pharmacologically distinct subtypes of 5-HT binding sites in the rat brain, we find that the pharmacological characteristics of the predominant electrophysiologically active receptor synthesized in Xenopus oocytes are most consistent with those of the 5-HT1C subtype. Additional electrophysiologically active 5-HT receptor types could not be detected. Injection of mRNA isolated from a number of rat brain regions shows that the choroid plexus is particularly enriched for 5-HT1C mRNA. Oocytes injected with RNA isolated from this region respond 16 or 8 times more strongly to serotonin than do oocytes injected with RNA isolated from cortex or substantia nigra, respectively. In addition, by fractionation of rat brain mRNA through agarose gels, we have identified a single RNA size class of about 5-6 kbase that encodes this serotonin receptor.

Animals↗
PubMed1986

Expression and modulation of voltage-gated calcium channels after RNA injection in Xenopus oocytes.

Calcium ions flow into cells through several distinct classes of voltage-dependent calcium-selective channels. Such fluxes play important roles in electrical signaling at the cell membrane and in chemical signaling within cells. Further information about calcium channels was obtained by injecting RNA isolated from rat brain, heart and skeletal muscle into Xenopus oocytes. Macroscopic currents through voltage-operated calcium channels were resolved when the endogenous calcium-dependent chloride current was blocked by replacing external calcium with barium and chloride with methanesulfonate. The resulting barium current was insensitive to tetrodotoxin but was completely blocked by cadmium or cobalt. With both heart and brain RNA at least two distinct types of calcium ion conductance were found, distinguishable by their time course and inactivation properties. In oocytes injected with heart RNA, the slowly inactivating component was selectively blocked by the calcium-channel antagonist nifedipine. Barium ion currents induced by heart RNA were modulated by isoproterenol, cyclic adenosine monophosphate, and acetylcholine.

Animals↗
PubMed1986

Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA.

Injection of poly(A)+ RNA from rat brain into Xenopus oocytes caused the appearance of Cl currents in response to serotonin (5-HT) and acetylcholine (ACh). Both neurotransmitters evoked two-component currents similar in their time course to the oocyte's endogenous cholinergic muscarinic response, which was shown in previous studies to be mediated by IP3 synthesis leading to Ca release from intracellular stores. The responses to ACh and 5-HT exhibited self- and cross-desensitization, i.e., application of either ACh or 5-HT inhibited the subsequent response to either one of the two transmitters. Intracellular injection of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) mimicked the 5-HT and ACh response, and also completely suppressed the response to the subsequent application of either ACh or 5-HT. Treatment of the oocytes with pertussis toxin (PTX) caused a 50% attenuation of ACh and 5-HT responses. In the membranes of both control and mRNA-injected oocytes, PTX catalyzed the ADP-ribosylation of a single Mr = approximately 40,000 protein. Injection of the purified beta gamma-subunits of transducin enhanced the 5-HT response. The 5-HT and GTP-gamma-S responses were inhibited by intracellular injection of the Ca2+ chelator, EGTA, as previously shown for the ACh response. These data suggest that ACh and 5-HT receptors, synthesized in the oocytes on the template of brain mRNA, act through a common pathway that involves (a) a guanine nucleotide binding protein and (b) IP3 production leading to Ca mobilization.

Acetylcholine↗
PubMed1986

Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes.

Several cDNA clones coding for the high molecular weight (alpha) subunit of the voltage-sensitive Na channel have been selected by immunoscreening a rat brain cDNA library constructed in the expression vector lambda gt11. As will be reported elsewhere, the amino acid sequence translated from the DNA sequence shows considerable homology to that reported for the Electrophorus electricus electroplax Na channel. Several of the cDNA inserts hybridized with a low-abundance 9-kilobase RNA species from rat brain, muscle, and heart. Sucrose-gradient fractionation of rat brain poly(A) RNA yielded a high molecular weight fraction containing this mRNA, which resulted in functional Na channels when injected into oocytes. This fraction contained undetectable amounts of low molecular weight RNA. The high molecular weight Na channel RNA was selected from rat brain poly(A) RNA by hybridization to a single-strand antisense cDNA clone. Translation of this RNA in Xenopus oocytes resulted in the appearance of tetrodotoxin-sensitive voltage-sensitive Na channels in the oocyte membrane. These results demonstrate that mRNA encoding the alpha subunit of the rat brain Na channel, in the absence of any beta-subunit mRNA, is sufficient for translation to give functional channels in oocytes.

