Probing the volume changes during voltage gating of Porin 31BM channel with nonelectrolyte polymers

AbstractTo probe the volume changes of the voltage-dependent anion-selective channel (VDAC), the nonelectrolyte exclusion technique was taken because it is one of the few existing methods that may define quite accurately the rough geometry of lumen of ion channels (in membranes) for which there is no structural data.Here, we corroborate the data from our previous study [FEBS Lett. 416 (1997) 187] that the gross structural features of VDAC in its highest conductance state are asymmetric with respect to the plane of the membrane, and state that this asymmetry is not dependent on sign of voltage applied. Hence, the plasticity of VDAC does not play a role in the determination of lumen geometry at this state and the asymmetry is an internal property of the channel.We also show that the apparent diameter of the cis segment of the pore decreases slightly from 2 to 1.8 nm when the channel's conductance decreases from its high to low state. However, the trans funnel segment undergoes a more marked change in polymer accessible volume. Specifically, its larger diameter decreases from ∼4 to 2.4 nm. Supposing the channel's total length is 4.6 nm, the apparent change in channel volume during this transition is estimated to be about 10 nm3, i.e. about 40% of the channel's volume in the high conductance state

Conductance and Ion Selectivity of a Mesoscopic Protein Nanopore Probed with Cysteine Scanning Mutagenesis

Nanometer-scale proteinaceous pores are the basis of ion and macromolecular transport in cells and organelles. Recent studies suggest that ion channels and synthetic nanopores may prove useful in biotechnological applications. To better understand the structure-function relationship of nanopores, we are studying the ion-conducting properties of channels formed by wild-type and genetically engineered versions of Staphylococcus aureus α-hemolysin (αHL) reconstituted into planar lipid bilayer membranes. Specifically, we measured the ion selectivities and current-voltage relationships of channels formed with 24 different αHL point cysteine mutants before and after derivatizing the cysteines with positively and negatively charged sulfhydryl-specific reagents. Novel negative charges convert the selectivity of the channel from weakly anionic to strongly cationic, and new positive charges increase the anionic selectivity. However, the extent of these changes depends on the channel radius at the position of the novel charge (predominately affects ion selectivity) or on the location of these charges along the longitudinal axis of the channel (mainly alters the conductance-voltage curve). The results suggest that the net charge of the pore wall is responsible for cation-anion selectivity of the αHL channel and that the charge at the pore entrances is the main factor that determines the shape of the conductance-voltage curves

Interaction of Heparins and Dextran Sulfates with a Mesoscopic Protein Nanopore

A mechanism of how polyanions influence the channel formed by Staphylococcus aureus α-hemolysin is described. We demonstrate that the probability of several types of polyanions to block the ion channel depends on the presence of divalent cations and the polyanion molecular weight and concentration. For heparins, a 10-fold increase in molecular weight decreases the half-maximal inhibitory concentration, IC50, nearly 104-fold. Dextran sulfates were less effective at blocking the channel. The polyanions are significantly more effective at reducing the conductance when added to the trans side of this channel. Lastly, the effectiveness of heparins on the channel conductance correlated with their influence on the ζ-potential of liposomes. A model that includes the binding of polyanions to the channel-membrane complex via Ca2+-bridges and the asymmetry of the channel structure describes the data adequately. Analysis of the single channel current noise of wild-type and site-directed mutant versions of α-hemolysin channels suggests that a single polyanion enters the pore due to electrostatic forces and physically blocks the ion conduction path. The results might be of interest for pharmacology, biomedicine, and research aiming to design mesoscopic pore blockers