Modulation of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica by divalent cations, protons and H2O2

AbstractElectrical properties of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica were analyzed after incorporation of BCC1 into black lipid bilayers. Single channel current fluctuations were modulated by divalent cations, protons and H2O2. Whereas the channel is permeable for Ca2+, Ba2+ and Sr2+, its conductance is strongly reduced in solutions containing MgCl2. Cu2+ and Zn2+ are potent inhibitors of BCC1 in micromolar concentrations. The open channel conductance of BCC1 increases with acidification of the electrolyte solution. H2O2 shows strong inhibitory effects on BCC1. The channel is almost completely closed at submillimolar concentrations of H2O2. The effects of pH and H2O2 on channel properties are directional and affect BCC1 at the Ca exit side, but not on the entry site. Thus, cytosolic pH and H2O2 levels may play an important role in the modulation of the cytoplasmic free calcium concentration through BCC1

Alamethicin induced single channel conductance fluctuations in biological membranes

Alamethicin, a polypeptide of molecular weight ∼2,000, induces voltage-dependent conductance phenomena in artificial lipid bilayer membranes that are in some ways similar to those found in excitable membranes1. Alamethicin-indueed conductance is probably a consequence of the formation of predominantly cation-selective channels which span the bilayer, and single open channels fluctuate at random between several well defined conductance states2–5. Statistical analysis of single channel conductance fluctuations in planar lipid bilayers of various compositions has revealed that, whereas the conductance values are relatively constant in a variety of membranes, the average lifetime of the individual conductance states depends strongly on the lipid used to form the bilayer. This variability has tentatively been attributed to differences in lipid fluidity6,7. Alamethicin is therefore a probe of some aspects of membrane composition. The study of single alamethicin channels in various natural membranes would allow comparison with the well defined bilayer systems and might yield information on lipid properties of natural membranes that could be important for the functioning of natural membrane channels and receptors. The experiments reported here show that alamethicin channels have similar conductances in the sarcolemmal (surface) membrane of frog and rat muscle and in lipid bilayers formed from synthetic lecithin. The average lifetime of the conductance states is, however, one order of magnitude larger in the biological membranes

Agonist activated ionic channels in acetylcholine receptor reconstituted into planar lipid bilayer

Planar lipid bilayers were formed with the mixed chain phospholipid 1-stearoyl-3-myristolglycero-2-phosphocholine. Acetylcholine receptor membrane fragments or the purified receptor protein was incorporated into these bilayers by fusing receptor-containing vesicles with the planar membranes a few degrees below the lipid phase transition temperature. Single-channel currents activated by nicotinic agonists in the reconstituted system resembled those observed in intact rat and frog muscle membrane as measured by the patch clamp technique. The observed channel characteristics did not depend on the degree of receptor purification. Thus, the receptor-enriched fragments and those depleted of nonreceptor peripheral peptides, the purified receptor monomer/dimer mixtures, and the isolated receptor monomer as defined by gel electrophoresis all shared similar electrochemical properties in the synthetic lipid bilayer. The agonist-activated ionic channel seems, therefore, to be contained within the receptor monomer