Biomedical subjects

W A Varanda

Publications and source records attributed to W A Varanda.

13 recordsLinked to original sources

PubMed1995

Hurst analysis in the study of ion channel kinetics.

Ion channels are protein molecules which can assume distinct open and closed conformational states. The transitions between these states can be controlled by the electrical field, ions and/or drugs. Records of unitary current events show that short open-time intervals are frequently adjacent to much longer closed-time intervals, and vice-versa, suggesting that the kinetic process has memory, i.e., the intervals are correlated in time. Here the rescaled range analysis (R/S Hurst analysis) is proposed as a method to test for correlation. Simulations were performed with a two-state Markovian model, which has no memory. The calculated Hurst coefficients (H) presented a mean +/- SD value of 0.493 +/- 0.025 (N = 100). For the Ca(2+)-activated K+ channels of Leydig cells, H was equal to 0.75, statistically different (1% level) from that calculated for the memoryless process. Randomly shuffling the experimental data resulted in an H = 0.55, not significantly different (1% level) from that found for the two-state Markovian model. For a linear three-state Markovian model, H was equal to 0.548 +/- 0.017 (N = 15), again not significantly different (1% level) from that of the memoryless process. Although the three-state Markovian model adequately describes the open- and closed-time distributions, it does not account for the correlation found in this Ca(2+)-activated K+ channel. Our results illustrate the efficacy of the R/S analysis in determining whether successive opening and closing events are correlated in time and can be of help in deciding which model should be used to describe the kinetics of ion channels.

Animals↗

PubMed1995

Calcium-activated potassium channels are involved in the response of mouse Leydig cells to human chorionic gonadotropin.

The patch-clamp technique was used to investigate the involvement of ion channels in the response of Leydig cells to gonadotropic hormones (viz. hCG). Recordings in the cell-attached configuration (pipette containing 140 mM KCl) showed unitary events with conductance of 187.9 +/- 5.2 pS (N = 24 patches) in about 70% of the cells. These channels were potassium selective and the open channel probability (Po) was always about 1% for displacement of potential from the resting value in the range of -20 to +60 mV. Treatment of the cells with hCG (2 ng/ml) led to a large increase in the frequency of openings, concomitant with a reduction in the mean closed time and there was essentially no effect on the mean open time of the channel. Dibutyryl cAMP (100 microM) produced an effect similar to that of hCG and both required external calcium for their action. No direct effect of either dibutyryl cAMP or hCG were observed in inside-out patches. Reversal potential measurements on excised inside-out patches demonstrated that the channels were highly potassium selective with unitary conductance of about 206.8 +/- 6.36 pS (mean +/- SEM of 6 measurements), and an estimated permeability of 3.6 x 10(-13) +/- 0.2 x 10(-13) cm3/s (mean +/- SEM for 6 measurements), in symmetrical 140 mM KCl. The activity of the channel in excised patches was very sensitive to the free-calcium concentration on the intracellular surface of the channel. Po evaluated at +60 mV increased from 3% at 10 nM to 47% at 100 nM free calcium. The Hill coefficient under these conditions was 1.1. These results demonstrate that Leydig cells have a Ca2(+)-activated K+ channel of large unitary conductance, which can be activated upon the binding of hCG to receptors in the cell membrane.

Animals↗

PubMed1994

Intercellular communication between mouse Leydig cells.

In this paper we describe the basic features of gap junctions in pairs of Leydig cells mechanically dissociated from mouse testes, studied with the double whole cell patch-clamp technique. These cells are extensively coupled with regard to dye injection and electrophysiological measurements. The mean junctional conductance (gj) measured in 61 pairs of cells was 10.6 +/- 1.5 (SE) nS. In most pairs gj was voltage dependent when transjunctional voltage exceeded +/- 50 mV. On imposition of a voltage gradient across the junction the transjunctional current decayed exponentially to a lower level, with a time constant of 3.3 s at 50 mV and 430 ms at 100 mV. As in other systems, octanol (600 microM final concentration) uncoupled the cells within approximately 2 min. In a few cell pairs, gj was low enough to permit recording of single channel currents without the use of uncoupling agents. Single channel conductance fluctuations measured using pipettes containing potassium aspartate were distributed mainly around three peaks, at 21, 39, and 60 pS, suggesting the presence of channels formed by connexin 43. Western blots of Percoll gradient purified Leydig cells using specific antibodies indicate that connexin 43 is indeed expressed in these cells, whereas connexin 26 and connexin 32 are not.

