Biomedical subjects

E Perozo

Publications and source records attributed to E Perozo.

28 records · Page 2Linked to original sources

PubMed1993

Voltage activation of reconstituted sodium channels: use of bacteriorhodopsin as a light-driven current source.

We present a general method for the development and control of transmembrane potentials (delta psi) in reconstituted vesicles. The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium halobium is the current source in the system, and the intensity of light controls the magnitude of delta psi at any given time. Transmembrane potentials were determined from the equilibrium distribution of hydrophobic ions, which was monitored by using either electron paramagnetic resonance spectroscopy and spin-labeled phosphonium ions or a tetraphenylphosphonium-selective electrode. A bias or holding potential was generated by using gradients of anions of limited permeability in the presence of an impermeable cation. Using n-methylglucamine or the polymer poly(ethylene imide) as the impermeable cation, the anions NO3- and SCN- were most effective in producing large (> -60 mV) negative diffusion potentials in egg phosphatidylcholine vesicles. In the presence of a NO(3-)-based negative holding potential (approx. -65 mV), bR is capable of depolarizing the membrane to at least the 0-mV level within a few seconds. More rapid depolarizations can be achieved by the application of a brief intense illumination preceding the preset illumination level (supercharging). The technique was successfully used to activate for the first time a population of unmodified voltage-dependent sodium channels purified from eel electroplax.

Bacteriorhodopsins↗

PubMed1993

Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels.

In voltage-dependent ion channels, a voltage sensor region is responsible for channel activation and an aqueous pore is responsible for ion conduction. These two processes have been traditionally considered to be independent. We describe here a mutation in the putative pore region (W434F) that completely abolishes ion conduction without affecting the gating charge of the channel. Gating currents in the nonconductive mutant were found to be identical in their kinetic and steady-state properties to those in conductive channels. Gating current measurements could be performed without subtracting pulses and in the presence of normal physiological solutions. Application of internal tetraethylammonium (an open channel blocker) induced Off charge immobilization for large depolarizations, suggesting that the internal tetraethylammonium-binding site becomes available upon depolarization. We concluded that for this mutant, although the conduction pathway is not functional, the channel can still undergo the closed-open conformation in response to voltage changes.

Animals↗

PubMed1992

Gating currents in Shaker K+ channels. Implications for activation and inactivation models.

We have studied ionic and gating currents in mutant and wild-type Shaker K+ channels to investigate the mechanisms of channel activation and the relationship between the voltage sensor of the channel and its inactivation particle. The turn on of the gating current shows a rising phase, indicating that the hypothetical identical activation subunits are not independent. Hyperpolarizing prepulses indicate that most of the voltage-dependence occurs in the transitions between closed states. The open-to-closed transition is voltage independent, as suggested by the presence of a rising phase in the off gating currents. In Shaker channels showing fast inactivation, the off gating charge is partially immobilized as a result of depolarizing pulses that elicit inactivation. In mutant channels lacking inactivation, the charge is recovered quickly at the end of the pulse. Internal TEA mimics the inactivation particle in its behavior but the charge immobilization is established faster and is complete. We conclude that the activation mechanism cannot be due to the movement of identical independent gating subunits, each undergoing first order transitions, and that the inactivation particle is responsible for charge immobilization in this channel.

Animals↗

PubMed1991

Molecular basis of gating charge immobilization in Shaker potassium channels.

Voltage-dependent ion channels respond to changes in the membrane potential by means of charged voltage sensors intrinsic to the channel protein. Changes in transmembrane potential cause movement of these charged residues, which results in conformational changes in the channel. Movements of the charged sensors can be detected as currents known as gating currents. Measurement of the gating currents of the Drosophila Shaker potassium channel indicates that the charge on the voltage sensor of the channels is progressively immobilized by prolonged depolarizations. The charge is not immobilized in a mutant of the channel that lacks inactivation. These results show that the region of the molecule responsible for inactivation interacts, directly or indirectly, with the voltage sensor to prevent the return of the charge to its original position. The gating transitions between closed states of the channel appear not to be independent, suggesting that the channel subunits interact during activation.

Amino Acid Sequence↗

PubMed1991

Phosphorylation of K+ channels in the squid giant axon. A mechanistic analysis.