Animals↗
PubMed1986

Dose-response of acetylcholine receptor channels opened by a flash-activated agonist in voltage-clamped rat myoballs.

Whole-cell or single-channel currents through acetylcholine (ACh) receptor channels were studied in voltage-clamped rat myoballs or in excised membrane patches from myoballs. The recording pipette contained CsCl to suppress outward currents, and tetrodotoxin was used to help suppress Na+ currents. To minimize problems associated with bath applied agonists, myoballs were bathed in a solution containing the inactive (cis) isomer of the photo-isomerizable azobenzene derivative, Bis-Q. Calibrated light flashes of varying intensity were presented to produce concentration jumps of agonist, trans-Bis-Q. The resulting whole-cell current relaxations through ACh channels approach a steady state along an exponential time course, then decline as the newly created agonist diffuses away over the next few seconds. The dose-response relationship was inferred from Hill (double-log) plots for myoballs bathed in 500 nM-cis-Bis-Q at three membrane potentials. At low agonist concentrations (less than 300 nM-trans-Bis-Q), the slope of the Hill plot averaged 1.62 at -150 mV, 1.89 at -100 mV, and 2.05 at +80 mV. These results are consistent with an apparent agonist affinity constant that decreases with membrane depolarization and shifts the responses further down on the dose-response curve. When the myoballs were bathed in higher concentrations of cis-Bis-Q (1.5-20 microM), the slope of the Hill plot was reduced at all membrane potentials, although it was still closer to two at positive potentials. This is expected from the known sigmoid shape of the dose-response relation. The shallow dependence of the Hill slope on agonist concentration suggests the presence of negative cooperativity in the over-all binding of agonist molecules. Following treatment of the membrane with dithiothreitol to reduce disulphide groups, the Hill slope for the reversibly bound agonist, trans-Bis-Q, remained near two. The kinetics of currents at hyperpolarized membrane potentials became complicated at higher agonist concentrations in a manner that was consistent with open-channel block by trans-Bis-Q; the currents showed a slow secondary increase in conductance. Averaged single-channel recordings at higher agonist concentrations resemble macroscopic relaxations under comparable conditions. Furthermore, those recordings also suggested that open channels are blocked by trans-Bis-Q at concentrations greater than 2 microM; the block depends strongly on membrane potential and increases with hyperpolarization. Currents at positive membrane potentials showed no evidence of open-channel block.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1986

Activation of acetylcholine receptor channels by covalently bound agonists in cultured rat myoballs.

Kinetic and equilibrium aspects of receptor activation by two irreversibly bound ('tethered') agonists, QBr and bromoacetylcholine (BrACh), were examined in cultured embryonic rat muscle. Myoballs were treated with dithiothretitol (2 mM), washed, exposed to BrACh or QBr, and then washed again. Voltage-clamp recordings were made both in the whole-cell mode and with excised outside-out patches at 15 degrees C. Whole-cell voltage-jump relaxations resembled those observed with reversibly bound agonists. The relaxation time constants were 5 ms for tethered QBr and 10 ms for tethered BrACh (-100 mV, 15 degrees C). At more positive membrane potentials, the relaxation rate constants increased and the conductance decreased. Whole-cell light-flash relaxations with tethered QBr were also studied. The conductance was increased and decreased, respectively, by cis----trans and trans----cis photoisomerizations. The relaxation time constants equalled those for voltage jumps. The functional stoicheiometry of tethered QBr was investigated by studying the relaxations in response to light flashes that produced known changes in the mole fractions of the two isomers. It is concluded that the open state of each receptor channel is controlled by the isomeric state of a single tethered QBr molecule. In single-channel recordings, tethered agonists opened channels with the same conductance as reversibly bound agonists (30 pS at 15 degrees C and -100 mV). More than 80% of the conductance was contributed by a population of openings with an average burst duration (lifetime) of 5 ms for QBr and 10 ms for BrACh. Thus the single-channel and macroscopic currents seem to be dominated by the same type of channel; these are presumably monoliganded receptors. About 30% of the openings belonged to a population with an average lifetime of about 0.5 ms. This population contributed less than 5% of the conductance. There were also more long openings (greater than 50 ms) than expected from a simple exponential distribution. A few patches from BrACh-treated cells showed openings with a conductance of 45 pS (-100 mV) and an average duration of approximately 2 ms. These data allow one to assess whether the agonist-receptor binding step plays a role in generating the brief openings. The main population of openings (burst durations 5 ms with QBr and 10 ms with BrACh) seem to be contributed by monoliganded receptors. One can therefore rule out the hypothesis that the brief channels arise exclusively from mono- and biliganded receptors, respectively.

