PubMed Health⌕ Search

Biomedical subjects

P Labarca

Publications and source records attributed to P Labarca.

At least 37 records · Page 2Linked to original sources
PubMed1993

Properties of whole cell currents in isolated olfactory neurons from the chilean toad Caudiverbera caudiverbera.

Isolated olfactory neurons from the chilean toad Caudiverbera caudiverbera were found to possess a same set of currents. Outward currents, made of a delayed rectifier and a Ca(2+)-dependent component, were blocked by replacing K+ by Cs+ in the patch pipette, in the presence of millimolar concentrations of tetraethylammonium and 4-aminopyridine in the external solution. Inward currents were made of a transient and a maintained component. The transient was abolished in the absence of external Na+ and was blocked by tetrodotoxin, with an apparent dissociation constant (KDapp) of 25.4 +/- 0.3 nM. The maintained inward currents were suppressed on removing external Ca2+, could be carried also by Ba2+, and were selectively blocked by Cd2+ (KDapp = 3.2 +/- 1.3 microM). A variety of agents found to block the maintained Ca2+ inward currents, including Co2+ and Ni2+, at millimolar concentrations, and nifedipine, verapamil, amiloride, and the amiloride analogue benzamil, at micromolar concentrations, were also effective in either modifying the gating of, or in blocking, the transient inward currents.

Animals↗
PubMed1992

K(+)-channel blockers restore synaptic plasticity in the neuromuscular junction of dunce, a Drosophila learning and memory mutant.

The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learning and memory mutant, were investigated. Larvae dnc mutants lack facilitation and post-tetanic potentiation (PTP) at their motor end-plates; dnc mutants are also deficient in a form of phosphodiesterase, and exhibit abnormally high levels of cyclic adenosine 3',5'-monophosphate (cAMP). A two-microelectrode voltage-clamp was used to record end-plate currents and spontaneous end-plate currents from longitudinal ventrolateral third-instar larval muscle. The K(+)-channel blockers 3,4-diaminopyridine (3,4-DAP) and tetraethylammonium (TEA), at micromolar concentrations, caused a reversible decrease in end-plate current amplitudes both in wild-type and mutant end-plates. In the presence of blockers, a period of high-frequency stimulation (tetanus) of the nerve gave way to a transient increase in the end-plate currents of dnc mutants resembling facilitation and PTP in normal end-plates; 3,4-DAP and TEA also restored facilitation and PTP in normal end-plates after incubation with a non-hydrolysable analogue of cAMP (8Br-cAMP). It is suggested that a specific K+ conductance might be relevant to the lack of synaptic plasticity at the dnc neuromuscular synapses.

4-Aminopyridine↗
PubMed1991

A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce.

Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Four cases of direct ion channel gating by cyclic nucleotides.

Four different nucleotide-gated ion channels are discussed in terms of their biophysical properties and their importance in cell physiology. Channels activated directly by cGMP are present in vertebrate and invertebrate photoreceptors. In both cases cGMP increases the fraction of time the channel remains in the open state. At least three cGMP molecules are involved in channel opening in vertebrate photoreceptors and the concentration of the cyclic nucleotide to obtain the half maximal effect is about 15 microM. The light-dependent channel of both vertebrates and invertebrates is poorly cation selective. The vertebrate channel allows divalent cations to pass through 10-15-fold more easily than monovalent ions. In agreement with their preference for divalent cations, this channel is blocked by l-cis Dialtazem, a molecule that blocks certain types of calcium channels. In olfactory neurons a channel activated by both cAMP and cGMP is found and, as in the light-dependent channel, several molecules of the nucleotide are needed to open the channel with a half maximal effect obtained in the range of 1-40 microM. The channel is poorly cationic selective. A K+ channel directly and specifically activated by cAMP is found in Drosophila larval muscle. At least three cAMP molecules are involved in the opening reaction. Half-maximal effect is obtained at about 50 microM. This channel is blocked by micromolar amount of tetraethylammonium applied internally. Interestingly, this channel has a probability of opening 10-20-fold larger in the mutant dunce, a mutant that possesses abnormally elevated intracellular cAMP level, than in the wild type.