Protein phosphorylation is an important mechanism in the modulation of voltage-dependent ionic channels. In squid giant axons, the potassium delayed rectifier channel is modulated by an ATP-mediated phosphorylation mechanism, producing important changes in amplitude and kinetics of the outward current. The characteristics and biophysical basis for the phosphorylation effects have been extensively studied in this preparation using macroscopic, single-channel and gating current experiments. Phosphorylation produces a shift in the voltage dependence of all voltage-dependent parameters including open probability, slow inactivation, first latency, and gating charge transferred. The locus of the effect seems to be located in a fast 20 pS channel, with characteristics of delayed rectifier, but at least another channel is phosphorylated under our experimental conditions. These results are interpreted quantitatively with a mechanistic model that explains all the data. In this model the shift in voltage dependence is produced by electrostatic interactions between the transferred phosphate and the voltage sensor of the channel.

Animals↗

PubMed1991

Single channel studies of the phosphorylation of K+ channels in the squid giant axon. I. Steady-state conditions.

Phosphorylation of the delayed rectifier channel of squid potentiates the macroscopic K+ current and slows its activation kinetics. We have studied this phenomenon at the single channel level using the cut-open axon technique under steady-state conditions. In 10 mM external K+/310 mM internal K+ there are predominantly two types of channels present, a 20-pS and a 40-pS channel. In steady state at depolarized potentials, the 40-pS channel was most active, whereas the 20-pS channel tended to disappear due to a slow inactivation process. Two methods were developed to shift the population of channels toward a dephosphorylated state. One method consisted of predialyzing a whole axon with solutions containing no ATP, while recording the currents under axial-wire voltage clamp. A piece of axon was then removed and cut open, and single channel currents were recorded from the cut-open axon. A second method was based on the difference in diffusion coefficients for ATP and proteins such as the endogenous phosphatase. The axon was cut open in a solution that did not contain Ca2+ or Cl- in order to maintain the axoplasm structurally intact and permit endogenous phosphatase to act on the membrane while ATP diffused away, before removing the axoplasm and forming a membrane patch. When dephosphorylating conditions were used, the steady-state open probability of the 40-pS channel at 42 mV was very low (less than 0.0002), and the channel openings appeared as a series of infrequent, short-duration events. The channel activity was increased up to 150-fold by photoreleasing caged ATP inside the patch pipette in the presence of the catalytic subunit of protein kinase A. The sharp increase in open probability could be accounted for by a decrease of the slow component of the closed time distribution from 23 s to 170 ms with little change in the distribution of open times (1-2 ms) and no change in the single channel current amplitude. In voltage-jump experiments the contribution of the 40-pS channel to the delayed rectifier current was often small due to the large values of the latency to the first opening.

Adenosine Triphosphate↗

PubMed1991

Single channel studies of the phosphorylation of K+ channels in the squid giant axon. II. Nonstationary conditions.

The effects of phosphorylation on the properties of the 20-pS channel of the squid giant axon were studied using the cut-open axon technique. Phosphorylation of the channel was achieved by photoreleasing caged ATP (inside the patch pipette) in the presence of the catalytic subunit of the protein kinase A. An inverted K+ gradient (500 K+ external parallel 5 K+ internal) was used to study the activation process. Phosphorylation decreased the frequency of openings of the channel at most potentials by shifting the probability vs. voltage curve toward more positive potentials. The mean open times showed no voltage dependence and were not affected by phosphorylation. The distribution of first latencies, on the other hand, displayed a sharp voltage dependence. Phosphorylation increased the latency to the first opening at all potentials, shifting the median first latency vs. voltage curve toward more positive potentials. The slow inactivation process was studied in the presence of a physiological K+ gradient (10 K+ external parallel 310 K+ internal). Pulses to 40 mV from different holding potentials were analyzed. Phosphorylation increases the overall ensemble probability by decreasing the number of blank traces. A single channel inactivation curve was constructed by computing the relative appearance of blank traces at different holding potentials before and after photoreleasing caged ATP. As determined in dialyzed axons, the effect of phosphorylation consisted in a shift of the inactivation curve toward more positive potentials. The 20-pS channel has the same characteristics as the delayed rectifier current in activation kinetics, steady-state inactivation, and phosphorylation effects.

