Ion-Induced PIP2 Clustering with Martini3: Modification of Phosphate-Ion Interactions and Comparison with CHARMM36.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a critical lipid for cellular signaling. The specific phosphorylation of the inositol ring controls protein binding as well as clustering behavior. Two popular models to describe ion-mediated clustering of PIP2 are Martini3 (M3) and CHARMM36 (C36). Molecular dynamics simulations of PIP2-containing bilayers in solutions of potassium chloride, sodium chloride, and calcium chloride, and at two different resolutions are performed to understand the aggregation and the model parameters that drive it. The average M3 clusters of PIP2 in bilayers of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and PIP2 bilayers in the presence of K+, Na+, or Ca2+ contained 2.2, 2.6, and 6.4 times more PIP2 than C36 clusters, respectively. Indeed, the Ca2+-containing systems often formed a single large aggregate. Reparametrization of the M3 ion-phosphate Lennard-Jones interaction energies to reproduce experimental osmotic pressure of sodium dimethyl phosphate (DMP), K[DMP], and Ca[DMP]2 solutions, the same experimental target as C36, yielded comparably sized PIP2 clusters for the two models. Furthermore, C36 and the modified M3 predict similar saturation of the phosphate groups with increasing Ca2+, although the coarse-grained model does not capture the cooperativity between K+ and Ca2+. This characterization of the M3 behavior in the presence of monovalent and divalent ions lays a foundation to study cation/protein/PIP2 clustering.

Control of artificial membrane fusion in physiological ionic solutions beyond the limits of electroformation.

Membrane fusion, merging two lipid bilayers, is crucial for fabricating artificial membrane structures. Over the past 40 years, in contrast to precise and controllable membrane fusion in-vivo through specific molecules such as SNAREs, controlling the fusion in-vitro while fabricating artificial membrane structures in physiological ionic solutions without fusion proteins has been a challenge, becoming a significant obstacle to practical applications. We present an approach consisting of an electric field and a few kPa hydraulic pressure as an additional variable to physically control the fusion, enabling tuning of the shape and size of the 3D freestanding lipid bilayers in physiological ionic solutions. Mechanical model analysis reveals that pressure-induced parallel/normal tensions enhance fusion among membranes in the microwell. In-vitro peptide-membrane assay, mimicking vesicular transport via pressure-assisted fusion, and stability of 38 days with in-chip pressure control via pore size-regulated hydrogel highlight the potential for diverse biological applications.

Tentonin 3 is a pore-forming subunit of a slow inactivation mechanosensitive channel.

Mechanically activating (MA) channels transduce numerous physiological functions. Tentonin 3/TMEM150C (TTN3) confers MA currents with slow inactivation kinetics in somato- and barosensory neurons. However, questions were raised about its role as a Piezo1 regulator and its potential as a channel pore. Here, we demonstrate that purified TTN3 proteins incorporated into the lipid bilayer displayed spontaneous and pressure-sensitive channel currents. These MA currents were conserved across vertebrates and differ from Piezo1 in activation threshold and pharmacological response. Deep neural network structure prediction programs coupled with mutagenetic analysis predicted a rectangular-shaped, tetrameric structure with six transmembrane helices and a pore at the inter-subunit center. The putative pore aligned with two helices of each subunit and had constriction sites whose mutations changed the MA currents. These findings suggest that TTN3 is a pore-forming subunit of a distinct slow inactivation MA channel, potentially possessing a tetrameric structure.