We investigate the physical principles of channel-facilitated transport of metabolites and other large solutes across cell and organelle membranes. To study channels under precisely controlled conditions, we reconstitute channel-forming proteins into planar lipid bilayers. Using this strategy, we elucidate the molecular mechanisms responsible for metabolite flux regulation under normal conditions and in pathology. I. Apoptosis: VDAC Regulation by Bcl-2 Family of Proteins. Mitochondria?s crucial role in the initiation of apoptosis is well established. Triggered by a number of different stimuli, the mitochondrial outer membrane (MOM) becomes permeable to apoptogenic factors such as cytochrome c. The Bcl-2 family proteins regulate the permeabilization of MOM: pro-apoptotic proteins such as Bax and Bid induce the release of apoptogenic factors, whereas anti-apoptotic proteins such as Bcl-xL prevent their release. There are two working models that associate these proteins with the existing major channel in the MOM, VDAC. Responsible for most of the metabolite flux across MOM, this channel is a very attractive candidate for a pathway for cytochrome c release. In the first model, the pro-apoptotic protein, Bax, induces formation of the large VDAC-based channels permeable to cytochrome c. In the second model it is the closure of VDAC channels that leads to MOM rupture. Therefore, though in both models the ability of Bcl-2 proteins to regulate the state and integrity of VDAC explains their function as either anti- or pro-apoptotic agents, the proposed mechanisms of VDAC channel regulation are diametrically opposite. To address this question we studied the effect of these proteins on the properties of VDAC channels reconstituted into the planar phospholipid membranes. First, we have demonstrated that, contrarily to general belief, there is no functionally significant interaction between VDAC channels and pro-apoptotic protein Bax in either monomeric or oligomeric forms. A detailed analysis of the characteristic properties of VDAC channels such as voltage gating, ion selectivity, single channel conductance, and water-soluble polymer exclusion did not show any change after Bax addition regardless of lipid composition, medium pH, or ionic content. However, we have found that another pro-apoptotic protein, tBid, affects the voltage-gating properties of VDAC by inducing channel closure. We have shown here that tBid induces closure of VDAC channels, both on single and multichannel membranes, in a dose-dependent manner. By decreasing the probability of VDAC opening, tBid would reduce the flux of adenine nucleotides and other negatively charged metabolites across the MOM, which may affect mitochondrial functions in different ways. One of the possible consequences of VDAC channel closure is the disruption of metabolite exchange across the MOM and the resultant accumulation of metabolites in the intermembrane space followed by mitochondria swelling and consequent loss of MOM integrity. The mechanism by which tBid alters the gating properties of VDAC remains to be understood. II. Water-Soluble Polymers as Molecular Probes. Polymer partitioning into nanoscale cavities is crucially important in many areas of science and technology. During the past year we have extended our studies in this direction to address the problem of polymer solution non-ideality. Thermodynamics of polymer partitioning in the regime of advanced solution non-ideality (de Cloizeaux regime) was studied with the alpha-Hemolysin channel. Using the change in conductance of a nanometer-wide protein pore of this channel to detect pore occupancy by polymers, we measured the equilibrium partitioning of differently sized linear poly(ethylene glycol)s (PEGs) as a function of polymer concentration in the bulk solution. In the semidilute regime, increased polymer concentration resulted in a sharp increase in polymer partitioning. Quantifying solution non-ideality by osmotic pressure and taking the free energy of polymer confinement by the pore at infinite dilution as an adjustable parameter allowed us to describe polymer partitioning only at low polymer concentrations. At larger concentrations the increase in partitioning is much sharper than the model predictions. The nature of this sharp transition between strong exclusion and strong partitioning might be rationalized within the concepts of scaling theory predicting this kind of behavior whenever the correlation length of the monomer density in the semidilute bulk solution becomes smaller than the pore radius. Specific attractive interactions between the protein pore and the polymer that exist in addition to the entropic repulsion accounted for in the present study may also play a role. III. Electrostatics in Ion Transport through Large Channels. Although the crystallographic structure of the bacterial porin OmpF has been known for a decade, the physical mechanisms of its ionic selectivity are still under investigation. We address this issue in a series of experiments with varied pH, salt concentrations, inverted salt gradient, and charged and uncharged lipids. Measuring reversal potential, we show that OmpF selectivity (traditionally regarded as slightly cationic) depends strongly on pH and salt concentration and is conditionally asymmetric, that is, sensitive to the direction of salt concentration gradient. At neutral pH and sub-decimolar salt concentrations the channel exhibits nearly ideal cation selectivity. Substituting neutral DPhPC with DPhPS, we demonstrate that the fixed charge of the host lipid has a small but measurable effect on the channel reversal potential. The available structural information allows for a qualitative explanation of our experimental findings. These findings now lead us to re-examine the ionization state of 102 titratable sites lining the OmpF channel. Using standard methods of continuum electrostatics tailored to our particular purpose, we find the charge distribution in the channel as a function of solution acidity and relate the pH-dependent asymmetry in channel selectivity to the pH-dependent asymmetry in charge distribution. In an attempt to find a simple phenomenological description of our results, we also discuss different macroscopic models of electrodiffusion through large channels.

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Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Khattab, Ahmed; Yuen, Tony; Al-Malki, Sultan et al. (2016) A rare CYP21A2 mutation in a congenital adrenal hyperplasia kindred displaying genotype-phenotype nonconcordance. Ann N Y Acad Sci 1364:5-10
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