Using Charge to Control the Functional Properties of Self‐Assembled Nanopores in Membranes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86890/1/smll_201100394_sm_suppl.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/86890/2/2016_ftp.pd

Proof of Kernel Work: a democratic low-energy consensus for distributed access-control protocols

We adjust the Proof of Work (PoW) consensus mechanism used in Bitcoin and Ethereum so that we can build on its strength while also addressing, in part, some of its perceived weaknesses. Notably, our work is motivated by the high energy consumption for mining PoW, and we want to restrict the use of PoW to a configurable, expected size of nodes, as a function of the local blockchain state. The approach we develop for this rests on three pillars: (i) Proof of Kernel Work (PoKW), a means of dynamically reducing the set of nodes that can participate in the solving of PoW puzzles such that an adversary cannot increase his attack surface because of such a reduction; (ii) Practical Adaptation of Existing Technology, a realization of this PoW reduction through an adaptation of existing blockchain and enterprise technology stacks; and (iii) Machine Learning for Adaptive System Resiliency, the use of techniques from artificial intelligence to make our approach adaptive to system, network and attack dynamics. We develop here, in detail, the first pillar and illustrate the second pillar through a real use case, a pilot project done with Porsche on controlling permissions to vehicle and data log accesses. We also discuss pertinent attack vectors for PoKW consensus and their mitigation. Moreover, we sketch how our approach may lead to more democratic PoKW-based blockchain systems for public networks that may inherit the resilience of blockchains based on PoW

Changes in single K+ channel behavior through the lipid phase transition

We show that the activity of an ion channel is strictly related to the phase state of the lipid bilayer hosting the channel. By measuring unitary conductance, dwell times, and open probability of the K+ channel KcsA as a function of temperature in lipid bilayers composed of POPE and POPG in different relative proportions, we obtain that all those properties show a trend inversion when the bilayer is in the transition region between the liquid disordered and the solid ordered phase. These data suggest that the physical properties of the lipid bilayer influence ion channel activity likely via a fine tuning of its conformations. In a more general interpretative framework, we suggest that other parameters such as pH, ionic strength, and the action of amphiphilic drugs can affect the physical behavior of the lipid bilayer in a fashion similar to temperature changes resulting in functional changes of transmembrane proteins

Membrane Cholesterol Content Modulates Activation of Volume-Regulated Anion Current in Bovine Endothelial Cells

Activation of volume-regulated anion current (VRAC) plays a key role in the maintenance of cellular volume homeostasis. The mechanisms, however, that regulate VRAC activity are not fully understood. We have examined whether VRAC activation is modulated by the cholesterol content of the membrane bilayer. The cholesterol content of bovine aortic endothelial cells was increased by two independent methods: (a) exposure to a methyl-β-cyclodextrin saturated with cholesterol, or (b) exposure to cholesterol-enriched lipid dispersions. Enrichment of bovine aortic endothelial cells with cholesterol resulted in a suppression of VRAC activation in response to a mild osmotic gradient, but not to a strong osmotic gradient. Depletion of membrane cholesterol by exposing the cells to methyl-β-cyclodextrin not complexed with cholesterol resulted in an enhancement of VRAC activation when the cells were challenged with a mild osmotic gradient. VRAC activity in cells challenged with a strong osmotic gradient were unaffected by depletion of membrane cholesterol. These observations show that changes in membrane cholesterol content shift VRAC sensitivity to osmotic gradients. Changes in VRAC activation were not accompanied by changes in anion permeability ratios, indicating that channel selectivity was not affected by the changes in membrane cholesterol. This suggests that membrane cholesterol content affects the equilibrium between the closed and open states of VRAC channel rather than the basic pore properties of the channel. We hypothesize that changes in membrane cholesterol modulate VRAC activity by affecting the membrane deformation energy associated with channel opening

Specificity of cholesterol and analogs to modulate BK channels points to direct sterol–channel protein interactions

The activity (Po) of large-conductance voltage/Ca2+-gated K+ (BK) channels is blunted by cholesterol levels within the range found in natural membranes. We probed BK channel–forming α (cbv1) subunits in phospholipid bilayers with cholesterol and related monohydroxysterols and performed computational dynamics to pinpoint the structural requirements for monohydroxysterols to reduce BK Po and obtain insights into cholesterol’s mechanism of action. Cholesterol, cholestanol, and coprostanol reduced Po by shortening mean open and lengthening mean closed times, whereas epicholesterol, epicholestanol, epicoprostanol, and cholesterol trisnorcholenic acid were ineffective. Thus, channel inhibition by monohydroxysterols requires the β configuration of the C3 hydroxyl and is favored by the hydrophobic nature of the side chain, while having lax requirements on the sterol A/B ring fusion. Destabilization of BK channel open state(s) has been previously interpreted as reflecting increased bilayer lateral stress by cholesterol. Lateral stress is controlled by the sterol molecular area and lipid monolayer lateral tension, the latter being related to the sterol ability to adopt a planar conformation in lipid media. However, we found that the differential efficacies of monohydroxysterols to reduce Po (cholesterol≥coprostanol≥cholestanol>>>epicholesterol) did not follow molecular area rank (coprostanol>>epicholesterol>cholesterol>cholestanol). In addition, computationally predicted energies for cholesterol (effective BK inhibitor) and epicholesterol (ineffective) to adopt a planar conformation were similar. Finally, cholesterol and coprostanol reduced Po, yet these sterols have opposite effects on tight lipid packing and, likely, on lateral stress. Collectively, these findings suggest that an increase in bilayer lateral stress is unlikely to underlie the differential ability of cholesterol and related steroids to inhibit BK channels. Remarkably, ent-cholesterol (cholesterol mirror image) failed to reduce Po, indicating that cholesterol efficacy requires sterol stereospecific recognition by a protein surface. The BK channel phenotype resembled that of α homotetramers. Thus, we hypothesize that a cholesterol-recognizing protein surface resides at the BK α subunit itself