Animals↗
PubMed1989

L-glutamate activates excitatory and inhibitory channels in Drosophila larval muscle.

Muscle fibers from Drosophila larvae show an L-glutamate-sensitive membrane potential. Bath-applied L-glutamate depolarizes the muscle in the range from 0.5 to 20 microM. Greater concentrations of the agonist repolarize the fibers. The repolarizing effect disappears if chloride is replaced by sulfate in the external medium. Intracellular recordings show the occurrence of depolarizing and hyperpolarizing spontaneous miniature postsynaptic potentials (smpp). Patch-clamp studies indicate the presence of two types of receptor channels: (i) an anion-selective channel activated by both L-glutamate and GABA. In outside out-patches, bathed in symmetrical 140 mM Cl- and 200 microM GABA, the channel displays conductance substates of 40, 80 and 110 pS. In the presence of 200 microM L-glutamate only the 40 and 80 pS substates are observed; (ii) a cation-selective channel activated only by L-glutamate that has a conductance of 104 pS in cell-attached patches (128 mM Na+ outside). The presence of these two types of receptor channels in Drosophila muscle may explain the effect of bath-applied L-glutamate on membrane potential and the presence of inhibitory and excitatory smpp.

Animals↗
PubMed1989

Activation by saccharides of a cation-selective pathway on canine lingual epithelium.

Responses of isolated canine lingual epithelium in an Ussing chamber to D-glucose and fructose reveal events associated with taste transduction. With the use of isotopic flux studies, together with ion substitution and pharmacological and voltage clamp measurements, it was found that the stimulation of ion transport by D-glucose arises from an increase in the influx of cations through a cation-selective pathway. This influx of cations is completely inhibited by 0.1 mM amiloride. The stimulation of transport by fructose in 0.05 M KCl and by D-glucose in 0.05 M RbCl was also inhibited by amiloride, demonstrating that the saccharide-stimulated entry pathway was specific for neither hexose sugars nor for Na. Saccharide stimulation of canine lingual epithelia does not appear to be modulated by increases in intracellular levels of adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, or Ca. The Na that enters taste cells on saccharide stimulation exits them via the ouabain inhibitable Na+-K+-adenosinetriphosphatase located in the serosal membranes.

Amiloride↗
PubMed1988

Ion channels from chemosensory olfactory neurons.

The olfactory epithelium has the ability to respond to a large number of volatile compounds of small molecular weight. Ultimately, such a property lies on a specialized type of neuron, the olfactory receptor cell. In the presence of odorants, the olfactory receptor neuron responds with action potentials whose frequency depends on odorant concentration. The primary events in the process of olfactory transduction are thought to occur at the cilia of olfactory receptor neurons and involve the binding of odorants to receptor molecules followed by the opening of ion channels. A crucial step in understanding olfactory transduction requires identifying the mechanisms that regulate the electrical activity of olfactory cells. In the last couple of years, patch-clamp recording from isolated olfactory cells and reconstitution of olfactory membranes in planar lipid bilayers have begun to shed light on some of these mechanisms. Although the information emerging from such studies is still preliminary, there are already well-defined hypotheses on the molecular events that might underlie the primary events in olfactory transduction. Currently, attention is being focused on the notions that second messengers might be involved in the activation of ion channels in olfactory cilia, and that odorant binding to a receptor molecule might lead directly to the gating of ion channels in chemosensory olfactory membranes. The coming years promise to be exciting ones in the field of olfactory transduction. We have now the necessary tools to be able to confront hypotheses and experimental facts.

Animals↗
PubMed1988

Activation by odorants of a multistate cation channel from olfactory cilia.

