Multi-scale Molecular Modeling and Simulations of Voltage-Gated Sodium Channels and their Modulation by the Proline-Rich Transmembrane Protein 2 (PRRT2)

Voltage-gated sodium (NaV) channels control neurons intrinsic excitability by a precise spatiotemporal regulation of ion fluxes through the plasmalemma. They trigger the initiation and propagation of action potentials, key events for the establishment of electrical signaling pathways that span both the central (CNS) and peripheral nervous systems. The experimental determination of NaV channels three dimensional structures has provided insights into the specific conformations associated with their function. Moreover, it paved the way for molecular dynamics (MD) investigations aimed at unraveling the mechanistic features of both ion permeation and conformational transitions between distinct functional states. Despite significant progress in the study of NaVs, several key questions remain unresolved, including the molecular determinants of ion selectivity and the role of modulatory binding proteins. In the past few years, our group contributed to the structural and functional characterization of PRRT2, an integral membrane protein with a large cytosolic domain. Variants of PRRT2 have been identified as a cause of several neurological disorders, including epilepsy and motor dysfunctions, suggesting its potential role in the modulation of neuronal excitability. Indeed, it was shown that when PRRT2 is co-expressed with NaV1.2 and NaV1.6, a reduction in channel conductance together with a decrement of their exposure to the cell membrane are recorded, advocating for a repressing role of NaV function by PRRT2. While the direct interaction between NaV1.2/NaV1.6 and PRRT2 has been confirmed by multiple experiments, structural details of the interaction interface are still lacking. Uncovering these features is of utmost importance to understand the molecular determinants of neuronal signaling and to boost the design of pharmaceuticals to compensate excitatory disfunctions. The central objective of this Ph.D. thesis has been to identify and analyze potential interaction interfaces between PRRT2 and the NaV1.2 voltage-gated sodium channel. The approach involved designing a multiscale computational pipeline that integrated all-atom and coarse- grained MD simulations, multi-software protein-protein docking analyses and machine learning approaches for predicting protein complexes. As a result, a ranked list of inferred hot spots for each NaV1.2 voltage sensor domain is disclosed, hierarchically ordered based on the predicted numerosity of residue-residue contacts formed with PRRT2. As the modulatory role ultimately a;ects ion conduction, we also used all-atom unbiased MD simulations to study Na+ permeation in the channel isoforms NaV1.1, 1.2 and 1.6, and enhanced sampling methods to map two-dimensional free energy landscapes for ion translocation through NaV1.2. By shedding light on the molecular basis of PRRT2-mediated modulation of NaV channels, this study provides a foundation for developing therapeutic strategies targeting excitability disorders linked to NaV-PRRT2 interactions. Additionally, our insights into Na+ permeation across di;erent NaV isoforms pave the way to a deeper understanding of the functional and structural implications of PRRT2 binding on ion conduction and neuronal signaling.