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R Villegas

Publications and source records attributed to R Villegas.

At least 37 records · Page 2Linked to original sources
PubMed1992

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers.

Alkaloid-modified, voltage-dependent sodium channels from lobster walking leg nerves were studied in planar neutral lipid bilayers. In symmetrical 0.5 M NaCl the single channel conductance of veratridine (VTD) (10 pS) was less than that of batrachotoxin (BTX) (16 pS) modified channels. At positive potentials, VTD- but not BTX-modified channels remained open at a flickery substate. VTD-modified channels underwent closures on the order of milliseconds (fast process), seconds (slow process), and minutes. The channel fractional open time (f(o)) due to the fast process, the slow process, and all channel closures (overall f(o)) increased with depolarization. The fast process had a midpoint potential (V(a)) of -122 mV and an apparent gating charge (z(a)) of 2.9, and the slow process had a V(a) of -95 mV and a z(a) of 1.6. The overall f(o) was predominantly determined by closures on the order of minutes, and had a V(a) of about -24 mV and a shallow voltage dependence (z(a) approximately 0.7). Augmenting the VTD concentration increased the overall f(o) without changing the number of detectable channels. However, the occurrence of closures on the order of minutes persisted even at super-saturating concentrations of VTD. The occurrence of these long closures was nonrandom and the level of nonrandomness was usually unaffected by the number of channels, suggesting that channel behavior was nonindependent. BTX-modified channels also underwent closures on the order of milliseconds, seconds, and minutes. Their characterization, however, was complicated by the apparent low BTX binding affinity and by an apparent high binding reversibility (channel disappearance) of BTX to these channels. VTD- but not BTX-modified channels inactivated slowly at high positive potentials (greater than +30 mV). Single channel conductance versus NaCl concentrations saturated at high NaCl concentrations and was non-Langmuirian at low NaCl concentrations. At all NaCl concentrations the conductance of VTD-modified channels was lower than that of BTX-modified channels. However, this difference in conductance decreased as NaCl concentrations neared zero, approaching the same limiting value. The permeability ratio of sodium over potassium obtained under mixed ionic conditions was similar for VTD (2.46)- and BTX (2.48)-modified channels, whereas that obtained under bi-ionic conditions was lower for VTD (1.83)- than for BTX (2.70)-modified channels. Tetrodotoxin blocked these alkaloid-modified channels with an apparent binding affinity in the nanomolar range.

Alkaloids↗
PubMed1988

The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein.

Solubilization and purification of the tetrodotoxin (TTX) binding protein of the lobster walking-leg nerve Na+ channel were carried out utilizing [3H]tetrodotoxin [( 3H]tetrodotoxin) as a marker. The nerve membrane was solubilized with Lubrol-PX and the Na+ channel protein was purified with diethylaminoethyl Bio-Gel A, Bio-Gel hydroxylapatite powder and two Sepharose 6B columns. Care was taken to keep the temperature of the Na+ channel preparation as close to 1 degrees C as possible and to use solutions (pH 7.5) that contain Na channel protectors, i.e., egg phosphatidylcholine/Lubrol-PX mixture, TTX, EDTA, EGTA, phenylmethylsulfonyl fluoride, pepstatin A, iodoacetamide, antipain, phosphoramidon, soybean trypsin inhibitor, leupeptin and bacitracin. From an initial specific binding of 20.1 pmol of [3H]TTX/mg protein for the solubilized membrane, the binding increased to 1241 pmol/mg protein for the most active fraction of the last Sepharose 6B column. The [3H]TTX specific binding of the Sepharose 6B fractions correlated with a large peptide of Mr 260,000 (240-280K), although other peptides were also present in lesser amounts.

Adsorption↗
PubMed1988

The interaction of homologous series of alkanols with sodium channels in nerve membrane vesicles.