Single-channel records were obtained after fusion of ciliary membranes from the olfactory epithelium of Rana catesbeiana to planar lipid bilayers, and odorant-activated cation-selective channels were identified. In addition, a 190-pS potassium-selective channel and a 40-pS cation-selective channel were found in a 0.2 M salt-containing buffer. Odorant-sensitive channels were directly and reversibly activated by nanomolar concentrations of the bell pepper odorant 3-isobutyl-2-methoxypyrazine and the citrus odorant 3,7,-dimethyl-2,6-octadienenitrile. These channels display burst kinetics, multiple conductance levels between 35 and 420 pS, and open times in the millisecond range. With increasing concentrations of odorant, the probability of populating the higher conductance levels increases. These results show that direct activation of channels by odorants may mediate excitation of the olfactory receptor cell.

Animals↗
PubMed1988

Voltage gating of conductance in lipid bilayers induced by porin from outer membrane of Neisseria gonorrhoeae.

Porins, polypeptides of approximately 35 kDa, are present as integral membrane proteins in the outer membranes of a variety of Gram-negative bacteria. As reported previously for a purified porin from Escherichia coli, voltage gating of conductance was found to be induced in a lipid bilayer by the solubilized purified porin, protein I, from Neisseria gonorrhoeae. The unitary response to an applied potential showed a cascade of current from an initial level through at least three levels, more or less equal, to a persisting lower level. The initial level of current corresponded to 1.0-1.3 nS for 0.2 M NaCl on either side of the bilayer. Briefly reducing the potential to zero restored the current to its initial level. Interpretation of the unitary response is suggested by electron microscopic data obtained on negatively stained outer membranes of E. coli indicating the presence of "pores" appearing in triplets. Moreover, low-resolution x-ray and neutron diffraction studies on crystals obtained with an E. coli porin show that three polypeptides associate to form a unit. Combining such structural data with the present electrical data lends support for the hypothesis that the unitary response results from three pores acting as a unit in response to an applied potential. Evidence obtained with the patch-clamp technique is mounting for a similar mechanism of many channels operating as a unit in a variety of cell membranes. The porin channel holds promise as a concrete model for the analysis of voltage gating of ionic conductance.

Bacterial Outer Membrane Proteins↗
PubMed1986

Native and chemically modified porin channels from Salmonella typhi Ty2 in planar lipid bilayers.

Native porins, from Salmonella typhi Ty2 outer membrane, and porins alkylated with pyridoxal phosphate (Plp) were studied in planar lipid bilayers. The conductance of bilayers exposed to native or chemically modified porins increases in discrete jumps. Conductance histograms for native porins displayed two major peaks at 1.7 and 6.7 nS (in 0.5 M KCl). On the other hand, Plp-treated porins exhibited a single major peak at 1 nS. The relation between bilayer conductance and native porin concentration was linear. However, this relation became logarithmic in the presence of modified porins. The results support the notion that alkaline reduction of S. typhi Ty2 porins with Plp dissociates porin channel trimers in a reversible fashion.

Alkylation↗
PubMed1986

Large cation-selective pores from rat liver peroxisomal membranes incorporated to planar lipid bilayers.

Fusion of a highly purified fraction of rat liver peroxisomal membranes to planar lipid bilayers incorporates large, cation-selective voltage-dependent pores. The PK/PCl ratio of these pores, estimated in KCl gradients, is close to 4. The pores display several conductance states and spend most of the time open at voltages near 0 mV, closing at more positive and negative voltages. At voltages near 0 mV the most frequent open state has a conductance of 2.4 nS in 0.3 M KCl. At voltages more positive and more negative than 10 mV the most frequent open state displays a conductance of 1.2 nS in 0.3 M KCl. With these results pore diameters of 3 and 1.5 nm, respectively, can be estimated. We suggest that these pores might account for the unusually high permeability of peroxisomes to low molecular weight solutes. Fusion also incorporates a perfectly anion-selective, two-open states channel with conductances of 50 and 100 pS in 0.1 M KCl.

