Pore Conformation Research Articles

Phospholipid chain length alters the equilibrium between pore and channel forms of gramicidin.

Gramicidin is an excellent model system for studying the passage of ions through biological membranes. The conformation of gramicidin is well defined in many different solvent and lipid systems, as are its conductance and spectroscopic properties. It is a polymorphic molecule that can adopt two different types of structure, the double helical "pore" and the helical dimer "channel". This study investigated the influence of the acyl chain length of membrane phospholipids on the conformations adopted by gramicidin. We used circular dichroism spectroscopy to examine the conformational equilibrium between the pore and channel forms in small unilamellar vesicles of phosphatidylcholine with acyl chain lengths of 18, 20 and 22 carbons. Our results show that in C18 and C20 lipids almost all the gramicidin is in the channel form, while in the longer C22 lipids the equilibrium shifts in favour of pore conformations, such that they form up to 43% of the total population. This change is attributed to the ability of the double helical conformation to tolerate more hydrophobic mismatch than the helical dimer, perhaps due to the greater number of stabilising intermolecular hydrogen bonds.

ATP-Induced Shape Change of Nuclear Pores Visualized with the Atomic Force Microscope

Bidirectional transport of molecules between nucleus and cytoplasm through the nuclear pore complexes (NPCs) spanning the nuclear envelope plays a fundamental role in cell function and metabolism. Nuclear import of macromolecules is a two-step process involving initial recognition of targeting signals, docking to the pore and energy-driven translocation. ATP depletion inhibits the translocation step. The mechanism of translocation itself and the conformational changes of the NPC components that occur during macromolecular transport, are still unclear. The present study investigates the effect of ATP on nuclear pore conformation in isolated nuclear envelopes from Xenopus laevis oocytes using the atomic force microscope. All experiments were conducted in a saline solution mimicking the cytosol using unfixed nuclear envelopes. ATP (1 mM) was added during the scanning procedure and the resultant conformational changes of the NPCs were directly monitored. Images of the same nuclear pores recorded before and during ATP exposure revealed dramatic conformational changes of NPCs subsequent to the addition of ATP. The height of the pores protruding from the cytoplasmic surface of the nuclear envelope visibly increased while the diameter of the pore opening decreased. The observed changes occurred within minutes and were transient. The slow-hydrolyzing ATP analogue, ATP-gamma-S, in equimolar concentrations did not exert any effects. The ATP-induced shape change could represent a nuclear pore "contraction."

Spontaneous oligomerization of a staphylococcal alpha-hemolysin conformationally constrained by removal of residues that form the transmembrane beta-barrel.

Staphylococcal alpha-hemolysin is a water soluble, monomeric, bacterial exotoxin, which forms heptameric pores in membranes. The rate determining step in assembly is the conversion of a heptameric prepore to the fully assembled pore in which the central glycine-rich domain of each subunit inserts into the membrane to form a 14 strand beta barrel. Barrel formation is accompanied by a conformational change in which each N terminus latches onto an adjacent subunit. In the monomer in solution, the central domain is loosely organized and exposed to solvent. In this study, 25 amino acids of the central domain were removed and replaced with the sequence Asp-Gly, which favors the formation of a type I' beta-turn, to yield a mutant devoid of hemolytic activity. Within minutes after synthesis in the absence of membranes, the mutant polypeptide spontaneously assembled into heptamers, as demonstrated by atomic force microscopy. Limited proteolysis suggested that the N termini of the subunits in the heptamers were in the fully assembled pore conformation rather than the prepore conformation. Based on these findings, the deletion is proposed to constrain the central domain and thereby force the creation of a shortened beta barrel, which in turn induces the additional structural changes that normally accompany pore formation. The truncated pore might make a useful framework for the construction of designed membrane active macromolecules.

Staphylococcal α-Toxin: Formation of the Heptameric Pore Is Partially Cooperative and Proceeds through Multiple Intermediate Stages

Staphylococcal alpha-toxin is a 293 residue polypeptide that assembles into pore-forming heptamers, residues 118-140, thereby inserting to form an amphipathic beta-barrel in the lipid bilayer. Fluorometric analyses were here conducted using cysteine-substitution mutants site-specifically-labeled at positions 35 or 130 with the environmentally-sensitive fluorophore acrylodan. In conjunction with functional assays, three conformational states of the heptamer were defined, which may represent transitional configurations of the toxin molecule along its way to membrane insertion and pore formation. The first was the freshly assembled, SDS-sensitive heptamer alpha7*a, where a minor alteration in the environment of H35 with no change in the environment of the membrane-inserting stem domain was observed. In transition stage alpha7*b, the stem domain moved from a hydrophilic to a more hydrophobic environment, due to protein-protein interaction. Transition to alpha7*c involved a cooperative effect, in which residue 35 was forced by a neighboring molecule into a markedly hydrophobic environment. At this stage, the heptamers acquired SDS stability. The final pore conformation alpha7 resulted when the stem domain inserted into the lipid bilayer, an event that was driven by H35 within the respective protomer. A model thus evolved in which cooperative forces first lever H35 into a position that subsequently drives the pore-forming sequence within each respective protomer into the membrane. In accord with this model, when hybrid heptamers were formed between a functionally defective H35 substitution mutant and active toxin, only the latter inserted their pore-forming domain into the membrane. In a satisfying functional correlate, pores of reduced size were then generated.