The potency of members of the homologous series of alkanols to block 22Na uptake through sodium channels stimulated by veratridine was studied in membrane vesicles obtained from lobster walking leg nerves. A cut-off was revealed at the level of 1-undecanol. However, secondary isomers of inactive primary homologues, such as 5-dodecanol and 5-tridecanol, were able to block ion flux. From the concentration required for an equipotent effect, it was calculated that the standard free energy for adsorption of primary alkanols is -725 cal/mol CH2. Furthermore, since the concentration required for an equipotent effect for primary isomer was found to be lower than that obtained for secondary isomers, it is concluded that the latter are less potent than the former. The similarity between this set of results and those obtained in intact frog sciatic nerve (J. Requena et al., J. Membrane Biol., 84:229-238, 1985) offers further support to the notion that the procedure employed to isolate the membrane vesicles does preserve the Na channels. However, the mechanism of alcohol inhibition of the Na channel in isolated membrane vesicles would seem to be somewhat different from that preferred in axons. While in vesicles the block needs to be thought in terms of a reduction in the number of conducting Na channel, in axons this is considered to be the less likely mode of action, mainly because under veratridine it is not possible to invoke a shift in the steady-state activation or inactivation.

Alcohols↗
PubMed1987

Anemone toxin II receptor site of the lobster nerve sodium channel. Studies in membrane vesicles and in proteoliposomes.

The receptor-site for the sea anemone toxin II from Anemonia sulcata (ATX) and its functional relationship with the Na+ channel were studied in plasma membrane preparations from lobster walking leg nerves. The modification of the 22Na influx by ATX was determined in membrane vesicles and in proteoliposomes prepared by reconstitution of detergent-extracted, unfractionated membrane particles into soybean liposomes. The effects of two other toxins, veratridine (VER) and tetrodotoxin (TTX), which bind to Na+ channel receptor-sites other than that for polypeptide toxins, were also studied, ATX and VER stimulated 22Na flux into membrane vesicles with K0.5 values in the order of 10(-7) and 10(-5) M, respectively. Positive cooperativity among these toxins was also seen; ATX displaces the K0.5 for VER towards lower VER concentrations. TTX abolishes the 22Na influx increment caused by ATX and/or VER with a K0.5 in the order of 10(-8) M. In proteoliposomes, in contrast, ATX modified the 22Na influx only at high concentrations (greater than 1 microM) and in the presence of VER. VER stimulation and TTX inhibition of the VER and the VER plus ATX modified fluxes, had the same characteristics as in the vesicle preparations. Measurable ATX and VER toxin effects were only seen in the presence of an outwardly directed K+ gradient for both vesicles and proteoliposomes. Detergent treatment and the reconstitution procedure seem to affect the functional properties of the ATX receptor site whereas the VER and the TTX sites remain unaltered.

Animals↗
PubMed1983

Sodium channel activity in brain membrane fractions isolated from rats of different ages.

The Na+ channel activity (tetrodotoxin sensitive 22Na+ flux induced by veratridine and/or anemone toxin II) was studied in two fractions of brain cell plasma membranes, named A and B, isolated by the method of Gray and Whittaker ((1962) J. Anat. 96, 79-87) from rats 5, 10, 30 and 60 days old. The 22Na+ flux was measured in membrane vesicles formed by the isolated membranes, in the absence of drugs (control), in the presence of veratridine, and in the presence of veratridine plus tetrodotoxin. Fraction A consists primarily of neuronal and glial membranes in rats of 5 and 10 days of age, while in the older rats this fraction becomes enriched in myelin. In Fraction A of 5-day-old and 10-day-old rats, veratridine (25 microM) increases the 22Na+ flux 2.4- and 1.6-fold, respectively, and the increment continues to diminish with age, until it becomes negligible in the 60-day-old rats. Fraction B consists of synaptosomes and membrane vesicles, and at the four ages studied veratridine (25 microM) causes an increment of the 22Na+ flux of about 2.5-fold. Fractions A and B from 10-day-old rats, and Fraction B from 60-day-old rats, which are sensitive to veratridine, also respond to anemone toxin II. When veratridine is used in presence of anemone toxin II (0.5 microM), the K0.5 for veratridine is diminished and the maximum 22Na+ flux is increased. The increments of 22Na+ flux caused by veratridine and/or anemone toxin II in Fractions A and B are blocked by tetrodotoxin (K0.5 approx. 5 nM). Fraction A from 60-day-old rats could be subfractionated by osmotic shock and sucrose gradient centrifugation to obtain three subfractions, two of which are enriched in axolemma and display Na+ channel activity. The other subfraction is enriched in myelin and shows no Na+ channel activity. The plasma membrane preparations from young rats (up to 10 days) are devoid of myelin and are useful for studies of Na+ channel activity.

