Large ion channels are key structural elements of metabolite exchange between different cellular compartments and between cells. To study these channels under precisely controlled conditions, we reconstitute channel-forming proteins into planar lipid bilayers. Our goal is to investigate the physical principles of channel-facilitated transport of metabolites and other large solutes across cell and organelle membranes. I. Apoptosis. Research on VDAC, the major channel from mitochondria outer membrane (MOM), has accelerated as evidence grows of its importance in mitochondrial function and in apoptosis. This small, ancient, highly-conserved protein appears to be involved in many cellular processes. The past year?s effort was to shed light on its role in apoptosis and to separate reliable information from more questionable claims. VDAC channels can exist in a variety of functional states that differ in their ability to pass non-electrolytes and conduct ions. The permeability of VDAC to small ions includes Ca. It is well established fact that cytosolic Ca triggers opening of the permeability transition pore (PTP) in the mitochondria inner membrane, which consequently allows passage of water and solutes up to approximately 1.5 kDa. Opening of PTP is one of the mechanisms responsible for MOM permeabilization, cytochrome c release, and, consequently, the apoptotic cell death. VDAC is thought to be one of the major components of the PTP. If VDAC is part of PTP, then it seems logical that closure of VDAC would close the PTP and protect mitochondria from cytosolic PTP activators, such as Ca ions. In experiments with VDAC channel reconstituted unto the planar lipid membranes we have shown that Ca permeates through both the open and ?closed? states of VDAC. The double positive charge does not exclude Ca cations from the open state because the anion selectivity is moderate. The closed state favors cations. The presence or absence of Ca does not change the conductance of the open state of VDAC. In 1 M NaCl the conductance is 3.3 +/- 0.3 nS in the presence of 0.1 mM EGTA and 3.4 +/- 0.1 nS in the presence of 1 mM CaCl2. Moreover, we have also shown that the voltage gating is unaffected by Ca presence. Closure of VDAC does not prevent Ca flux and the closed state of VDAC has a similar molecular size cut-off as PTP. Our conclusion is that the closure of VDAC cannot protect against PTP opening. Thus, Ca cannot regulate PTP by opening or closing VDAC. The notion of a supramolecular PTP complex is fashionable but seems unnecessary considering the large number of VDAC channels in the outer membrane and the higher permeability of VDAC than that measured for the PTP. Correspondingly, the influx of metabolites that leads to the swelling of the matrix must flow through VDAC whether the supramolecular complex exists or not. These data and the analysis of the results obtained by other groups confirm our previously suggested model wherein closure of VDAC, not VDAC opening, leads to MOM permeabilization and apoptosis. II. Water-Soluble Polymers as Molecular Probes. The past year?s progress in quantitative understanding of polymer probing of ion channels resulted in a theoretical model explaining the observed polymer concentration dependence of the partition coefficient. We achieved this by recognizing that non-ideality of polymer solution in the pore is weaker than in the bulk because the overlap volume fraction of the polymer in the pore is higher than that in the bulk. The reason is that polymer molecules in the pore form cigars with high intra-molecular monomer density. Therefore, the observed concentration dependence of the partition coefficient cannot be explained using the standard approach that assumes non-ideality of the polymer solution in the pore to be identical to non-ideality in the bathing solution: measured partitioning is a much sharper function of polymer concentration than identical non-ideality predicts. In a separate study, we also analyzed the data on the electrical conductivity and viscosity of aqueous solutions of polyethylene glycol in order to use them as the reference information in studying channels. Over wide ranges of concentration and polymer molecular weight, conductivity is independent of the molecular weight for long chains and weakly dependent on the molecular weight for short chains. The processes responsible for a decrease in the mobility of ions were qualitatively analyzed to explain the weak conductivity sensitivity to the length of polymer chains. It was shown that experiments can be interpreted using the microviscosity concept. Microviscosity increases with the addition of a polymer much less rapidly than usual (macroscopic) viscosity. A simple empirical formula describing the dependence of conductivity on the polymer concentration was suggested. III. Transport Properties of Toxin Channels. Understanding of the physical principles and structural aspects involved in large-channel permeability and selectivity is still far from being satisfactory, and our progress relies on the detailed knowledge of the transport phenomenology. This knowledge can be obtained in experiments with single channels reconstituted into planar bilayers by studying the effect of penetrating molecules on the ionic current through the channel. High-resolution conductance recording and appropriate statistical analyses allow one to quantitatively evaluate solute partitioning into, and dynamics within, the confines of ion channel aqueous pores. During the past year we studied how molecular topology of polyethylene glycol (PEG) influences its interaction with large channels. We found that closing linear PEG into a circular ?crown? molecule dramatically changes its dynamics in the alpha-Hemolysin channel. In the electrically neutral crown ether, six ethylene oxide monomers are linked into a circle that gives the molecule ion-complexing capacity and increases its rigidity. As with linear PEG, addition of the crown to the membrane-bathing solution decreases the ionic conductance of the channel and generates additional conductance noise. However, in contrast to linear PEG, both the conductance reduction (reporting on crown partitioning into the channel pore) and the noise (reporting on crown dynamics in the pore) now depend strongly and non-monotonically on the applied voltage. Within the whole frequency range accessible by channel reconstitution experiments, the noise power spectrum is ?white?, showing that crown exchange between the channel and the bulk solution is fast. Analyzing these data in the framework of a Markovian two-state model, we are able to characterize the process quantitatively. We show that the lifetime of the crown in the channel pore reaches its maximum (a few microseconds) at about the same voltage (approximately 100 mV, negative from the side of protein addition) where the crown?s reduction of the channel conductance is most pronounced. Our interpretation is that, because of its rigidity, the crown feels an effective steric barrier in the narrowest part of the channel pore. This barrier together with crown-ion complexing and resultant interaction with the applied field leads to behavior usually associated with voltage-dependent binding in the channel pore. Studies of crown ether effects on the conductance of ion channels are important because the crown ether superfamily has been shown to exhibit pharmacological effects.
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