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Biomedical subjects

R Latorre

Publications and source records attributed to R Latorre.

At least 109 records · Page 6Linked to original sources
PubMed1985

Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers.

A Ca-activated, K-selective channel from plasma membrane of rat skeletal muscle was studied in artificial lipid bilayers formed from either phosphatidylethanolamine (PE) or phosphatidylserine (PS). In PE, the single-channel conductance exhibited a complex dependence on symmetrical K+ concentration that could not be described by simple Michaelis-Menten saturation. At low K+ concentrations the channel conductance was higher in PS membranes, but approached the same conductance observed in PE above 0.4 m KCl. At the same Ca2+ concentration and voltage, the probability of channel opening was significantly greater in PS than PE. The differences in the conduction and gating, observed in the two lipids, can be explained by the negative surface charge of PS compared to the neutral PE membrane. Model calculations of the expected concentrations of K+ and Ca2+ at various distances from a PS membrane surface, using Gouy-Chapman-Stern theory, suggest that the K+-conduction and Ca2+-activation sites sense a similar fraction of the surface potential, equivalent to the local electrostatic potential at a distance of 9 A from the surface.

Animals↗
PubMed1984

Alkali metal ion selectivity of the hemocyanin channel.

The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permeability ratios relative to K+ measured from biionic potentials were: NH4+ (1.52) greater than Rb+ (1.05) greater than K+ (1.0) greater than Cs+ (0.89) greater than Na+ (0.81) greater than Li+ (0.35). Single-channel ion conductance was a saturating function of ion concentration regardless of the cation present in the bathing medium. Maximal conductances were 270, 267, 215, 176, 170 and 37 ps for K+, Rb+, NH4+, Cs+, Na+ and Li+, respectively. Current-voltage curves for the different monovalent cations were measured and described using a three-barrier model previously used to explain the voltage dependence of the "instantaneous" channel conductance (Cecchi, Alvarez & Latorre, 1981). In this way, binding and peak energies were estimated for the different ions. Considering the energy peaks as transition states between the ion and the channel, it is concluded that they follow Eisenman's selectivity sequences XI (cis peak, i.e., Li+ greater than Na+ greater than K+ greater than Rb+ greater than Cs+; highest field strength), VII (central peak) and II (trans peak). The cis side was that to which hemocyanin was added and was electrically ground. The binding energies, on the other hand, follow Eisenman's series XI for strong electric field sites. Binding of NH4+ to the cis-well suggests that the orientation of the ligands in the site is tetrahedric.

Hemocyanins↗
PubMed1984

Single potassium channels with delayed rectifier behavior from lobster axon membranes.

Single-channel potassium currents from lobster axon membranes were studied in planar bilayers made from monolayers. Channel-opening events are grouped by time, forming bursts with an average duration of 4.5 ms. The mean open time at 0 mV is 1.8 ms. The frequency of bursts is voltage dependent, increasing e-fold per 12-16 mV. At sufficiently high positive voltages, channels inactivate. Measured from reversal potentials, channels discriminate against Na+ by a permeability ratio PNa/PK of 1:30. The channel is blocked by tetraethylammonium and nonyltrimethylammonium in a voltage-dependent manner and at concentrations similar to those used in whole-axon experiments. Voltage-dependent block by Cs+ suggests that more than one ion may occupy the channel simultaneously. The kinetics and selectivity of this channel suggest that purified axolemma contains active K+ channels that are likely to participate in delayed rectification in the lobster axon membrane.

Animals↗
PubMed1983

Saxitoxin and ouabain binding activity of isolated skeletal muscle membrane as indicators of surface origin and purity.

A simple biochemical method for identifying and distinguishing transverse tubule and sarcolemma membranes in preparations of skeletal muscle microsomes is proposed and evaluated. This method is based on the previous observation that the ratio of ouabain to saxitoxin binding sites is five-fold higher in the sarcolemma than the transverse tubule. We measured [3H]saxitoxin and [3H]ouabain binding to microsomes of frog, rat and rabbit muscle in the presence of detergents to expose latent sites. A high density fraction (30--40% sucrose) of the membranes was identified as transverse tubule on the basis of a low ouabain/saxitoxin ratio and its association with sarcoplasmic reticulum. A low density fraction (20--30% sucrose) was identified as transverse tubule containing variable amounts of sarcolemma as judged by a higher ratio of ouabain/saxitoxin sites. Our results suggest that this ratio can be used to determine the surface origin of muscle membrane preparations. Several different methods for purifying transverse tubules were compared by this technique.