Animals↗

PubMed1992

Toad bladder amiloride-sensitive channels reconstituted into planar lipid bilayers.

In the present study we used established methods to obtain apical membrane vesicles from the toad urinary bladder and incorporated these membrane fragments to solvent-free planar lipid bilayer membranes. This resulted in the appearance of a macroscopic conductance highly sensitive to the diuretic amiloride added to the cis side. The blockage is voltage dependent and well described by a model which assumes that the drug binds to sites in the channel lumen. This binding site is localized at about 15% of the electric field across the membrane. The apparent inhibition constant (K(0)) is equal to 0.98 microM. Ca2+, in the micromolar range on the cis side, is a potent blocker of this conductance. The effect of the divalent has a complex voltage dependence and is modulated by pH. At the unitary level we have found two distinct amiloride-blockable channels with conductances of 160 pS (more frequent) and 120 pS. In the absence of the drug the mean open time is around 0.5 sec for both channels and is not dependent on voltage. The channels are cation selective (PNa/PCl = 15) and poorly discriminate between Na+ and K+ (PNa/PK = 2). Amiloride decreases the lifetime in the open state of both channels and also the conductance of the 160-pS channel.

Amiloride↗

PubMed1992

Glomerular permeability to macromolecules in gentamicin-treated rats.

1. To determine the effect of gentamicin on the functional properties of the glomerular barrier, 44 Wistar rats received daily doses of 80 mg/kg body weight for 6 days. Glomerular permeability to neutral dextrans and albumin was evaluated by day 6 and albuminuria was determined on the 1st, 3rd and 5th days of treatment. 2. Treatment induced an intense increase in albuminuria from 74 micrograms/24 h to 11.5 mg/24 h on the 5th day of treatment (N = 11). This increase was associated with the presence of large amounts of albumin in elements of the glomerular filter and in the apical region of the proximal tubular cells (N = 4). Fractional clearances of neutral dextrans having molecular radii in the range of 18-41 A were not significantly different in control (N = 5) and gentamicin-treated rats (N = 7). 3. These results show that gentamicin, a polycation at pH 7.4, produces an increase in the glomerular permeability to negatively charged macromolecules in rats, probably due to interaction of the polycation with negative changes in the glomerular filter.

Albumins↗

PubMed1991

Ca(2+)-induced down-regulation of epithelial amiloride-sensitive channels reconstituted into planar lipid bilayers.

Multichannel experiments were carried out to investigate the Ca(2+)-induced down-regulation of epithelial Na+ channels reconstituted into planar lipid bilayer membranes. Reconstitution was achieved by fusion of vesiculated apical membrane fragments to solvent-free planar lipid bilayers. We found that the presence of micromolar concentrations of Ca2+ on the side to which the vesicles were added substantially lowered the channel-mediated current. The inhibition was strongly influenced by pH. At pH 8.0, all the current was blocked by 1 mM calcium, whereas at pH 7.1 the inhibition was about 80%. The blocking kinetics was clearly voltage-dependent. The mechanism of blocking cannot be explained either in terms of interactions with a single site, or by a model in which two blocking sites are assumed.

Amiloride↗

PubMed1988

Gating properties of channels formed by Colicin Ia in planar lipid bilayer membranes.