Aging↗
PubMed1982

Ion selectivity of the nerve membrane sodium channel incorporated into liposomes.

Tetrodotoxin-sensitive sodium channels of lobster nerve membranes were incorporated into soybean liposomes by the freeze-thaw-sonication procedure and their ionic selectivity was studied. Veratridine and grayanotoxin-I were used to activate the sodium channels and the increment of the ionic flux through them was specifically abolished by tetrodotoxin. The drug-sensitive 22Na+, 42K+, 86Rb+ and 137Cs+ influxes were measured. The permeability ratios calculated directly from ion fluxes showed that the channels preferably allow the passage of Na+. No anion influx ([32P]phosphate, [35S]sulfate, 36Cl) sensitive to the drugs was observed. The data reveal that the sodium channels incorporated into liposomes remain cation-selective and discriminate among different cations.

Animals↗
PubMed1981

Reconstitution of the sodium channel with partially solubilized lobster nerve membrane.

Reconstitution experiments were carried out with particles obtained from lobster nerve plasma membrane preparations by detergent treatment, differential centrifugation and ammonium sulfate fractionation. The NA channel activity of the three fractions obtained, which have different amounts of the same peptides present in the original membrane, appears related to their content in a large component which does not enter the 9% polyacrylamide gel and in peptides with 220,000 and 110,000 apparent molecular weight. Other reconstitution experiments made with two fractions obtained by detergent treatment, differential centrifugation and gel exclusion chromatography, revealed that the Na channel active fraction contains the material which does not enter the gel in addition to the 220,000 and 110,000 molecular weight peptides. The other fraction was inactive and does not contain those components. The 220,000 dalton peptide has a molecular weight similar to those determined for the tetrodotoxin-saxitoxin receptor and the scorpion toxin receptor of the Na channel. Whether any of the other peptides is a Na channel constituent is unknown at present.

Animals↗
PubMed1979

Studies on the incorporation of the sodium channel of lobster nerve into soybean liposomes.

Na+ channels of lobster nerve membrane were incorporated into soybean liposomes by the freeze-thaw-sonication procedure. Electron microscopy showed that the vesicular pattern of membranes and liposomes was broken and reassembled during the process. The amount of membrane protein incorporated depended linearly on the amount of membrane protein added to the liposomes for reconstitution. The 22Na flux into the reconstituted vesicles was increased by veratridine (0.5 mM) or grayanotoxin I (150 microM) and the increment was abolished by tetrodotoxin (Ki = 4 to 5 nM). The drug-sensitive 22Na influx depended linearly on the amount of membrane protein incorporated. No response to the drugs was found in protein-free vesicles or in vesicles reconstituted with membrane heated at 50 degrees C for 15 min. A fraction of digitonin-treated membranes had a higher Na+ channel activity. The Na+ channel was able to discriminate between Na+ and K+ or Rb+.

Animals↗
PubMed1976

The polypeptide and the phospholipid components of axon plasma membranes.