Amphibian Proteins↗
PubMed1983

Large divalent cations and electrostatic potentials adjacent to membranes. Experimental results with hexamethonium.

A simple extension of the Gouy-Chapman theory predicts that the ability of a divalent cation to screen charges at a membrane-solution interface decreases significantly if the distance between the charges on the cation is comparable with the Debye length. We tested this prediction by investigating the effect of hexamethonium on the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes. The distance between the two charges of an extended hexamethonium molecule is approximately 1 nm, which is the Debye length in the 0.1 M monovalent salt solutions used in these experiments. Six different experimental approaches were utilized. We measured the electrophoretic mobility of multilamellar vesicles to determine the zeta potential, the line width of the 31P nuclear magnetic resonance (NMR) signal from sonicated vesicles to calculate the change in potential at the phosphodiester moiety of the lipid, and the conductance of planar bilayer membranes exposed to either carriers (nonactin) or pore formers (gramicidin) to estimate the change in potential within the membrane. We also measured directly the effect of hexamethonium on the potential above a monolayer formed from negative lipids, and attempted to calculate the change in the surface potential of a bilayer membrane from capacitance measurements. With the exception of the capacitance calculations, each of the techniques gave comparable results: hexamethonium exerts a smaller effect on the potential than that predicted by the classic screening theory. The results are consistent with the predictions of the extended Gouy-Chapman theory and are relevant to the interpretation of physiological and pharmacological experiments that utilize hexamethonium and other large divalent cations.

Animals↗
PubMed1983

Phospholipid bilayers made from monolayers on patch-clamp pipettes.

Phospholipid bilayers were made from phospholipid monolayers at the air/water interface on patch-clamp pipettes. Lipid bilayers were characterized using the K+ carrier nonactin and the channel formers gramicidin and alamethicin. Bilayers were also formed from monolayers spontaneously assembled in a suspension of native vesicles from cardiac sarcolemma and lobster axonal membranes and an excess of lipids. In these types of bilayers we observed several different channels including one contained in the axonal membrane that shows delayed rectifier behavior. This technique permits the study of reconstituted channels on a time scale and noise comparable to cellular patch-clamp standards.

Alamethicin↗
PubMed1983

Sodium flux ratio through the amiloride-sensitive entry pathway in frog skin.

The sodium flux ratio of the amiloride-sensitive Na+ channel in the apical membrane of in vitro Rana catesbeiana skin has been evaluated at different sodium concentrations and membrane potentials in sulfate Ringer solution. Amiloride-sensitive unidirectional influxes and effluxes were determined as the difference between bidirectional 22Na and 24Na fluxes simultaneously measured in the absence and presence of 10(-4) M amiloride in the external bathing solution. Amiloride-sensitive Na+ effluxes were induced by incorporation of cation-selective ionophores (amphotericin B or nystatin) into the normally Na+-impermeable basolateral membrane. Apical membrane potentials (Va) were measured with intracellular microelectrodes. We conclude that since the flux ratio exponent, n', is very close to 1, sodium movement through this channel can be explained by a free-diffusion model in which ions move independently. This result, however, does not necessarily preclude the possibility that this transport channel may contain one or more ion binding sites.

Amiloride↗
PubMed1983

Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage-dependent Ca2+ binding reactions.

The gating kinetics of a Ca2+-activated K+ channel from adult rat muscle plasma membrane are studied in artificial planar bilayers. Analysis of single-channel fluctuations distinguishes two Ca2+- and voltage-dependent processes: (a) short-lived channel closure (less than 1 ms) events appearing in a bursting pattern; (b) opening and closing events ranging from one to several hundred milliseconds in duration. The latter process is studied independently of the first and is denoted as the primary gating mode. At constant voltage, the mean open time of the primary gating mode is a linear function of the [Ca2+], whereas the mean closed time is a linear function of the reciprocal [Ca2+]. In the limits of zero and infinite [Ca2+], the mean open and the mean closed times are, respectively, independent of voltage. These results are predicted by a kinetic scheme consisting of the following reaction steps: (a) binding of Ca2+ to a closed state; (b) channel opening; (c) binding of a second Ca2+ ion. In this scheme, the two Ca2+ binding reactions are voltage dependent, whereas the open-closed transition is voltage independent. The kinetic constant derived for this scheme gives an accurate theoretical fit to the observed equilibrium open-state probability. The results provide evidence for a novel regulatory mechanism for the activity of an ion channel: modulation by voltage of the binding of an agonist molecule, in this case, Ca2+ ion.