Colicin Ia forms voltage-dependent channels when incorporated into planar lipid bilayers. A membrane containing many Colicin Ia channels shows a conductance which is turned on when high positive voltages (greater than +10 mV) are applied to the cis side (side to which the protein is added). The ionic current flowing through the membrane in response to a voltage step shows at first an exponential and then a linear rise with time. The relationship between the steady-state conductance, achieved immediately after the exponential portion, and voltage is S-shaped and is adequately fit by a Boltzmann distribution. The time constant (tau) of the exponential is also dependent on voltage, and the relation between these two parameters is asymmetric around Vo (voltage at which half of the channels are open). In both cases the steepness of the voltage dependence, a consequence of the number of effective gating particles (n) present in the channel, is greatly influenced by the pH of the bathing solutions. Thus, increasing the pH leads to a reduction in n, while acidic pH's have the opposite effects. This result is obtained either by changing the pH on both sides of the membrane or on only one side, be it cis or trans. On the other hand, changing pH on only one side by addition of an impermeant buffer fails to induce any change in n. At the single-channel level, pH had an effect both on the unitary conductance, doubling it in going from pH 4.5 to 8.2, as well as on the fraction of time the channels stay open, F(v). For a given voltage, F(v) is clearly diminished by increasing the pH. This titration of the voltage sensitivity leads to the conclusion that gating in the Colicin Ia molecule is accomplished by charged amino- acid residues present in the protein molecule. Our results also support the notion that these charged groups are inside the aqueous portion of the channel.

Colicins↗

PubMed1985

The acetylcholine receptor of the neuromuscular junction recognizes mecamylamine as a noncompetitive antagonist.

The secondary amine, mecamylamine, interacts with the nicotinic receptor ionic channel complex as a noncompetitive antagonist. Mecamylamine (1-10 microM) blocked indirect muscle twitches with no discernible effect on the membrane potential, overshoot, or amplitude of the action potential. It also produced a voltage- and concentration-dependent depression of the peak amplitude of the endplate currents (EPC) and induced nonlinearity in the current-voltage relationship. The decay time constant of the EPC (TEPC) was significantly shortened. The linear relationship between the reciprocal of TEPC and the drug concentration suggested an open channel blockade. Patch-clamp studies, in agreement with the noise analysis results, revealed that mecamylamine (1-8 microM) shortened the lifetime of the open channels. Further, the single channel studies showed that at high concentrations mecamylamine reduced the double exponential nature of the distribution of open times characteristic of channels recorded from myoballs. Closed times had a complex distribution that could not be fitted to a single exponential function because of the presence of short closures or "flickers" during the open state. Although the frequency of channel openings progressively decreased with increasing drug concentration, the single channel conductance remained unchanged at all the concentrations tested. Biochemical studies showed that mecamylamine (up to 100 microM) did not block [3H]acetylcholine binding to the nicotinic receptor of the Torpedo electroplax, but inhibited the binding of [3H]perhydrohistionicotoxin to its channel site, both in the resting and the activated state. These results suggested that, at the nicotinic receptors of the neuromuscular junction, mecamylamine acted as a noncompetitive blocker, binding primarily to the receptor's open channel conformation. Most of the alterations of EPCs were consistent with the predictions of a sequential model for open channel blockade. Biochemical and patch-clamp results, however, could not be fully explained by this model and provided some evidence of the existence of additional blocked states most likely through pathways into desensitized species. In contrast to a competitive antagonism of acetylcholine receptors reported at autonomic ganglia, there was no such action of the drug at the neuromuscular junction; thus, mecamylamine is a useful tool to characterize the nicotinic receptors from different synapses.

Amphibian Venoms↗

PubMed1984

Interactions of gephyrotoxin with the acetylcholine receptor-ionic channel complex. I. Blockade of the ionic channel.