The axon plasma membrane fraction isolated from garfish olfactory nerve was analyzed for its polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were present over 20 well-resolved polypeptide components in this membrane, and eleven of them, with an apparent molecular weight range of 22,000-130,000, accounted for most of the membrane proteins. None of the major polypeptide species present in the membrane appeared to be glycoprotein. Based on electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel, eight of the major polypeptides found in garfish nerve membrane appeared to be also present in the axon plasma membrane isolated from lobster walking leg nerve. Both garfish and lobster nerve membranes contained high concentration of lipids (66-76%) which were essentially cholesterol and phospholipids. The classes of phospholipids present were phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol and sphingomyelin. Lobster nerve membrane also contained about 3% phosphatidic acid. Assays for acetylcholinesterase in axon plasma membrane fractions isolated from different nerve sources showed a wide variation, ranging from a specific activity of 2.4 for garfish nerve to 312.5 for lobster nerve membrane.

Acetylcholinesterase↗
PubMed1976

Sodium flux through the sodium channels of axon membrane fragments isolated from lobster nerves.

The efflux of 22Na from vesicles formed by axolemma fragments isolated from lobster nerves was studied in the presence and in the absence of drugs having well-known action on the sodium channels. The vesicles were equilibrated 12-14 h at 4 degrees C with 22Na in lobster solution containing 1 mM ouabain. Afterwards the suspension was divided: one portion was used as control and the others were treated with veratrine (0.025-0.50 mg/ml), tetrodotoxin (1-2,000 nM) in the presence of veratrine, or tetrodotoxin alone. After 3 h at 20-22 degrees C, the suspensions were diluted into nonradioactive solutions and the 22Na efflux followed by a rapid filtration technique. The results revealed that veratrine increases the efflux rate and the additional application of tetrodotoxin abolishes it, e.g., 0.50 mg of veratrine/ml increases the rate, expressed in 10(-2) min(-1), from 0.59 +/- 0.04 (mean +/- SEM; n = 13) to 0.86 +/- 0.05 (n = 13), and the addition of 100 nM tetrodotoxin diminishes it to 0.48 +/- 0.07 (n = 4). This increase and diminution are statistically significant (P less than 0.005), but this is not the case between the control and the veratrine plus tetrodotoxin values (P greater than 0.05). 50% of the diminution is produced by 11.9 +/- 2.4 nM tetrodotoxin. Tetrodotoxin alone produces a slight diminution of the 22Na efflux. Batrachotoxin (0.50 muM) has an action similar to veratrine's. These findings are considered evidence of the presence of functioning sodium channels in the isolated axolemma fragments.

Animals↗
PubMed1976

Grayanotoxin, veratrine, and tetrodotoxin-sensitive sodium pathways in the Schwann cell membrane of squid nerve fibers.

The actions of grayanotoxin I, veratrine, and tetrodotoxin on the membrane potential of the Schwann cell were studied in the giant nerve fiber of the squid Sepioteuthis sepioidea. Schwann cells of intact nerve fibers and Schwann cells attached to axons cut lengthwise over several millimeters were utilized. The axon membrane potential in the intact nerve fibers was also monitored. The effects of grayanotoxin I and veratrine on the membrane potential of the Schwann cell were found to be similar to those they produce on the resting membrane potential of the giant axon. Thus, grayanotoxin I (1-30 muM) and veratrine (5-50 mug-jl-1), externally applied to the intact nerve fiber or to axon-free nerve fiber sheaths, produce a Schwann cell depolarization which can be reversed by decreasing the external sodium concentration or by external application of tetrodotoxin. The magnitude of these membrane potential changes is related to the concentrations of the drugs in the external medium. These results indicate the existence of sodium pathways in the electrically unexcitable Schwann cell membrane of S. sepioidea, which can be opened up by grayanotoxin I and veratrine, and afterwards are blocked by tetrodotoxin. The sodium pathways of the Schwann cell membrane appear to be different from those of the axolemma which show a voltage-dependent conductance.

Animals↗