Animals↗
PubMed1983

Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade.

The interaction of Ca2+ and Ba2+ with a Ca2+-activated K+ channel from rabbit skeletal muscle membranes is studied in planar lipid bilayers. At [Ca2+] greater than or equal to 100 microM in the cis side (the side to which the vesicles are added) and at positive voltages, the channel kinetics consisted of bursts of activity interrupted by long periods of quiescence. We found that the reciprocal of the mean burst time increases linearly with [Ca2+], whereas the mean time for the quiescent (closed) periods is independent of [Ca2+]. The number of quiescent periods is reduced by increasing [K+]. Micromolar amounts of cis Ba2+ do not activate the channel, but induce similar "slow" closings. Also, in this case, the mean burst time is inversely proportional to the [Ba2+] and the mean closed time is independent of [Ba2+]. Raising [K+] either symmetrically or only in the trans side relieved the Ba2+ effect. trans Ba2+ also induces changes in the slow kinetics, but in millimolar amounts. These results suggest that the quiescent periods correspond to a channel blocked by a Ba ion. The voltage dependence of the cis blockade indicates that the Ba2+ binding site is past the middle of the membrane field. The similarities in the slow kinetics induced by Ca2+ and Ba2+ suggest that Ca2+ blocks the channel by binding to the same site. However, binding of Ca2+ to the site is 10(5)-fold weaker.

Animals↗
PubMed1982

Detection of K+ and Cl-channels from calf cardiac sarcolemma in planar lipid bilayer membranes.

The ionic currents underlying the cardiac action potential are believed to be much more complex than those in nerve. During the cardiac action potential, various membrane channels control the flow of K+, Na+, Ca2+ and Cl- across the sarcolemma of cardiac muscle cells. Thus, it has become increasingly clear that a detailed knowledge of the mechanisms that activate (or inactivate) heart channels is required to understand cardiac excitability. We report here the use of planar lipid bilayer techniques to detect and characterize K+ and Cl- channels in purified heart sarcolemma membrane vesicles. We have identified four different types of channel on the basis of their selectivity, conductance and gating kinetics. We present in some detail the properties of a K+ channel and a Cl- channel. We have tentatively identified the K+ channel with the ix type of current found in Purkinje, myocardial ventricular and atrial fibres. The chloride channel might be related to the transient chloride current found in Purkinje fibres.

Action Potentials↗
PubMed1982

Influence of cholesterol on water penetration into bilayers.

X-ray diffraction and capacitance measurements have been used to calculate the depth to which water penetrates in fully hydrated bacterial phosphatidylethanolamine bilayers in the presence and absence of cholesterol. The data indicate that cholesterol decreases in depth of water penetration by about 2.5 angstroms.

Cholesterol↗
PubMed1982

Reconstitution in planar lipid bilayers of a Ca2+-dependent K+ channel from transverse tubule membranes isolated from rabbit skeletal muscle.

Addition of membrane vesicles prepared from transverse tubule (T-tubule) membranes of rabbit skeletal muscle to the aqueous phase of a planar lipid bilayer induces a stepwise increase in conductance. This conductance is both voltage and Ca2+ dependent. At 1 mM Ca2+, the steady-state conductance is maximal at voltages higher than +20 mV and decreases for more negative voltages. (Voltages refer to the side to which the vesicles are added, cis) Decreasing the Ca2+ concentration reversibly shifts the conductance-voltage curve toward the right along the voltage axis. Furthermore, Ca2+ can activate the conductance only if added to the cis compartment. Neither Mg2+, Ba2+, nor Cd2+ can activate the conductance induced by T-tubule vesicles. Addition of 5 mM tetraethylammonium ion to the trans, but not the cis, side abolishes the T-tubule-induced conductance. The Ca2+-dependent conductance appears as a consequence of ionic channel formation. Single-channel activity appears in bursts followed by periods of time in which the channel remains "silent". The conductance of the open channel averages 226 pS in 0.1 M KC1 and is voltage and Ca2+ independent. However, the fraction of time that the channel remains in the open state is voltage and Ca2+ dependent in a manner that parallels the voltage and Ca2+ dependence of the multichannel membrane. The channel is 6.6 times more permeable to K+ than to Na+ and is impermeable to C1-.

Animals↗