Animals↗

PubMed1990

Phosphorylation affects voltage gating of the delayed rectifier K+ channel by electrostatic interactions.

The delayed rectifier K+ channel of the squid axon undergoes a series of modifications in its kinetic and conductive parameters when it is phosphorylated as the result of shifts in its voltage-dependent parameters. These effects can be interpreted as due to electrostatic interaction between the voltage sensor of the channel and the transferred phosphate from ATP. Using different concentrations of intracellular Mg2+, we determined the density of surface charges seen by the K+ channel voltage sensor before and after phosphorylation. Values for the surface charge density in the cytoplasmic side of the membrane were between 1/350 and 1/250 e-/A2 in the absence of ATP and between 1/160 and 1/155 e-/A2 under phosphorylating conditions. Incorporation of a surface potential into a kinetic model for the delayed rectifier channel can predict quantitatively phosphorylation-like changes in K+ currents. These results provide evidence for the importance of electrostatic interactions as one of the mechanisms by which phosphorylation modulates the behavior of voltage-dependent channels.

Animals↗

PubMed1989

Modulation of K channels in dialyzed squid axons. ATP-mediated phosphorylation.

In squid axons, internally applied ATP potentiates the magnitude of the potassium conductance and slows down its activation kinetics. This effect was characterized using internally dialyzed axons under voltage-clamp conditions. Both amplitude potentiation and kinetic slow-down effects are very selective towards ATP, other nucleotides like GTP and ITP are ineffective in millimolar concentrations. The current potentiation Km for ATP is near 10 microM with no further effects for concentrations greater than 100 microM. ATP effect is most likely produced via a phosphorylative reaction because Mg ion is an obligatory requirement and nonhydrolyzable ATP analogues are without effect. In the presence of ATP, the K current presents more delay, resembling a Cole-Moore effect due to local hyperpolarization of the channel. ATP effect induces a 10-20 mV shift in both activation and inactivation parameters towards more depolarized potentials. As a consequence of this shift, conductance-voltage curves with and without ATP cross at approximately -40 mV. This result is consistent with the hyperpolarization observed with ATP depletion, which is reversed by ATP addition. At potentials around the resting value, addition of ATP removes almost completely K current slow inactivation. It is suggested that a change in the amount of the slow inactivation is responsible for the differences in current amplitude with and without ATP, possibly as a consequence of the additional negative charge carried by the phosphate group. However, a modification of the local potential is not enough to explain completely the differences under the two conditions.

Adenosine Triphosphate↗

PubMed1987

Spin-labeled amphotericin B: synthesis, characterization, biological and spectroscopic properties.

A biologically active spin-labeled derivative of amphotericin B has been synthesized by the nucleophilic addition of amphotericin B to 4-(2-iodoacetamido)-2,2',6,6'-tetramethylpiperadine-N-oxyl in dimethyl-sulphoxide at 40 degrees C. The derivative is a moderately water-soluble compound which displays the same biological activity of the parental compound against the sensitive organism Leishmania mexicana; also, the rates of proton-cation exchange induced by the two compounds in large unilamellar liposomes are indistinguishable. The ESR spectra of spin-labeled amphotericin B in lipid vesicles indicate a high degree of motion, very similar to that encountered for the compound in aqueous solutions at neutral pH and in deoxycholate micelles, and suggest that the structures formed by the antibiotic in membranes are composed by a small number of molecules. In contrast, the spectra of the labeled antibiotic in ethanol, diethyl ether and dimethylformamide indicate restricted motion and exchange interactions, probably resulting from the micellar aggregation induced in these media. Ascorbate at 10 mM is able to reduce completely the nitroxide group of the labeled antibiotic in lipid vesicles in less than 30 s, indicating that an asymmetric disposition of the antibiotic molecules across the membrane is capable of inducing its biological and ionophoric properties. Ni2+ and Cu2+ produce moderate exchange broadening of the ESR signal of spin-labeled amphotericin B in lipid vesicles; the comparison of this phenomenom with the exchange broadening produced by the same ions in the ESR spectrum of 2,2',6,6'-tetramethylpiperidine-N-oxyl in water solution suggests an specific Cu2+-amphotericin B interaction in membranes.

Amphotericin B↗