A long-sought, and thus far elusive, goal has been to develop drugs to manage diseases of excitability. One such disease that affects millions each year is cardiac arrhythmia, which occurs when electrical impulses in the heart become disordered, sometimes causing sudden death. Pharmacological management of cardiac arrhythmia has failed because it is not possible to predict how drugs that target cardiac ion channels, and have intrinsically complex dynamic interactions with ion channels, will alter the emergent electrical behavior generated in the heart. Here, we applied a computational model, which was informed and validated by experimental data, that defined key measurable parameters necessary to simulate the interaction kinetics of the anti-arrhythmic drugs flecainide and lidocaine with cardiac sodium channels. We then used the model to predict the effects of these drugs on normal human ventricular cellular and tissue electrical activity in the setting of a common arrhythmia trigger, spontaneous ventricular ectopy. The model forecasts the clinically relevant concentrations at which flecainide and lidocaine exacerbate, rather than ameliorate, arrhythmia. Experiments in rabbit hearts and simulations in human ventricles based on magnetic resonance images validated the model predictions. This computational framework initiates the first steps toward development of a virtual drug-screening system that models drug-channel interactions and predicts the effects of drugs on emergent electrical activity in the heart.

Supplementary data are available at Bioinformatics online.

Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein β 1 γ 2 subunits (Gβγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gβγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gβγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.

Ion channels play crucial roles in transport and regulatory functions of living cells. Understanding the gating mechanisms of these channels is important to understanding and treating diseases that have been linked to ion channels. One potential model peptide for studying the mechanism of ion channel gating is alamethicin, which adopts a split alpha/310 helix structure and responds to changes in electric potential. In this study, sum frequency generation vibrational spectroscopy (SFG-VS), supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), has been applied to characterize interactions between alamethicin (a model for larger channel proteins) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers in the presence of an electric potential across the membrane. The membrane potential difference was controlled by changing the pH of the solution in contact with the bilayer, and measured using fluorescence spectroscopy. The orientation angle of alamethicin in POPC lipid bilayers was then determined at different pH values using polarized SFG amide I spectra. Assuming that all molecules adopt the same orientation (a δ distribution), at pH=6.7 the α-helix at the N-terminus and the 310 helix at the C-terminus tilt at about 72° (θ1) and 50° (θ2) versus the surface normal, respectively. When pH increases to 11.9, θ1 and θ2 decrease to 56.5° and 45°, respectively. The δ distribution assumption was verified using a combination of SFG and ATR-FTIR measurements, which showed a quite narrow distribution in the angle of θ1 for both pH conditions. This indicates that all alamethicin molecules at the surface adopt a nearly identical orientation in POPC lipid bilayers. The localized pH change in proximity to the bilayer modulates the membrane potential and thus induces a decrease in both the tilt and bend angles of the two helices in alamethicin. This is the first reported application of SFG to the study of model ion channel gating mechanisms in model cell membranes.

Antimicrobial peptides (AMPs) selectively disrupt bacterial cell membranes to kill bacteria whereas they either do not or weakly interact with mammalian cells. The orientations of AMPs in lipid bilayers mimicking bacterial and mammalian cell membranes are related to their antimicrobial activity and selectivity. In order to better understand the role of AMP-lipid interactions on the functional properties of AMPs, we determined the membrane orientation of an AMP (MSI-78 or pexiganan) in various model membranes using sum frequency generation (SFG) vibrational spectroscopy. Solid supported single 1,2-Dipalmitoyl-an-Glycero-3-[Phospho-rac-(1-glycerol)] (DPPG) bilayer or 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (POPG) bilayer was used as model bacterial cell membrane. Supported 1,2-Dipalmitoyl-an-Glycero-3-Phosphocholine (DPPC) bilayer or 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) bilayer was used as model mammalian cell membrane. Our SFG results indicate that the helical MSI-78 molecules are associated with the bilayer surface with ~70° deviation from the bilayer normal in the negatively charged gel-phase DPPG bilayer at 400 nM peptide concentration. On the other hand, when the concentration was increased to 600 nM, MSI-78 molecules change orientation to make a 25° tilt from the lipid bilayer normal; whereas multiple orientations were observed for even higher peptide concentration in agreement with toroidal-type pore formation as reported from a previous solid-state NMR study. In contrary, no interaction between MSI-78 and a zwitterionic DPPC bilayer was observed even at a much higher peptide concentration (~12000 nM). These results demonstrate that SFG can provide insights into the antibacterial activity and selectivity of MSI-78. Interestingly, the peptide exhibits a concentration-dependent membrane orientation in lamellar phase POPG bilayer and also found to be inducing toroidal-type pore formation. The deduced lipid flip-flop from SFG signals observed from lipids also supports MSI-78 induced toroidal-type pore formation.