Acetylcholine↗
PubMed1985

Photochemically produced intracellular concentration jumps of cAMP mimic the effects of catecholamines on excitation-contraction coupling in frog atrial fibers.

Previously, we reported that concentration jumps of cAMP produced by light flashes in the presence of a photosensitive analogue of cAMP increase the amplitude of the slow inward current (Isi) in isolated bullfrog atrial trabeculae (Nargeot et al. 1983). Here, using newly designed photolabile cyclic nucleotides (Nerbonne et al. 1984a), we have examined the effects of intracellular concentration jumps of cAMP and cGMP on excitation-contraction coupling in frog heart. Concentration jumps of cAMP increase the amplitude and the duration of action potentials, increase Isi and twitch tension. Following single flashes, maximum responses are observed in 10-30 s and recovery times are 30-120 s. The time courses of the cAMP-induced increases in Isi and phasic tension amplitudes are parallel, implying a direct correlation between Ca2+ influx through the slow channels and the development of phasic tension. Although the amplitudes are increased severalfold, cAMP jumps do not measurably alter the kinetics or voltage dependences of the current or tension. cAMP concentration jumps increase the delayed K+ current (IK) and decrease tonic tension; relaxation of contraction is not, however, influenced by cAMP jumps. Concentration jumps of cGMP, on the other hand, have no measurable effects on the action potential, Isi, IK or tension in this preparation.

Action Potentials↗
PubMed1985

Software for electrophysiological experiments with a personal computer.

These programs were written for the IBM personal computer equipped with a Tecmar Labmaster analog interface board. The software operates under the MS-DOS operating system; it is written in compiled BASIC and employs short machine-language subroutines for crucial functions. Details are presented on the analog interface routines which make special use of the 9513 counter/timer chip on the Labmaster. Time resolution is about 32 microseconds per sample on the PC and 15 microseconds on the PC/AT. One series of programs performs traditional voltage- and current-clamp experiments on macroscopic currents in whole cells. A second series interfaces with a hardware window discriminator to capture single-channel events in a recirculating buffer; a stimulus can be triggered to fire during an open channel. Off-line programs perform standard analyses, and further processing can be performed with standard spreadsheet programs.

Animals↗
PubMed1985

Photoactivation and dissociation of agonist molecules at the nicotinic acetylcholine receptor in voltage-clamped rat myoballs.

The photochemical properties of the azobenzene derivative, Bis-Q, were exploited to carry out an agonist concentration jump followed by a molecular rearrangement of bound agonist molecules at acetylcholine (ACh) receptor channels of voltage-clamped rat myoballs. Myoballs were bathed in solutions containing low concentrations of cis-Bis-Q, the inactive isomer. Whole-cell current relaxations were studied following a light flash that produced a concentration jump of agonist, trans-Bis-Q, followed by a second flash that produced net trans----cis photoisomerizations of Bis-Q molecules. The concentration-jump relaxation provided a measure of the mean burst duration for ACh receptor channels occupied by trans-Bis-Q (7.7 ms, 22 degrees C). The second current relaxation was a more rapid conductance decrease (phase 1, tau = 0.8 ms). Phase 1 may represent either the burst duration for receptors initially occupied by a single cis- and a single trans-Bis-Q molecule or that for unliganded receptors. Single-channel current recordings from excised outside-out membrane patches showed that single channels open following an agonist concentration jump comparable to that used in the whole-cell experiments; when many such records were averaged, a synthetic macroscopic relaxation was produced. Individual open channels closed faster following a flash that promoted trans----cis photoisomerizations of the bound ligand, thus confirming the whole-cell observations of phase 1.