The novel tricyclic alkaloid, gephyrotoxin ( GyTX ), found in the skin secretions of the frog Dendrobates histrionicus , potentiates and blocks the indirectly elicited muscle twitch in a concentration-dependent manner. GyTX prolongs the falling phase of the muscle action potential and decreases delayed rectification, supporting the idea that the alkaloid blocks the voltage-sensitive potassium conductance of the electrically excitable membrane. The peak amplitude of the end-plate currents (EPC) and miniature end-plate currents ( MEPC ) were depressed, but no significant deviation from linearity relative to control was seen in the current-voltage relationship. The decay time constant of the EPC (tau EPC) was markedly shortened by GyTX , the effect being greater at 10 degrees than at 22 degrees. The relationship between the log of tau EPC and membrane potential disclosed a linear relationship at all concentrations tested, but a progressive loss of voltage sensitivity of tau EPC was seen when GyTX concentrations were increased. Also, the plot of 1/tau EPC against GyTX concentration revealed a linear relationship. The lack of voltage and time dependence suggests that GyTX has little effect on the ACh receptor-ionic channel complex in the closed conformation. Single-channel conductance studied by means of fluctuation analysis did not change after GyTX application, but the channel lifetime decreased by about 40% at clamp potentials of -105 mV and at a toxin concentration of 7.5 microM. Repetitive nerve stimulation led to a pronounced " rundown " in the EPCs which was frequency-dependent. These findings were taken as evidence that GyTX interacts with the acetylcholine receptor complex, causing a blockade of its channel mainly in the open conformation.

Action Potentials↗

PubMed1984

Interactions of gephyrotoxin with the acetylcholine receptor-ionic channel complex. II. Enhancement of desensitization.

The actions of the tricyclic alkaloid gephyrotoxin ( GyTX ) on the extrajunctional and junctional acetylcholine (ACh) sensitivity and desensitization were studied in the chronically denervated rat soleus muscle and cutaneous pectoris muscle of the frog. At low concentrations, GyTX greatly depressed the extrajunctional ACh sensitivity of the chronically denervated soleus muscles. In addition, GyTX produced a strong inhibition of junctional end-plate potentials evoked by ACh. Junctional and extrajunctional desensitizations induced by microiontophoretically applied ACh were greatly enhanced by the alkaloid in a frequency-dependent manner. These effects were readily reversible. The interaction of GyTX with binding sites on the acetylcholine receptor-channel (AChR) complex was studied on electroplax membranes from Torpedo californica. GyTX binds to the AChR complex at a site distinct from the ACh binding site, as revealed by its lack of inhibition of [125I]alpha-bungarotoxin ( [125I]BGT) binding. On the other hand, GyTX at a concentration range between 1 microM and 100 microM significantly increased the potency of the agonist carbamylcholine as an antagonist of binding of [125I]BGT. At low micromolar concentrations, GyTX inhibited the binding of [3H]perhydrohistrionicotoxin and [3H] phencyclidine to sites associated with the ionic channel of the AChR complex. The affinity of GyTX for these sites was increased 3- to 5-fold by carbamylcholine. Results of electrophysiological and binding studies indicate that GyTX not only blocks the open channel of the AChR but also enhances desensitization of the AChR complex by increasing receptor affinity for agonists.

Acetylcholine↗

PubMed1982

A quartz cell for studying planar lipid bilayer membranes.

A quartz chamber is proposed for use in experiments with planar lipid bilayer membranes. Membranes are formed in a hole made on the lateral wall of a fused quartz test tube, immersed in an electrolyte solution. The quartz cell is easy to clean, chemically inert and easily made. Membranes formed in this chamber had specific resistances higher than 10(8) omega.cm2 and excellent mechanical stability.

Lipid Bilayers↗

PubMed1979

Transient potassium fluxes in toad skin.

