Probing Membrane Association of α-Synuclein Domains with VDAC Nanopore Reveals Unexpected Binding Pattern

Abstract

It is well established that α-synuclein (α-syn) binding from solution to the surface of membranes composed of negatively charged and/or non-lamellar lipids can be characterized by equilibrium dissociation constants of tens of micromolar. Previously, we have found that a naturally occurring nanopore of the mitochondrial voltage-dependent anion channel (VDAC), reconstituted into planar bilayers of a plant-derived lipid, responds to α-syn at nanomolar solution concentrations. Here, using lipid mixtures that mimic the composition of mitochondrial outer membranes, we show that functionally important binding does indeed take place in the nanomolar range. We demonstrate that the voltage-dependent rate at which a membrane-embedded VDAC nanopore captures α-syn is a strong function of membrane composition. Comparison of the nanopore results with those obtained by the bilayer overtone analysis of membrane binding demonstrates a pronounced correlation between the two datasets. The stronger the binding, the larger the on-rate, but with some notable exceptions. This leads to a tentative model of α-syn-membrane interactions, which assigns different lipid-dependent roles to the N- and C-terminal domains of α-syn accounting for both electrostatic and hydrophobic effects. As a result, the rate of α-syn capture by the nanopore is not simply proportional to the α-syn concentration on the membrane surface but found to be sensitive to the specific interactions of each domain with the membrane and nanopore.

A lower affinity to cytosolic proteins reveals VDAC3 isoform-specific role in mitochondrial biology

“…To maximize binding of α-syn and maintain the open state of VDAC, a PLM lipid composition of DOPC/DOPE/2DOPG was used, which virtually abolishes voltage gating at applied voltages <60 mV (Queralt-Martín et al, 2019). Furthermore, the higher content of anionic DOPG in the lipid mixture increases the on-rate of α-syn with VDAC (Jacobs et al, 2019). Single-channel recordings of hVDAC3 and mVDAC1 in the presence of 10 nM of α-syn are shown in Fig.…”

“…5 B) for all applied potentials. The on-rate constant, k on , (the inverse of < τ on > normalized over the α-syn concentration) is highly voltage dependent where it increases exponentially with the applied voltage (Rostovtseva et al, 2015; Hoogerheide et al, 2017; Jacobs et al, 2019). For hVDAC3, the k on is 10- to 100-fold lower than mVDAC1 for all voltages and at both polarities (Fig.…”

VDAC1 is essential for neurite maintenance and the inhibition of its oligomerization protects spinal cord from demyelination and facilitates locomotor function recovery after spinal cord injury

“…These events subsequently lead to the alteration of mitochondrial permeability through the interaction of external proteins, such as B-cell lymphoma 2 (Bcl-2) protein family and α-synuclein, with mitochondrial transmembrane proteins 17–19 , disturbing its metabolism and triggering the apoptosis intrinsic pathway 20,21 . Activation of this pathway starts when pro-apoptotic signals overcome anti-apoptotic signals, leading to the release of proteins to the cytosol 22 , such as cytochrome-C (Cyto-C) 23 , which can interact with apoptotic protease-activating factor 1 (Apaf-1), forming the apoptosome.…”

α-Synuclein emerges as a potent regulator of VDAC-facilitated calcium transport

“…Recombinant mouse VDAC1 was a kind gift of Dr. Adam Kuszak (NIDDK, NIH). Protocols for expression, refolding, and purification of VDAC1 were based on the work of Dr. Jeff Abramson’s lab (10) as described previously (36). Recombinant mouse VDAC1 E73Q mutant and human VDAC3 were the kind gifts of Jeff Abramson (UCLA).…”