Animals↗
PubMed1985

Mouse-Torpedo hybrid acetylcholine receptors: functional homology does not equal sequence homology.

The nicotinic acetylcholine (AcCho) receptor (AcChoR) is a multisubunit protein complex of stoichiometry alpha 2 beta gamma delta. The several subunits show homology with each other within a given species; in addition, homology is found between analogous subunits between species. We have used the phage SP6 RNA polymerase transcription system to produce single-species RNA in vitro for various AcChoR subunits from cDNAs. Injection of an equimolar mixture of RNA for the alpha, beta, gamma, and delta subunits of Torpedo californica AcChoR into Xenopus oocytes results in the appearance of functional receptors in the oocyte membrane. No response to AcCho is detected when the beta or gamma subunit RNA is omitted, and a small response is seen when the delta subunit RNA is omitted. Replacement of Torpedo delta subunit RNA by the mouse BC3H-1 cell line AcChoR delta subunit RNA leads to the formation of functional receptors that show a 3-4-fold greater response to AcCho than does the full Torpedo complex. No response is seen when the mouse delta RNA replaces Torpedo gamma RNA. By amino acid homology profile comparisons, the mouse delta subunit appears to be moderately but not highly similar to the Torpedo delta subunit; the apparent similarity to the Torpedo gamma subunit is only slightly less. Therefore, the features of the primary sequence that determine the functional delta character of the mouse polypeptide are not revealed by simple homology comparisons.

Amino Acid Sequence↗
PubMed1985

Acetylcholine and phorbol esters inhibit potassium currents evoked by adenosine and cAMP in Xenopus oocytes.

In Xenopus laevis oocytes, adenosine and other purinergic agonists induce a K+-conductance increase that is fully mimicked by intracellular application of cAMP. Acetylcholine suppresses the K+-conductance increase caused by adenosine, by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, or by intracellular injection of cAMP. This effect of acetylcholine is not mimicked by intracellular injection of Ca2+ or of the Ca-mobilizing agent inositol 1,4,5-trisphosphate. However, adenosine and cAMP responses are inhibited by 4 beta-phorbol 12,13-dibutyrate and 4 beta-phorbol 12-myristate 13-acetate. These results suggest that, in Xenopus oocytes, the muscarinic inhibition of purinergic and cAMP responses is mediated through the activation of the phospholipid-dependent, Ca-activated protein kinase (protein kinase C).

Acetylcholine↗
PubMed1985

Rates and equilibria for a photoisomerizable antagonist at the acetylcholine receptor of Electrophorus electroplaques.

Voltage-jump and light-flash experiments have been performed on isolated Electrophorus electroplaques exposed simultaneously to nicotinic agonists and to the photoisomerizable compound 2,2'-bis-[alpha-(trimethylammonium)methyl]-azobenzene (2BQ). Dose-response curves are shifted to the right in a nearly parallel fashion by 2BQ, which suggests competitive antagonism; dose-ratio analyses show apparent dissociation constants of 0.3 and 1 microM for the cis and trans isomers, respectively. Flash-induced trans----cis concentration jumps produce the expected decrease in agonist-induced conductance; the time constant is several tens of milliseconds. From the concentration dependence of these rates, we conclude that the association and dissociation rate constants for the cis-2BQ-receptor binding are approximately 10(8) M-1 s-1 and 60 s-1 at 20 degrees C; the Q10 is 3. Flash-induced cis----trans photoisomerizations produce molecular rearrangements of the ligand-receptor complex, but the resulting relaxations probably reflect the kinetics of buffered diffusion rather than of the interaction between trans-2BQ and the receptor. Antagonists seem to bind about an order of magnitude more slowly than agonists at nicotinic receptors.

Animals↗