Experiments were carried out in the isolated short-circuited skin of the toad Bufo marinus ictericus. 42K influx and efflux experiments were carried out with skins bathed on both sides by NaCl-Ringer's solution. Those fluxes showed very similar kinetics of equilibration with time and the results could be fitted by equations of a model of two intraepithelial compartments and the bathing solutions. In the steady state K influx is 3.99 +/- 0.36 nmol cm-2 hr-1 (n = 7) and efflux 3.62 +/- 0.38 nmol cm-2 hr-1 (n = 7) and are not statistically different, indicating that no net K flux is present across the epithelium. Different kinds of perturbations affecting the rates of 42K discharge into the bathing solutions were studied. Immediately after addition of amiloride (10(-4) M) to the outer solution, a sharp decline is observed in the rate of 42K discharge into the bathing solution, JK21, which falls from 3.62 +/- 0.38 nmol cm-2 hr-1 to 2.02 +/- 0.04 nmol cm-2 hr-1 (n = 7) 2 min after addition of the drug, followed by a partial recuperation with time. A complete Na by K substitution in the outer bathing solution induces a prompt and marked decline in JK21 which is similar to that induced by amiloride. Increase in the outer bathing solution Na concentration from zero Na concentration induces a nonlinear increase in JK21 and a linear relationship was observed between JK21 and short-circuit current in the range of 0 to 115 mM external Na concentration. The decline in JK21 induced by amiloride or by lowering external Na concentration was interpreted as being caused by electrical hyperpolarization of the external barrier of the epithelium induced by these procedures. Depolarization of the epithelial barriers by inner Na by K substitution in the short-circuited state (when the potential barriers are equal) drastically interfere with the rate of 42K discharge from the epithelium into the bathing solutions. Thus, transient increases are observed both in the rate of 42K discharge to the outer and to the inner bathing solutions upon depolarization of the barriers. These results indicate that at least the most important component of transepithelial K unidirectional fluxes goes through a transcellular route with a negligible paracellular component. Addition of ouabain (10(-3) M) to the inner bathing solution induces a transient rise in the rate of 42K discharge to the outer bathing solution with a peak on the order of 200% of the stationary value previous to the action of the inhibitor, followed by a return to new stationary values not statistically different from those observed previously to the effect of ouabain. The behavior of JK21 upon the effect of ouabain, as suggested by comparison with predictions from computer simulation, strongly supports the notion of a rheogenic Na pump in the inner barrier of the epithelium against the notion of a nonrheogenic 1:1 Na--K pump.

Amiloride↗

PubMed1978

Transients in toad skin: short circuit current and ionic fluxes related to inner sodium substitution by monovalent cations.

When the Na electrochemical potential difference across the skin (delta muNa) is altered by perturbing the transmembrane electrical potential difference or the external Na concentration, effects on transport and associated oxygen consumption can be described by the formalism of linear nonequilibrium thermodynamics (Vieira, Caplan & Essig, 1972, J. Gen. Physiol. 59:77; Danisi & Lacaz-Vieira, 1974, J. Gen. Physiol. 64:372; Procópio and Lacaz-Vieira, 1977, J. Membrane Biol. 35:219). We now show that with modifications of delta muNa by substitution of Li or choline for Na in the inner bathing solution, this formalism is no longer applicable. Inner Na by K substitution ((Na X K)i) causes profound alterations in short-circuit current (SCC), JinNa, K efflux (JeffK) and PD. SCC drops transiently after (Na X K)i in Cl and in SO4 media, increasing subsequently. In Cl medium, following the initial transient, there is a late decline in SCC toward a steady state. The rate of SCC decline in Cl medium is more pronounced than that observed in SO4 medium. (Na X K)i causes a transient increase in JinNa with a peak synchronous to the minimum in SCC, both in Cl and in SO4 media. This was interpreted as due to depolarization of the inner membrane. In SO4 medium, following the peak observed after (Na X K)i, JimNa drops, to increase again toward a steady state in which SCC and JinNa are not statistically different, resembling the control condition before (Na X K)i. In Cl medium, however, the JinNa steady state is approximately 100% higher than SCC. This difference is due to an important K efflux (JeffK), which builds up progressively after the substitution. The apparent K permeability [JeffK/(Ki)] is of comparable magnitude in Cl and in SO4 media before (Na X K)i and also in SO4 medium after (Na X K)i. However, in Cl medium, after (Na X K)i, the apparent K permeability increases one order of magnitude as compared to the control condition before the ionic substitution. In Cl medium, the high levels of JinNa and of Jeff(K) observed in the steady state after (Na X K)i were interpreted as being a consequence of cell swelling. SCC and PD follow very different temporal patterns after (Na X K)i which are characterized by transients in SCC and a simple fall in PD. Reasons for these differences are discussed.

Animals↗