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X Cecchi

Publications and source records attributed to X Cecchi.

9 recordsLinked to original sources
PubMed1993

Microcin E492 forms ion channels in phospholipid bilayer membrane.

Microcin E492, a polypeptide antibiotic, has been shown to have an M(r) of 6,000 by urea-SDS-polyacrylamide gel electrophoresis of the fluorescently labelled compound. It is known that the bactericidal action of microcin involves a loss of the transmembrane potential. In this study we show that microcin forms cation-selective channels in planar phospholipid bilayers. The channels have two main conductance states the current-voltage curves of which rectify. The reversal potentials measured under biionic conditions indicate a permeability sequence of NH4+ > K+ = Rb+ = Cs+ > Na+ = Na+ = Li+ > Tris+. The results suggest that membrane potential dissipation induced by microcin is a consequence of the formation of pores in the bacterial membrane.

Anti-Bacterial Agents↗
PubMed1989

Streaming potential measurements in Ca2+-activated K+ channels from skeletal and smooth muscle. Coupling of ion and water fluxes.

Streaming potentials arising across large-conductance Ca2+-activated K+ channels incorporated into planar lipid bilayers were measured. Ca2+-activated channels obtained either from skeletal muscle or from smooth muscle membranes were used. Streaming potentials were extracted from the current-voltage relationship for the open channel obtained in the presence of an osmotic gradient. The osmotic gradient was established by adding glucose to one side of the membrane. At 300 mM KCl, the average streaming potential was 0.72 mV/osmol per kg for t-tubule channels and 0.83 mV/osmol per kg for smooth muscle channels. Streaming potential values depend on KCl concentration, they decrease as KCl concentration increases, and the value obtained by extrapolation to zero KCl concentration is 0.85 mV/osmol per kg. Assuming that water and ions cannot pass each other, at least in a region of the channel, the streaming potential values obtained indicate that this region contains a minimum of two and a maximum of four water molecules. It is concluded that the channel has a narrow region with a length of 0.6-1.2 nm.

Animals↗
PubMed1988

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: individual differences in the number of channels.

We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K+ (Ca-K) channel of human red cells (RBC). Cytosolic Ca2+ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na+ media containing CaCl2. The Ca-K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8 +/- 2.4 mmol/liter cell.hr, to 3.7 +/- 1.0 mmol/liter cell.hr by 6 nM CTX (n = 3). The kinetic of Ca-K efflux was studied by increasing cell ionized Ca2+ using A23187 (60 mumol/liter cell), and buffering with EGTA or citrate; initial rates of net K+ efflux (90 mmol/liter cell K+) into Na+ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca-K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca2+ was raised, with a Km of 0.37 microM and saturating between 2 and 10 microM Ca2+. Ca-K efflux was partially blocked (71 +/- 7.8%, mean +/- SD, n = 17) by CTX with high affinity (IC50 0.8 nM), a finding suggesting that is a high affinity ligand of Ca-K channels. CTX also blocked 72% of the Ca-activated K+ efflux into 75 mM K+ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K+ channel in Na+ media. CTX did not block Valinomycin-activated K+ efflux into Na+ or K+ medium and therefore it does not inhibit K+ movement coupled to anion conductive permeability. The Vmax, but not the Km-Ca of Ca-K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell.min (FU). In red cells with Hb A, Vmax was 9.36 +/- 3.0 FU (mean +/- SD, n = 17). The Vmax of the CTX-sensitive, Ca-K efflux was 6.27 +/- 2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75 +/- 3.2 FU, n = 8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of the Vmax of Ca-K+ efflux. Estimation of the number of CTX-sensitive Ca-activated K+ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K+ efflux (2.7 +/- 0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.

Calcium↗
PubMed1987

Mechanisms of Cs+ blockade in a Ca2+-activated K+ channel from smooth muscle.

Large unitary conductance Ca2+-activated K+ channels from smooth muscle membrane were incorporated into phospholipid planar bilayers, and the blockade induced by internally and externally applied Cs+ was characterized. Internal Cs+ blockade is voltage dependent and can be explained on the basis of a Cs+ binding to a site that senses 54% of the applied voltage, with an apparent dissociation constant, Kd(0), of 70 mM. On the other hand, external Cs+ blocks the channel in micromolar amounts, and the voltage dependence of blockade is a function of Cs+ concentration. The fractional electrical distance can be as large as 1.4 at 10 mM Cs+. This last result suggests that the channel behaves as a multi-ion pore. At large negative voltages the I-V relationships in the presence of external Cs+ show an upturn, indicating relief of Cs+ block. External Cs+ blockade is relieved by increasing the internal K+ concentration, but can be enhanced by increasing the external K+. All the characteristics of external Cs+ block can be explained by a model that incorporates a "knock-on" of Cs+ by K+.

Animals↗
PubMed1986

Characterization of a calcium-activated potassium channel from rabbit intestinal smooth muscle incorporated into planar bilayers.

Interaction of vesicles from a microsomal fraction of rabbit intestinal smooth muscle with planar bilayers promotes the incorporation of a large conductance potassium-selective channel. The channel conductance fluctuates between two states: closed and open and the fraction of time the channel dwells in the open state is a function of the electric potential difference and the calcium concentrations. This channel seems to correspond to a Ca-activated K channel described by other authors in smooth muscle cells with the patch-clamp technique. Single-channel conductance is a saturating function of the potassium concentration. The relationship between conductance and concentration cannot be described by a hyperbolic function, suggesting multiple occupancy of the channel. The single-channel conductance is 230 pS in symmetrical 0.1 M KCl. Current is a linear function of the applied voltage in the range between -100 and +100 mV, at concentrations of 0.1 M KCl or higher. At lower concentrations, current-to-voltage curves bend symmetrically to the voltage axis. Sodium, lithium and cesium ions do not pass through the channel and the permeability for Rb is 66% that of potassium. All these alkali cations and Ca2+ block the channel in a voltage-dependent manner. A two-site three-barrier model on Eyring absolute reaction rate theory can account for the conduction and blocking characteristics.

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↗
PubMed1982

Probing the pore size of the hemocyanin channel.

We have studied single-channel conductance for different monovalent cations and streaming potentials caused by osmotic gradients of non-electrolytes in hemocyanin-treated membranes. We have found that the smaller ion, which cannot pass through the channel, is tetramethylammonium and that acetamide is the smaller non-electrolyte excluded from the pore. From the streaming potentials measured, we calculated that no more than three water molecules can accompany the ion through the channel in a row. From these results we conclude that the hemocyanin channel has in its structure a narrow portion which can be represented as a cylinder 6 A long and 5 A in diameter.

Animals↗
PubMed1981

A three-barrier model for the hemocyanin channel.

Keyhole limpet hemocyanin forms ion-conducting channels in planar lipid bilayer membranes. Ionic current through the open hemocyanin channel presents the following characteristics: (a) it is carried mainly by cations; (b) it is a nonlinear function of membrane potential; (c) channel conductance is a saturating function of ion activity; (d) it shows ionic competition. A model for the open hemocyanin channel is developed from absolute reaction rate theory. The model calls for three energy barriers in the channel. Two energy barriers represent the entrance and exit of the ion into and out of the channel. The third barrier separates two energy minima that represent two binding sites. Furthermore, only one ion is allowed inside the channel at a given time. This model is able to recreate all the hemocyanin characteristics found experimentally in negatively charged and neutral membranes.

Hemocyanins↗