Membrane biochemistry最新文献

Inactivation of single Ca channels in snail neurons was examined to test the idea that entering Ca ions react directly with the channel to produce this effect. Simulations of specific models were used for comparison with the experimental data. The Ca-dependent model predicts time-dependent changes in the single-channel events which were not found experimentally. It is possible that Ca-dependent inactivation is mediated by a Ca-binding protein that is associated with but not part of the channel itself.

The sterol content of human erythrocyte membranes was modified by polyvinylpyrrolidone (PVP)-mediated enrichment or depletion of cholesterol (CHL) or incorporation of cholesteryl hemisuccinate (CHS). The effects of these modifications on osmotic fragility and anion exchange protein (AEP) disposition and function were evaluated. CHS enrichment was fast (1 hr, 37 degrees C) and led to a concentration-dependent crenation as well as a decrease in osmotic cell fragility, in parallel with increased membrane microviscosity. CHL caused similar but considerably less marked effects due to slower incorporation rates into membranes. CHS enrichment of cells induced susceptibility of AEP to trypsin, a protease which otherwise does not affect AEP in intact cells. Although transport rates of monosaccharides, nucleosides, and anions were markedly slowed down by CHS enrichment of cells in parallel with increased membrane viscosity, anion transport was the most affected. The temperature profile of anion transport in CHS-enriched cells revealed a 10-kcal/mol increase in the enthalpy of activation relative to normal cells. Anion transport measured in heteroexchange conditions (Cl in--pyruvate out) and (Cl in-sulfate out) was relatively more susceptible to CHS modification than when it was measured in homoexchange conditions (Cl in-Cl out). The results of these measurements indicate that CHS-mediated increase in membrane viscosity affects AEP translocation capacity and transmembrane disposition via changes in lipid compressibility. Specific effects of CHS on AEP function, however, could not be ruled out.

Notexin belongs to a class of snake venom neurotoxins and myotoxins that have phospholipase A2 activity. Previous studies have shown that these toxins affect target cells differently from phospholipases that are not neurotoxic or myotoxic. Notexin inhibited the Ca2+ uptake into fragmented sarcoplasmic reticulum from rabbit skeletal muscle, but it did not cause an efflux of previously accumulated Ca2+ or inhibit the Ca2+--ATPase activity. It is suggested that notexin specifically binds to and decreases the conductance for Ca2+ of the Ca2+ pump and/or the conductance of a channel for an ion that facilitates Ca2+ transport. The K+ ionophore valinomycin reversed the notexin-induced inhibition of Ca2+ uptake into sarcoplasmic reticulum, suggesting that the molecular target of notexin could be a K+ channel. Two types of reconstitution experiments make it unlikely that notexin acts by degrading a minor lipid that is resistant to hydrolysis by nontoxic phospholipases A2. Notexin-inactivated sarcoplasmic reticulum vesicles were reactivated (with respect to Ca2+ uptake) by simple solubilization with detergent and subsequent reconstitution by detergent removal. Second, notexin was still active on sarcoplasmic reticulum vesicles after greater than 94% of the lipids were replaced by soybean phosphoglycerides during the reconstitution procedure.

Changes in the charge of sarcoplasmic reticulum (SR) vesicles are studied using lipophilic ions, which are adsorbed by the membrane phase. Upon addition of MgATP, phenyldicarbaundecaborane (PCB-) and tetraphenylboron (TPB-) are taken up by the SR vesicles, while tetraphenylphosphonium (TPP+) is released into the water phase. The PCB- uptake occurs as well under conditions when SR membrane is shunted by high Cl- concentration. MgATP induces minor additional binding of PCB- in the presence of oxalate and it is followed by release of the lipophilic anion from the vesicles. EGTA partly reverses the ATP effect, and calcium ionophore A23187 plus EGTA reverses it completely. Vesicles that were preliminarily loaded by Ca2+ demonstrated higher passive and lower ATP-dependent PCB- binding. Activation of isolated Ca2+-ATPase in the presence of 0.1 mM EGTA results in PCB- release into the medium and additional TPP+ binding to the enzyme. We suggest that the redistribution of the lipophilic ions between the water phase and SR membrane reflects charge changes in Ca2+-binding sites inside both SR vesicles and Ca2+-ATPase molecules in the course of Ca2+ translocation.