Animals↗
PubMed1985

Kinetic analysis of channel gating. Application to the cholinergic receptor channel and the chloride channel from Torpedo californica.

Identification of the minimum number of ways in which open and closed states communicate is a crucial step in defining the gating kinetics of multistate channels. We used certain correlation functions to extract information about the pathways connecting the open and closed states of the cation channel of the purified nicotinic acetylcholine receptor and of the chloride channel of Torpedo californica electroplax membranes. Single channel currents were recorded from planar lipid bilayers containing the membrane channel proteins under investigation. The correlation functions are conveniently computed from single channel current records and yield information on E, the minimum number of entry/exit states into the open or closed aggregates. E gives a lower limit on the numbers of transition pathways between open and closed states. For the acetylcholine receptor, the autocorrelation analysis shows that there are at least two entry/exit states through which the open and closed aggregates communicate. The chloride channel fluctuates between three conductance substates, here indentified as C, M, and H for closed, intermediate, and high conductance, respectively. Correlation analysis shows that E is greater than or equal to 2 for the M aggregate, indicating that there are at least two distinct entry/exit states in the M aggregate. In contrast, there is no evidence for the existence of more than one entry/exit state in the C or H aggregates. Thus, these correlation functions provide a simple and general strategy to extract information on channel gating kinetics.

Animals↗
PubMed1985

The occurrence of long openings in the purified cholinergic receptor channel increases with acetylcholine concentration.

To establish the relationship between cholinergic ligand occupancy and channel open states, we recorded single-channel currents activated by different acetylcholine (ACh) concentrations from Torpedo californica ACh receptors reconstituted in lipid bilayers. Inspection of single-channel records shows that the frequency of occurrence of long openings increases with ACh concentration. Analysis of the probability distribution of open dwell times indicates that the ACh receptor channel has two distinct channel open states, short- and long-lived. The frequency of occurrence of the long openings over the short increased with ACh concentration, whereas the corresponding time constants were virtually unaltered. The extent of agonist occupancy at the ACh-binding sites in the purified cholinergic receptor appears, therefore, to correlate with an increased probability of the long-lived open state. These results are consistent with the notion that the two open-channel states arise from different extents of ligand occupancy at the receptor molecule.

Acetylcholine↗
PubMed1984

Channel properties of the purified acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayer membranes.

The electrophysiological properties of the cation channel of the purified nicotinic acetylcholine receptor (AChR) reconstituted in planar lipid bilayers were characterized. Single-channel currents were activated by acetylcholine, carbamylcholine and suberyldicholine. The single channel conductance (28 pS in 0.3 M NaCl) was ohmic and independent of the agonist. Single channel currents increased with Na+ concentration to a maximum conductance of 95 pS and showed a half-saturation point of 395 mM. The apparent ion selectivity sequence, derived from single-channel current recordings, is: NH+4 greater than Cs+ greater than Rb+ greater than or equal to Na+ Cl-, F-, SO2-(4). The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of at least two distinct open states. The time constants depend on the choice of agonist, being consistently longer for suberyldicholine than for carbamylcholine. Similar channel properties were recorded in bilayers formed from monolayers at the tip of patch pipets . Single-channel currents occur in paroxysms of channel activity followed by quiescent periods. This pattern is more pronounced as the agonist concentration increases, and is reflected in histograms of channel-opening frequencies. Computer simulations with a three-state model, consisting of two closed (unliganded and liganded) and one open state, do not resemble the recorded pattern of channel activity, especially at high agonist concentration. Inclusion of a desensitized liganded state reproduces the qualitative features of channel recordings. The occurrence of paroxysms of channel activity thus seems to result from the transit of AChR through its active conformation, from which it can open several times before desensitizing.

Animals↗
PubMed1984

Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers.

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.

Animals↗