The basic amino acids lysine (Lys) and arginine (Arg) play important roles in membrane protein activity, the sensing of membrane voltages, and the actions of antimicrobial, toxin, and cell-penetrating peptides. These roles are thought to stem from the strong interactions and disruptive influences of these amino acids on lipid membranes. In this study, we employ fully atomistic molecular dynamics simulations to observe, quantify, and compare the interactions of Lys and Arg with saturated phosphatidylcholine membranes of different thickness. We make use of both charged (methylammonium and methylguanidinium) and neutral (methylamine and methylguanidine) analogue molecules, as well as Lys and Arg side chains on transmembrane helix models. We find that the free energy barrier experienced by a charged Lys crossing the membrane is strikingly similar to that of a charged Arg (to within 2 kcal/mol), despite the two having different chemistries, H-bonding capability, and hydration free energies that differ by ∼10 kcal/mol. In comparison, the barrier for neutral Arg is higher than that for neutral Lys by around 5 kcal/mol, being more selective than that for the charged species. This can be explained by the different transport mechanisms for charged or neutral amino acid side chains in the membrane, involving membrane deformations or simple dehydration, respectively. As a consequence, we demonstrate that Lys would be deprotonated in the membrane, whereas Arg would maintain its charge. Our simulations also reveal that Arg attracts more phosphate and water in the membrane, and can form extensive H-bonding with its five H-bond donors to stabilize Arg-phosphate clusters. This leads to enhanced interfacial binding and membrane perturbations, including the appearance of a trans-membrane pore in a thinner membrane. These results highlight the special role played by Arg as an amino acid to bind to, disrupt, and permeabilize lipid membranes, as well as to sense voltages for a range of peptide and protein activities in nature and in engineered bionanodevices.

Voltage-gated Na(+) channels play an essential role in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. The recent solution of X-ray structures for the bacterial channel NavAb has provided an opportunity to study functional mechanisms at the atomic level. This channel's selectivity filter exhibits an EEEE ring sequence, characteristic of mammalian Ca(2+), not Na(+), channels. This raises the fundamentally important question: just what makes a Na(+) channel conduct Na(+) ions? Here we explore ion permeation on multimicrosecond timescales using the purpose-built Anton supercomputer. We isolate the likely protonation states of the EEEE ring and observe a striking flexibility of the filter that demonstrates the necessity for extended simulations to study conduction in this channel. We construct free energy maps to reveal complex multi-ion conduction via knock-on and "pass-by" mechanisms, involving concerted ion and glutamate side chain movements. Simulations in mixed ionic solutions reveal relative energetics for Na(+), K(+), and Ca(2+) within the pore that are consistent with the modest selectivity seen experimentally. We have observed conformational changes in the pore domain leading to asymmetrical collapses of the activation gate, similar to proposed inactivated structures of NavAb, with helix bending involving conserved residues that are critical for slow inactivation. These structural changes are shown to regulate access to fenestrations suggested to be pathways for lipophilic drugs and provide deeper insight into the molecular mechanisms connecting drug activity and slow inactivation.

A wealth of experimental data exists describing the elementary building blocks of complex physiological systems. However, it is increasingly apparent in the biomedical sciences that mechanisms of biological function cannot be observed or readily predicted via study of constituent elements alone. This is especially clear in the longstanding failures in prediction of effects of drug treatment for heart rhythm disturbances. These failures stem in part from classical assumptions that have been made in cardiac antiarrhythmic drug development - that a drug operates by one mechanism via one target receptor that arises from one gene.

Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the "hinged lid"-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water-protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors.

We have explored the mechanisms of uncatalyzed membrane ion permeation using atomistic simulations and electrophysiological recordings. The solubility-diffusion mechanism of membrane charge transport has prevailed since the 1960s, despite inconsistencies in experimental observations and its lack of consideration for the flexible response of lipid bilayers. We show that direct lipid bilayer translocation of alkali metal cations, Cl(-), and a charged arginine side chain analog occurs via an ion-induced defect mechanism. Contrary to some previous suggestions, the arginine analog experiences a large free-energy barrier, very similar to those for Na(+), K(+), and Cl(-). Our simulations reveal that membrane perturbations, due to the movement of an ion, are central for explaining the permeation process, leading to both free-energy and diffusion-coefficient profiles that show little dependence on ion chemistry and charge, despite wide-ranging hydration energies and the membrane's dipole potential. The results yield membrane permeabilities that are in semiquantitative agreement with experiments in terms of both magnitude and selectivity. We conclude that ion-induced defect-mediated permeation may compete with transient pores as the dominant mechanism of uncatalyzed ion permeation, providing new understanding for the actions of a range of membrane-active peptides and proteins.

Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.