Freshly dissociated cells from the stomach muscularis of the toad Bufo marinus have been employed to carry out a systematic set of electrophysiological studies on the membrane properties of smooth muscle. The existence of Ca2+-activated K+ channels became apparent during the first studies under current clamp. In subsequent studies under voltage clamp, a Ca2+-activated. TEA-sensitive outward current was evident, and it was more than an order of magnitude larger than any other current observed in the cells. The channel responsible, at least in part, for this large outward current has been identified on the basis of single-channel records, and some of its main characteristics have been studied. It is similar in many respects to the large-conductance, Ca2+-activated K+ channel seen in other preparations. This channel has now been found in a considerable diversity of smooth muscle types.

The effects of various pharmacological agents that block single batrachotoxin-activated Na channels from rat muscle can be described in terms of three modes of action that correspond to at least three different binding sites. Guanidinium toxins such as tetrodotoxin, saxitoxin, and a novel polypeptide, mu-conotoxin GIIIA, act only from the extra-cellular side and induce discrete blocked states that correspond to residence times of individual toxin molecules. Such toxins apparently do not deeply penetrate the channel pore since the voltage dependence of block is insensitive to toxin charge and block is not relieved by internal Na+. Many nonspecific organic cations, including charged anesthetics, exhibit a voltage-dependent block that is enhanced by depolarization when present on the inside of the channel. This site is probably within the pore, but binding to this site is weak, as indicated by fast blockade that often appears as lowered channel conductance. A separate class of neutral and tertiary amine anesthetics such as benzocaine and procaine induce discrete closed states when added to either side of the membrane. This blocking effect can be explained by preferential binding to closed states of the channel and appears to be due to a modulation of channel gating.

We report here the first evidence in intact epithelial cells of unit conductance events from amiloride-sensitive Na+ channels. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). Two types of channels were observed: one with a high selectivity to Na+ and one with relatively low selectivity. The characteristics of the low-selectivity channel are as follows: single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette) and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed times as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches. The characteristics of the high-selectivity channel are: a single-channel conductance of 1-3 pS (mean = 2.8 +/- 1.2), the current-voltage relationship is markedly nonlinear with a Na+/K+ selectivity greater than 20:1. The mean open and closed times for the two types of channels are quite different, the high-selectivity channel being open only about 10% of the time while the low-selectivity channel is open about 30% of the time.

Delipidation of beef heart electron transport particles with phospholipase A2 has been examined. When the particles were treated with the lipase and subjected to a low bovine serum albumin wash, ATPase activity was unaffected as was the lipid/protein ratio of the particles. However, energisation by ATP/Mg2+ was abolished. Furthermore, unsaturated but not saturated fatty acids discharged the steady-state ATP-driven membrane potential of control samples. When the phospholipase A2 hydrolysis products were removed, inhibition of energy-linked reactions in the lipid-depleted particles was still observed and was interpreted in terms of non-specific leaks in the vesicle membranes, and 'specific' leaks through impaired H+-ATPase complexes. ATPase activity was less susceptible to delipidation than energisation but was, nevertheless, strongly inhibited at 50 percent lipid depletion. Spin label studies indicated a decrease in the fluidity of particle membranes accompanying delipidation. Moreover, the discontinuity seen in Arrhenius plots of ATPase activity was shifted from 17 degrees C (control) to 22 degrees C at 50 percent phospholipid depletion. The data are consistent with a release of unsaturated fatty acids by phospholipase A2 rendering the transport particles both leakier and the membranes less fluid than controls.