Activation-Dependent Subconductance Levels in the drk1 K Channel Suggest a Subunit Basis for Ion Permeation and Gating

Ion permeation and channel opening are two fundamental properties of ion channels, the molecular bases of which are poorly understood. Channels can exist in two permeability states, open and closed. The relative amount of time a channel spends in the open conformation depends on the state of activation. In voltage-gated ion channels, activation involves movement of a charged voltage sensor, which is required for channel opening. Single-channel recordings of drk1 K channels expressed in Xenopus oocytes suggested that intermediate current levels (sublevels) may be associated with transitions between the closed and open states. Because K channels are formed by four identical subunits, each contributing to the lining of the pore, it was hypothesized that these sublevels resulted from heteromeric pore conformations. A formal model based on this hypothesis predicted that sublevels should be more frequently observed in partially activated channels, in which some but not all subunits have undergone voltage-dependent conformational changes required for channel opening. Experiments using the drk1 K channel, as well as drk1 channels with mutations in the pore and in the voltage sensor, showed that the probability of visiting a sublevel correlated with voltage- and time-dependent changes in activation. A subunit basis is proposed for channel opening and permeation in which these processes are coupled.

Kinetic Analysis of the Mitochondrial Permeability Transition

The permeability transition process in rat liver mitochondria was studied by following the swelling consequent to external solute equilibration through the membrane pore. The kinetics of the transition was analyzed according to a model based on the assumptions that the transition rate follows a first-order process and that the solute diffusion rate strongly depends on the pore conformation. Three kinetic parameters, easily calculated from absorbance measurements during mitochondrial swelling, were used to determine whether changes of the swelling rate are due to changes of (i) the transition rate, (ii) the amount of permeabilized mitochondria, (iii) the mean pore dimension, or (iv) the number of pores per mitochondrion. The model was tested for transitions induced either by phenylarsine oxide (PhAsO) or by Ca2+ and Pi. Under both conditions, only a definite fraction of mitochondria was permeabilized, and the transition always followed a first-order reaction, indicating that mitochondria behaved as a homogeneous population. However, the equilibration of external solutes was rapid only in a fraction of permeabilized mitochondria and slow in the remaining fraction, due to restricted solute diffusion through narrower pores. With 0.2 mM PhAsO as the inducer, the fraction of permeabilized mitochondria was about 0.8. Sucrose diffusion was rapid only in 15% of this fraction (half-time less than 1 s) and restricted in the remaining 85% (half-time of about 60 s). Increasing PhAsO concentrations increased the number of pores per mitochondrion and the rate constant of the permeability transition, but not the mean pore diameter and the fraction of permeabilized mitochondria. With 0.28 microM free [Ca2+] and 1 mM Pi as the inducers, the fraction of permeabilized mitochondria was about 0.43. Sucrose diffusion was rapid in 60% of this fraction and restricted in the remaining 40% (half-time of about 9 s). The permeabilization process started above a threshold- free [Ca2+] of 0.15 microM, and the rate constant and the fraction of permeabilized mitochondria reached a maximum at about 0.2 microM, while both parameters were inhibited at higher free [Ca2+].

Three-dimensional lipophilicity characterization of molecular pores and channel-like cavities

Molecular lipophilicity is a useful property for assessing molecular similarity or complementarity within the context of computer-aided drug design. As well, local contributions to solvent affinity help us to understand both dynamics and conformational stability in biomolecules. In this work, we discuss an approach to characterize the local contributions to hydrophobicity by using one- and two-dimensional representations of molecular channel-like cavities. The method monitors how a phenomenological lipophilicity potential (based on fragmental atom contributions) changes over a continuum of “molecular tubes” used for modeling channels and pores. Our results convey a relatively detailed picture of the spatial distribution of water affinity. The procedure can then be used as a complement to the hydrophobicity scales based on averaging contributions from single amino acids. In addition, we can study how the water affinity changes for inner and outer regious of the pores. As an application, we compute the 3D distribution of lipophilicity in the “pore conformation” of gramicidin A. The qualitative trends indicated by our results are broadly consistent with computer simulations of the gramicidin channel in the presence of hydrated ions. The behavior revealed by the simulations can then be incorporated to produce an improved, simple 2D model for water-channel interactions.

Structural conservation of ion conduction pathways in K channels and glutamate receptors.

Single channel recordings demonstrate that ion channels switch stochastically between an open and a closed pore conformation. In search of a structural explanation for this universal open/close behavior, we have uncovered a striking degree of amino acid homology across the pore-forming regions of voltage-gated K channels and glutamate receptors. This suggested that the pores of these otherwise unrelated classes of channels could be structurally conserved. Strong experimental evidence supports a hairpin structure for the pore-forming region of K channels. Consequently, we hypothesized the existence of a similar structure for the pore of glutamate receptors. In ligand-gated channels, the pore is formed by M2, the second of four putative transmembrane segments. A hairpin structure for M2 would affect the subsequent membrane topology, inverting the proposed orientation of the next segments, M3. We have tested this idea for the NR1 subunit of the N-methyl-D-aspartate receptor. Mutations that affected the glycosylation pattern of the NR1 subunit localize both extremes of the M3-M4 linker to the extracellular space. Whole cell currents and apparent agonist affinities were not affected by these mutations. Therefore it can be assumed that they represent the native transmembrane topology. The extracellular assignment of the M3-M4 linker challenged the current topology model by inverting M3. Taken together, the amino acid homology and the new topology suggest that the pore-forming M2 segment of glutamate receptors does not transverse the membrane but, rather, forms a hairpin structure, similar to that found in K channels.

A Computational Model of the HBK2 Potassium Channel Ion Pore

A computational model of the putative ion pore region of the HBK2 potassium channel was developed. Utilizing experimentally derived constraints, conformations corresponding to both the open and closed states of the ion pore were determined. Also, a conformational basis for the different sensitivities of the internal and external tetraethylammonium binding sites has been proposed. The model presented here suggests a role for other regions of the HBK2 protein in determining the ion pore conformation.