Ximena Cecchi

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

SummaryWe 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 6nm CTX (n=3). The kinetic of Ca−K efflux was studied by increasing cell ionized Ca2+ using A23187 (60 μmol/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 aKm of 0.37 μm and saturating between 2 and 10 μm Ca2+. Ca−K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC500.8nm), a finding suggesting that is a high affinity ligand of Ca−K channels. CTX also blocked 72% of the Ca-activated K+ efflux into 75mm 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.TheVmax, but not theKm−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). TheVmax 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 theVmax 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.

Microcin E492 forms ion channels in phospholipid bilayer membranes

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+ = Li+ > Tris+. The results suggest that membrane potential dissipation induced by microcin is a consequence of the formation of pores in the bacterial membrane.

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.

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

SummaryInteraction 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 0.1m KCl. Current is a linear function of the applied voltage in the range between −100 and +100 mV, at concentrations of 0.1m 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.

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.

Alkali metal ion selectivity of the hemocyanin channel.

SummaryThe 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)>Rb+ (1.05)>K+ (1.0)>Cs+ (0.89)>Na+ (0.81)>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 threebarrier 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 Eisenmans selectivity sequences XI (cis peak, i.e., Li+>Na+>K+>Rb+>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 Eisenmans 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.

The molecular organization of nerve membranes: II. Glycolytic enzymes and ATP synthesis by plasma membranes of squid retinal axons

Abstract The following glycolytic enzyme activities were demonstrated in membrane fractions isolated from squid retinal nerve: phosphoglyceric kinase, pyruvic kinase, glyceraldehyde-3-phosphate dehydrogenase, aldolase, glucose-6-phosphate dehydrogenase, and hexokinase. After two additional washings with a hypotonic solution, only two of them, glucose-6-phosphate dehydrogenase and pyruvic kinase, were solubilized. The incubation of these membranes with ADP or GDP and P i resulted in the incorporation of P i into the diphosphate. The reaction product was identified as ATP or GTP. No esterification was obtained with CDP or UDP as P i acceptor. The enzyme system responsible for the synthesis of the nucleotide triphosphate was demonstrated to be membrane bound, and not due to soluble or mitochondrial contamination of the membrane fraction. The possible role of this system is discussed.

Characterization of Large-Unitary-Conductance Calcium-Activated Potassium Channels in Planar Lipid Bilayers

In this chapter we discuss the data obtained for a calcium-activated potassium channel of large unitary conductance. These channels are thought to be involved in the regulation of different cellular functions such as repetitive firing of neurons, hormonal secretion, potassium secretion in renal tubular cells, cyclic activity in smooth muscle, and others (Latorre et al., 1985).