The specific complexes of human alpha-lactalbumin (alpha-LA) with oleic acid (OA), HAMLET and LA-OA-17 (OA-complexes), possess cytotoxic activity against tumor cells but the mechanism of their cell penetration remains unclear. To explore the molecular mechanisms underlying interaction of the OA-complexes with the cell membrane, their interactions with small unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles and electroexcitable plasma membrane of internodal native and perfused cells of the green alga Chara corallina have been studied. The fractionation (Sephadex G-200) of mixtures of the OA-complexes with the vesicles shows that OA-binding increases the affinity of alpha-LA to DPPC vesicles. Calcium association decreases protein affinity to the vesicles; the effect being less pronounced for LA-OA-17. The voltage clamp technique studies show that LA-OA-17, HAMLET, and their constituents produce different modifying effects on the plasmalemmal ionic channels of the Chara corallina cells. The irreversible binding of OA-complexes to the plasmalemma is accompanied by changes in the activation-inactivation kinetics of developing integral transmembrane currents, suppression of the Ca(2+) current and Ca(2+)-activated Cl(-) current, and by increase in the nonspecific K(+) leakage currents. The latter reflects development of nonselective permeability of the plasma membrane. The HAMLET-induced effects on the plasmalemmal currents are less pronounced and potentiated by LA-OA-17. The control experiments with OA and intact alpha-LA show their qualitatively different and much less pronounced effects on the transmembrane ionic currents. Thus, the modification of alpha-LA by OA results in an increase in the protein association with the model lipid bilayer and in drastic irreversible changes in permeability of several types of the plasmalemmal ionic channels.
The voltage-clamp technique was used to study Ca(2+) and Cl(-) transient currents in the plasmalemma of tonoplast-free and intact Chara corallina cells. In tonoplast-free cells [perfused medium with ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] long-term inward and outward currents through Ca channels consisted of two components: with and without time-dependent inactivation. The voltage dependence of the Ca channel activation ratio was found to be sigmoid-shaped, with about -140-mV activation threshold, reaching a plateau at V>50 mV. As the voltage increased, the characteristic activation time decreased from approximately 10(3) ms in the threshold region to approximately 10 ms in the positive region. The positive pulse-activated channels can then be completely deactivated, which is recorded by the Ca(2+) tail currents, at below-threshold negative voltages with millisecond-range time constants. This tail current is used for fast and brief Ca(2+) injection into tonoplast-free and intact cells, to activate the chloride channels by Ca(2+) . When cells are perfused with EDTA-containing medium in the presence of excess Mg(2+), this method of injection allows the free submembrane Ca(2+) concentration, [Ca(2+)](c), to be raised rapidly to several tens of micromoles per liter. Then a chloride component is recorded in the inward tail current, with the amplitude proportional to [see text]. When Ca(2+) is thus injected into an intact cell, it induces an inward current in the voltage-clamped plasmalemma, having activation-inactivation kinetics qualitatively resembling that in EDTA-perfused cells, but a considerably higher amplitude and duration (approximately 10 A m(-2) and tau(inact)~0.5 s at -200 mV). Analysis of our data and theoretical considerations indicate that the [Ca(2+)](c) rise during cell excitation is caused mainly by Ca(2+) entry through plasmalemma Ca channels rather than by Ca(2+) release from intracellular stores.
We studied the influence of Bacillus cereus bacteria on cells of the freshwater alga Chara corallina. These bacteria and recombinant Bacillus subtilis strains are capable of producing the secreted toxin HlyII, which changes the electrophysiological parameters of the algal electrically excitable plasma membrane by forming pores. Cooperative incubation of bacterial cells, which carry active hlyII gene, and Chara corallina cells caused a decrease in the resting potential (V(m)) and plasma membrane resistance (R(m)) of algal cells. The efficiency of each strain was commensurable with its ability to produce HlyII. Purified hemolysin II caused a similar effect on V(m) and R(m) of intact and perfused cells. This protein changed the kinetics and magnitude of transient voltage-dependent calcium and calcium-activated chloride currents owing to the formation of additional Ca(2+)-permeable pores in algal cell membrane. Occurrence of the cellulose cell wall with pores 2.1 to 4.6nm in diameter suggests that HlyII molecules reach the plasma membrane surface strictly as monomers.
Effects of D2O were studied on internodal cells of the freshwater alga Nitellopsis obtusa under plasmalemma perfusion (tonoplast-free cells) with voltage clamp, and on Ca2+ channels isolated from the alga and reconstituted in bilayer lipid membranes (BLM). External application of artificial pond water (APW) with D2O as the solvent to the perfused plasmalemma preparation led to an abrupt drop of membrane resistance (Rm = 0.12 +/- 0.03 k omega.cm2), thus preventing further voltage clamping. APW with 25% D2O caused a two-step reduction of Rm: first, down to 2.0 +/- 0.8 k omega.cm2, and then further to 200 omega.cm2, in 2 min. It was shown that in the first stage, Ca2+ channels are activated, and then, Ca2+ ions entering through them activate the Cl- channels. The Ca2+ channels are activated irreversibly. If 100 mM CsCl was substituted for 200 mM sucrose (introduced for iso-osmoticity), no effect of D2O on Rm was observed. Intracellular H2O/D2O substitution also did not change Rm. In experiments on single Ca2+ channels in BLM H2O/D2O substitution in a solution containing 100 mM KCl (trans side) produced no effect on channel activity, while in 10 mM KCl, at negative voltage, the open channel probability sharply increased. This effect was irreversible. The single channel conductance was not altered after the H2O/D2O substitution. The discussion of the possible mechanism of D2O action on Ca2+ and Cl- channels was based on an osmotic-like stress effect and the phenomenon of higher D-bond energy compared to the H-bond.
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