The conduction pore of a cardiac potassium channel.

Ion channels form transmembrane water-filled pores that allow ions to cross membranes in a rapid and selective fashion. The amino acid residues that line these pores have been sought to reveal the mechanisms of ion conduction and selectivity. The pore (P) loop is a stretch of residues that influences single-channel-current amplitude, selectivity among ions and open-channel blockade and is conserved in potassium-channel subunits previously recognized to contribute to pore formation. To date, potassium-channel pores have been shown to form by symmetrical alignment of four P loops around a central conduction pathway. Here we show that the selectivity-determining pore region of the voltage-gated potassium channel of human heart through which the I(Ks) current passes includes the transmembrane segment of the non-P-loop protein minK. Two adjacent residues in this segment of minK are exposed in the pore on either side of a short barrier that restricts the movement of sodium, cadmium and zinc ions across the membrane. Thus, potassium-selective pores are not restricted to P loops or a strict P-loop geometry

Analyzing the effects of surface distribution of pores in cell electroporation for a cell membrane containing cholesterol

This paper presents a model and numerical analysis (simulations) of transmembrane potential induced in biological cell membrane under the influence of externally applied electric field (i.e., electroporation). This model differs from the established models of electroporation in two distinct ways. Firstly, it incorporates the presence of cholesterol (~20% mole-fraction) in biological membrane. Secondly, it considers the distribution of pores as a function of the variation of transmembrane potential from one region of the cell to another. Formulation is based on the role of membrane tension and electrical forces in the formation of pores in a cell membrane, which is considered as an infinitesimally thin insulator. The model has been used to explore the process of creation and evolution of pores and to determine the number and size of pores as a function of applied electric field (magnitude and duration). Results show that the presence of cholesterol enhances poration by changing the membrane tension. Analyses indicate that the number of pores and average pore radii differ significantly from one part of the cell to the other. While some regions of the cell membrane undergo rapid and dense poration, others remain unaffected. The method can be a useful tool for a more realistic prediction of pore formation in cells subjected to electroporation.Comment: 11 pages, 3 figures. v2: added new references, grammatical changes, corrected typo

Computational design of transmembrane pores.

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications

Elucidating mysteries of phase-segregated membranes: Mobile-lipid recruitment facilitates pores' passage to the fluid phase

Phase segregation of multicomponent lipid bilayers leads to, under phase-coexistence conditions, domain formation, featuring delimitation by essentially one-dimensional borders. (Micro-)phase segregation of bilayers is proposed to influence the physiological behaviour of cell membranes and provides the driving force for lipid-raft formation. Experiments show a maximum in the electrical-conductivity of membranes at the phase-transition point, which has been conjectured to arise from border-nucleated transmembrane-conducting defects or pores. However, recent electroporation experiments on phase-segregated bilayers demonstrate electro-pore detection in the liquid disordered phase (Ld), wherein they diffuse over macroscopic periods without absorption into the liquid ordered phase (Lo). Here, we scrutinise transmembrane-pore formation via molecular dynamics simulations on a multicomponent phase-segregated bilayer. We find that pores created in Lo domains always migrate spontaneously to the Ld phase, via 'recruitment' of unsaturated lipids to the pore's rim to transport the pore to the fluid phase under a large stress-field driving force. Once in Ld domains, pores migrate towards their centre, never returning or pinning to Lo. These findings are explained by thermodynamics. By comparing the free-energy cost for creating pores in the bulk of Ld and Lo membranes, and in the phase-segregated system, we show that it is always more energetically tractable to create pores in Ld domains, independent of the pore size.Fil: López Martí, Jesús María. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: English, Niall J.. University College Dublin; IrlandaFil: del Popolo, Mario Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentin

Single-channel electrophysiology reveals a distinct and uniform pore complex formed by α-synuclein oligomers in lipid membranes.

Synucleinopathies such as Parkinson's disease, multiple system atrophy and dementia with Lewy bodies are characterized by deposition of aggregated α-synuclein. Recent findings indicate that pathological oligomers rather than fibrillar aggregates may represent the main toxic protein species. It has been shown that α-synuclein oligomers can increase the conductance of lipid bilayers and, in cell-culture, lead to calcium dyshomeostasis and cell death. In this study, employing a setup for single-channel electrophysiology, we found that addition of iron-induced α-synuclein oligomers resulted in quantized and stepwise increases in bilayer conductance indicating insertion of distinct transmembrane pores. These pores switched between open and closed states depending on clamped voltage revealing a single-pore conductance comparable to that of bacterial porins. Pore conductance was dependent on transmembrane potential and the available cation. The pores stably inserted into the bilayer and could not be removed by buffer exchange. Pore formation could be inhibited by co-incubation with the aggregation inhibitor baicalein. Our findings indicate that iron-induced α-synuclein oligomers can form a uniform and distinct pore species with characteristic electrophysiological properties. Pore formation could be a critical event in the pathogenesis of synucleinopathies and provide a novel structural target for disease-modifying therapy

Modelling single cell electroporation with bipolar pulse parameters and dynamic pore radii

We develop a model of single spherical cell electroporation and simulate spatial and temporal aspects of the transmembrane potential and pore radii as an effect of any form of applied electric field. The extent of electroporation in response to sinusoidal electric pulses of two different frequencies in a range of extracellular conductivity for two different cell radii are compared. Results show that pore radii tend to be more normalized for AC fields. The relative difference in fractional pore area is reduced by the use of a 1 MHz sinusoidal applied electric field over a 100 kHz field

Defect formation of lytic peptides in lipid membranes and their influence on the thermodynamic properties of the pore environment

We present an experimental study of the pore formation processes of small amphipathic peptides in model phosphocholine lipid membranes. We used atomic force microscopy to characterize the spatial organization and structure of alamethicin- and melittin- induced defects in lipid bilayer membranes and the influence of the peptide on local membrane properties. Alamethicin induced holes in gel DPPC membranes were directly visualized at different peptide concentrations. We found that the thermodynamic state of lipids in gel membranes can be influenced by the presence of alamethicin such that nanoscopic domains of fluid lipids form close to the peptide pores, and that the elastic constants of the membrane are altered in their vicinity. Melittin-induced holes were visualized in DPPC and DLPC membranes at room temperature in order to study the influence of the membrane state on the peptide induced hole formation. Also differential scanning calorimetry was used to investigate the effect of alamethicin on the lipid membrane phase behavior.Comment: 11 pages, 7 figures, 1 tabl

Free energy barrier for melittin reorientation from a membrane-bound state to a transmembrane state

An important step in a phospholipid membrane pore formation by melittin antimicrobial peptide is a reorientation of the peptide from a surface into a transmembrane conformation. In this work we perform umbrella sampling simulations to calculate the potential of mean force (PMF) for the reorientation of melittin from a surface-bound state to a transmembrane state and provide a molecular level insight into understanding peptide and lipid properties that influence the existence of the free energy barrier. The PMFs were calculated for a peptide to lipid (P/L) ratio of 1/128 and 4/128. We observe that the free energy barrier is reduced when the P/L ratio increased. In addition, we study the cooperative effect; specifically we investigate if the barrier is smaller for a second melittin reorientation, given that another neighboring melittin was already in the transmembrane state. We observe that indeed the barrier of the PMF curve is reduced in this case, thus confirming the presence of a cooperative effect