Biophysics of Large Membrane Channels

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

I. Voltage-activated complexation of alpha-synuclein with beta-barrel channels and its inhibition as a potential therapeutic target for Parkinsons disease treatment Voltage-activated protein complexation is the process by which a transmembrane potential drives complex formation between a membrane-embedded channel and a soluble or membrane-peripheral target protein. Metabolite and calcium flux across the mitochondrial outer membrane was shown to be regulated by voltage-activated complexation of the voltage-dependent anion channel (VDAC) and either dimeric tubulin or alpha-synuclein (aSyn). However, the roles played by VDACs characteristic attributes its anion selectivity and voltage gating behavior have remained unclear. During this reporting period we have conducted a comparative analysis of in vitro measurements of voltage-activated complexation of aSyn with three well-characterized beta-barrel channels VDAC, MspA, and alpha-Hemolysin that differ widely in their organism of origin, structure, geometry, charge density distribution, and voltage gating behavior. The voltage dependences of the complexation dynamics for the different channels were observed to differ quantitatively but have similar qualitative features. In each case, energy landscape modeling describes the complexation dynamics in a manner consistent with the known properties of the individual channels, while voltage gating does not appear to play any role. The reaction free energy landscapes thus calculated reveal a common physical mechanism of complexation for all three channels, together with a non-trivial dependence of the complex stability on the surface density of aSyn. It is well-recognized that involvement of aSyn in Parkinsons disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that aSyn can regulate mitochondrial function by voltage-activated complexation with VDAC described above. When complexed with aSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites though the mitochondrial outer membrane. Further, aSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of aSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. This year we studied an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on aSyn membrane binding, and hence on aSyn complex formation with VDAC and translocation through it. In electrophysiology experiments, the addition of HK2p at micromolar concentrations to the same side of the membrane as aSyn results in a dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of aSyn from lipid membranes. Experiments with HeLa cells using proximity ligation assay confirmed that HK2p impedes aSyn entry into mitochondria. Our results demonstrate that it is possible to regulate aSynVDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate aSyn mitochondrial toxicity in PD and other synucleinopathies. II. The single residue K12 governs the exceptional voltage sensitivity of VDAC gating VDAC is the most abundant protein in the mitochondrial outer membrane (MOM) and is the primary conduit for ions and water-soluble metabolites such as ATP and ADP to cross the MOM. As such, VDAC plays a central role in the regulation of MOM permeability and mitochondrial metabolism, and in communication between mitochondria and the rest of the cell. VDAC responds to a transmembrane potential by gating, i.e. transitioning to one of a variety of low-conducting states of unknown structure. The gated state results in nearly complete suppression of multivalent mitochondrial metabolite (such as ATP and ADP) transport while enhancing calcium transport. Voltage gating is a common property of beta-barrel channels and has been observed in bacterial outer membrane porins as well as in anthrax, aerolysin, and alpha-Hemolysin toxins, but VDAC gating is anomalously sensitive to transmembrane potential. This reporting period we have shown that a single residue in the pore interior, K12, is responsible for most of VDACs voltage sensitivity. Using the analysis of over 40 microseconds of atomistic molecular dynamics (MD) simulations, we explored correlations between motions of charged residues inside the VDAC pore and geometric deformations of the beta-barrel. Residue K12 is bistable; its motions between two widely separated positions along the pore axis enhance the fluctuations of the beta-barrel and augment the likelihood of gating. Single channel electrophysiology of various K12 mutants reveals a dramatic reduction of the voltage-induced gating transitions. The crystal structure of the K12E mutant at a resolution of 2.6 indicates a similar architecture of the K12E mutant to the wild type; however, 60 microseconds of atomistic MD simulations using the K12E mutant showed restricted motion of residue 12, due to enhanced connectivity with neighboring residues, and diminished amplitude of barrel motions. We thus conclude that beta-barrel fluctuations, governed particularly by residue K12, drive VDAC gating transitions. III. Intrinsic diffusion resistance of a membrane channel, mean first-passage times between its ends, and equilibrium unidirectional fluxes Diffusion resistance is an important characteristic of channel-facilitated membrane transport that is widely used in chemical engineering, electrochemistry, and cell biophysics. It is a diffusion analog of the electrical resistance, relating the steady-state diffusive flux of solute molecules through a membrane channel with the driving force of the transport process, the solute concentration difference in the two reservoirs separated by the membrane. This reporting period we derived analytical expressions for the diffusion resistance in the case of a cylindrically symmetric blocker whose axis coincides with the axis of a cylindrical nanopore in two limiting cases where the blocker radius changes either smoothly or abruptly. Comparison of our theoretical predictions with the results obtained from Brownian dynamics simulations shows good agreement between the two. We have also established a general relation between the channel diffusion resistance and the mean first-passage times of the solute molecules between the channel openings. Specifically, we have shown that this direction-independent characteristic of transport is equal to the sum of the direction-dependent mean first-passage times, divided by the molecule partition function in the channel. Our analysis is based on the consideration of the equilibrium unidirectional fluxes flowing through the channel in opposite directions. The approach is quite general in the sense that it does not appeal to any specific model of the channel and, therefore, is universally applicable to transport in channels of arbitrary shape and tortuosity, at arbitrary interaction strength of solute molecules with the channel walls. This result promises to be of great value in computing the intrinsic diffusion resistance of the channel numerically, as it allows researchers to avoid dealing with multiple problems in analyzing transport under non-equilibrium conditions.