Biophysics of Large Membrane Channels

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

I. Alpha-Synuclein emerges as a potent regulator of VDAC-facilitated calcium transport Voltage-dependent anion channel (VDAC) is the most ubiquitous channel at the mitochondrial outer membrane and is believed to be the pathway for calcium entering or leaving the mitochondria. Therefore, understanding the molecular mechanisms of how VDAC regulates calcium influx and efflux from the mitochondria is of particular interest for mitochondrial physiology. When the Parkinsons disease (PD) related neuronal protein, alpha-synuclein (aSyn), is introduced to the reconstituted VDAC, it reversibly and partially blocks VDAC conductance by its acidic C-terminal tail. Using single-molecule electrophysiology of reconstituted VDAC we now show that, at CaCl2 concentrations below 150 mM, aSyn reverses the channels selectivity from anionic to cationic. Importantly, we find that the decrease in channel conductance upon its blockage by aSyn is hugely overcompensated by a favorable change in the electrostatic environment for calcium, making the blocked state orders-of-magnitude more selective for calcium and thus increasing its net flux. Our findings with higher calcium concentrations also demonstrate that the phenomenon of charge inversion is taking place at the level of a single polypeptide chain. Measurements of ion selectivity of three VDAC isoforms in CaCl2 gradient show that VDAC3 exhibits the highest calcium permeability among them, followed by VDAC2 and VDAC1, thus pointing to isoform-dependent physiological function. Mutation of the E73 residue VDAC1 purported calcium binding site shows that there is no measurable effect of the mutation in either open or aSyn-blocked VDAC1 states. Our results confirm VDACs involvement in calcium signaling and reveal a new regulatory role of aSyn, with clear implications for both normal calcium signaling and PD-associated mitochondrial dysfunction. Highlighting the emerging evidence that cytosolic proteins interact with VDAC and regulate its calcium permeability, we advocate for continued investigations into the VDAC interactome at the contact sites between mitochondria and organelles and its role in mitochondrial calcium transport. II. Tunable electromechanical nanopore trap reveals populations of peripheral membrane protein binding conformations We demonstrate that VDAC can play a dual role, as a mitochondrial channel implicated in a plethora of pathologies including developmental, and as a nanopore, an informative sensor for membrane-catalyzed processes. In particular, in its latter role, VDAC can be used to electromechanically trap and interrogate proteins bound to a membrane surface at the single molecule level. Such electromechanical probing of aSyn reveals wide variation in the time required for individual proteins to unbind from the same membrane surface. Binding of aSyn to the lipid bilayer is a prerequisite of the channel-protein interaction; surprisingly, however, we find that the strength of aSyn binding to the membrane does not correlate in any simple way with its efficiency of blocking VDAC, suggesting that the lipid-dependent conformations of the membrane-bound aSyn control the interaction. The observed distributions of unbinding times span up to 3 orders of magnitude and depend strongly on the lipid composition of the membrane. It turned out that lipid membranes to which aSyn binds weakly are most likely to contain subpopulations in which electromechanically driven unbinding is very slow, the result that one would expect in the case of strong binding. Quantitative physical models of the free energy landscape governing the capture and release processes allow us to rationalize these counter-intuitive findings and discriminate between several aSyn (sub-) conformations on the membrane surface. These findings, combined with known structural features of aSyn on anionic lipid membranes, point to a picture in which the lipid composition determines the fraction of aSyn molecules for which the negatively charged C terminal domain is constrained to be close, but not tightly bound, to the membrane surface and thus readily captured by the VDAC nanopore. We speculate that changes in the mitochondrial membrane lipid composition may be among key regulators of the aSyn-VDAC interaction and consequently of VDAC-facilitated transport of ions and metabolites in and out of mitochondria and, i.e., of mitochondrial function. III. Capturing single molecules by nanopores: Measured times and thermodynamics Nanopores are successfully used in numerous applications, including measurements of chemical kinetics and DNA and protein sequencing. The source of information in nanopore sensing is analysis of transient interruptions in ion current through single nanopores induced by capturing solute molecules. Nanopore sensing belongs to a broader realm of single-molecule experiments, where researchers face challenges not encountered in bulk measurements. One of the issues is that they have to measure weak signals with high time resolution. Intrinsic noise of these measurements is a crucial factor that grossly limits experimental possibilities and has to be minimized. This minimization is often achieved by artificially restricting the measurement bandwidth by either digital or analog electronic filtering. As a consequence, certain events are lost since they are too short to be recorded. This may result in an erroneous interpretation of the experimental data. This year we addressed this issue analytically in a case study of the reversible capture of solute molecules by nanopores. We have shown that under typical conditions the distribution of time spent by a single captured solute molecule in a nanopore is bimodal with the majority of capture events being too fast to be experimentally resolved. As a result, the exact mean durations of the event and inter-event interval are orders of magnitude shorter than their measured values. Moreover, the exact and measured mean durations have qualitatively different dependences on the molecule diffusivity. This leads to a formal contradiction with the thermodynamics of molecule partitioning between the bulk and the nanopore, wherein the capture on-rate depends on the molecules bulk diffusivity while the off-rate on its diffusivity in the nanopore. In the present study we resolve this controversy. We also demonstrate that, surprisingly, the probability of finding a molecule in the nanopore, obtained from the ratio of the measured mean durations of the capture event and interevent interval, is essentially identical to the exact equilibrium thermodynamic probability.