Biophysics of Large Membrane Channels

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

I. Regulation of mitochondrial respiration by membrane-bound peripheral proteins with disordered polyanionic C-terminal domains The crucial role of mitochondrial outer membrane (MOM) permeability in maintaining an efficient metabolite exchange between mitochondria and cytoplasm in normal respiration is well-established, with the voltage-dependent anion channel (VDAC) recognized as the key metabolite pathway and regulator in MOM. It is believed that the uniqueness of this relatively simple monomeric beta-barrel channel mainly arises from its crucial position at the interface between a mitochondrion and the cytosol. When reconstituted into lipid membranes, VDAC responds to sufficiently large transmembrane potentials by transitioning to gated states in which ATP/ADP flux is reduced and calcium flux is increased. However, a major reason for uncertainty regarding the physiological role of the VDAC voltage gating for the regulation of MOM permeability is the source and magnitude of the outer membrane potential in vivo. We found that two otherwise unrelated cytosolic proteins, tubulin, and alpha-synuclein (a-Syn), dock with VDAC by a newly established mechanism in which the transmembrane potential draws their disordered, polyanionic C-terminal domains into and through the VDAC channel, thus physically blocking the pore. Remarkably, for both tubulin and a-Syn, the blocked states are observed at much lower transmembrane potentials than VDAC gated states with their bulk concentrations as small as 10 nanomoles. Therefore, in the presence of these cytosolic docking proteins VDACs sensitivity to transmembrane potential is dramatically increased. The features of the VDAC voltage-gated states relevant for bioenergeticsreduced metabolite flux and increased calcium fluxare preserved in the blocked states induced by either docking protein. The ability of tubulin and a-Syn to modulate mitochondrial potential and ATP production in vivo is now supported by many studies. The common physical origin of the interactions of both tubulin and a-Syn with VDAC leads to a general model of a VDAC inhibitor, facilitates predictions of the effect of post-translational modifications of known inhibitors, and, because of its generality, points the way toward the development of novel therapeutics targeting VDAC. II. Trapping of single diffusing particles by a circular disk on a reflecting flat surface Recent progress in cell biophysics (for example, in studies of chemical sensing and spatiotemporal cell signaling) poses new challenges to the statistical theory of trapping of single diffusing particles. We offered an analytical solution to one of them, namely, the trapping kinetics of single particles diffusing in a half-space bounded by a reflecting flat surface containing an absorbing circular disk. Our analysis is quite general and is thus relevant for various applications involving not only signal transduction problems in biology but operation of different kinds of chemical sensors. Signaling within and between cells is the key phenomenon underlying the development and functional behavior of cells and multicellular organisms. The so-called spatiotemporal dynamic cell-signaling modeling focuses on signaling processes both in individual cells and the spatial coupling between different cells within a tissue. As follows from the name, the efficiency and characteristic duration of signal transduction between cells are studied as functions of their relative spatial location. Researchers point out that the response strength could depend on a vector distance between the signal origin and the receptor, rather than a scalar distance alone, thus exhibiting directional behavior. This trapping problem is essentially two dimensional and the question of the angular dependence of the kinetics on the particle starting point is highly nontrivial. We proposed an approximate approach to the problem that replaces the absorbing disk with an absorbing hemisphere of a properly chosen radius. This replacement makes the problem angular independent and essentially one-dimensional. After the replacement, one can find an exact solution for the particle propagator (Greens function) that allows one to completely characterize the kinetics. Extensive testing of the theoretical predictions based on the absorbing hemisphere approximation against three-dimensional Brownian dynamics simulations shows excellent agreement between the analytical and simulation results when the particle starts sufficiently far away from the disk. It works well for the distances of the particle starting point from the disk center comparable with the disk diameter or larger. This condition reflects actual situations in multiple signaling pathways at the cell and organism levels. III. Analytical theory for the permeability and diffusion resistance of porous membranes and its numerical test Channels of cell and organelle membranes are often clustered, forming domains of tightly packed transport proteins. A well-known example is the voltage-dependent anion channel, VDAC, the major gateway for metabolites and ions in the outer mitochondrial membrane. Indeed, electron micrographs of MOM fragments from Neurospora crassa and high-resolution atomic force imaging of MOM fragments from Saccharomyces Cerevisiae show that, in both cases, VDAC beta-barrels form dense arrays of hundreds of channels. The characteristic distances between barrel centers are as small as 4 to 5 nm, which are comparable to the barrel outer diameter of about 3 nm. Estimates demonstrate that in these high-density domains, VDAC protein occupies about 80% of the membrane surface. Previously we considered the effect of clustering on particle trapping by absorbing disks and channel-facilitated transport, assuming that the size of the cluster is much smaller than the total membrane area. This year, we addressed a different problem. Specifically, we studied the case when the channels are spread over the entire membrane, with a focus on the effects of channel crowding on membrane transport. We considered the transport of neutral solutes through porous flat membranes, driven by the solute concentration difference in the reservoirs separated by the membrane. Transport occurs through membrane channels, which are assumed to be non-overlapping, identical, straight cylindrical pores connecting the reservoirs. The key quantities characterizing transport are membrane permeability and diffusion resistance. Such transport problems, arising in very different contexts ranging from plant physiology and cell biology to chemical engineering, have been studied for more than a century. Nevertheless, an expression giving the permeability for a membrane of arbitrary thickness at arbitrary surface densities of the channel openings has been unknown. Now we filled this gap and derived such an expression. Since this expression involves approximations, we compared its predictions with the results obtained from Brownian dynamics simulations and found excellent agreement between the two.