Christine L. Henry

Ion channel formation and membrane-linked pathologies of misfolded hydrophobic proteins: The role of dangerous unchaperoned molecules

1. Protein–membrane interaction includes the interaction of proteins with intrinsic receptors and ion transport pathways and with membrane lipids. Several hypothetical interaction models have been reported for peptide‐induced membrane destabilization, including hydrophobic clustering, electrostatic interaction, electrostatic followed by hydrophobic interaction, wedge × type incorporation and hydrophobic mismatch.

Diversity of Amyloid β Protein Fragment [1–40]-Formed Channels

Abstract1. The lipid bilayer technique was used to characterize the biophysical and pharmacological properties of several ion channels formed by incorporating amyloid beta protein fragment (AβP) 1–40 into lipid membranes. Based on the conductance, kinetics, selectivity, and pharmacological properties, the following AβP[1–40]-formed ion channels have been identified: (i) The AβP[1–40]-formed “bursting” fast cation channel was characterized by (a) a single channel conductance of 63 pS (250/50 mM KCl cis/trans) at +140 mV, 17 pS (250/50 mM KCl cis/trans) at −160 mV, and the nonlinear current–voltage relationship drawn to a third-order polynomial, (b) selectivity sequence PK > PNa > PLi = 1.0:0.60:0.47, (c) Po of 0.22 at 0 mV and 0.55 at +120 mV, and (d) Zn2+-induced reduction in current amplitude, a typical property of a slow block mechanism. (ii) The AβP[1–40]-formed “spiky” fast cation channel was characterized by (a) a similar kinetics to the “bursting” fast channel with exception for the absence of the long intraburst closures, (b) single channel conductance of 63 pS (250/50 KCl) at +140 mV 17 pS (250/50 KCl) at −160 mV, the current–voltage relationship nonlinear drawn to a third-order polynomial fit, and (c) selectivity sequence PRb > PK > PCs > PNa > PLi = 1.3:1.0:0.46:0.40:0.27. (iii) The AβP[1–40]-formed medium conductance channel was charcterized by (a) 275 pS (250/50 mM KCl cis/trans) at +140 mV and 19 pS (250/50 mM KCl cis/trans) at −160 mV and (b) inactivation at Vms more negative than −120 and more positive than +120 mV. (iv) The AβP[1–40]-formed inactivating large conductance channel was characterized by (a) fast and slow modes of opening to seven multilevel conductances ranging between 0–589 pS (in 250/50 mM KCl) at +140 mV and 0–704 pS (in 250/50 mM KCl) at −160 mV, (b) The fast mode which had a conductance of <250 pS was voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.2 s at +36 mV. The slow mode which had a conductance of >250 pS was also voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.0 s at −76 mV, (c) the value of PK/Pcholine for the fast mode was 3.9 and selectivity sequence PK > PCs > PNa > PLi = 1.0:0.94:0.87:0.59. The value of PK/Pcholine for the slow mode was 2.7 and selectivity sequence PK > PNa > PLi > PCs = 1.0:0.59:0.49:0.21, and (d) asymmetric blockade with 10 mM Zn2+-induced reduction in the large conductance state of the slow mode mediated via slow block mechanism. The fast mode of the large conductance channel was not affected by 10 mM Zn2+.2. It has been suggested that, although the “bursting” fast channel, the “spiky” fast channel and the inactivating medium conductance channel are distinct, it is possible that they are intermediate configurations of yet another configuration underlying the inactivating large conductance channel. It is proposed that this heterogeneity is one of the most common features of these positively-charged cytotoxic amyloid-formed channels reflecting these channels ability to modify multiple cellular functions.3. Furthermore, the formation of β-sheet based oligomers could be an important common step in the formation of cytotoxic amyloid channels.

The link between ion specific bubble coalescence and Hofmeister effects is the partitioning of ions within the interface.

Specific ion effects are ubiquitous in soft matter systems and are most readily observed at high salt concentrations where long-range electrostatic forces are screened. In biological systems, ion-specificity is universal and is necessary to introduce the complexity required to carry out the processes of life. Many specific ion effects fall within the Hofmeister paradigm, whereby the strengths of action of the anions and cations follow a well-defined order, independent of the counterion. In contrast, specific ion effects evident in bubble coalescence inhibition depend on the combination of ions, and this phenomenon can be codified using simple ion-combining rules not evident in the Hofmeister systems. Here we show that these disparate specific ion effects have the same origin: They result from the variation in ion affinity for the solution interface. Equilibrium affinities explain Hofmeister effects, whereas we argue that the cation/anion combination controls bubble coalescence inhibition because of dynamic interfacial processes occurring at the more deformable gas-water interface.

Ion-Specific Influence of Electrolytes on Bubble Coalescence in Nonaqueous Solvents

We report the effects of electrolytes on bubble coalescence in nonaqueous solvents methanol, formamide, propylene carbonate, and dimethylsulfoxide (DMSO). Results in these solvents are compared to the ion-specific bubble coalescence inhibition observed in aqueous electrolyte solutions, which is predicted by simple, empirical ion combining rules. Coalescence inhibition by electrolytes is observed in all solvents, at a lower concentration range (0.01 M to 0.1M) to that observed in water. Formamide shows ion-specific salt effects dependent upon ion combinations in a way analogous to the combining rules observed in water. Bubble coalescence in propylene carbonate is also consistent with ion-combining rules, but the ion assignments differ to those for water. In both methanol and DMSO all salts used are found to inhibit bubble coalescence. Our results show that electrolytes influence bubble coalescence in a rich and complex way, but with notable similarities across all solvents tested. Coalescence is influenced by the drainage of fluid between two bubbles to form a film and then the rupture of the film and one might expect that these processes will vary dramatically between solvents. The similarities in behavior we observe show that coalescence inhibition is unlikely to be related to the surface forces present but is perhaps related to the dynamic thinning and rupture of the liquid film through the hydrodynamic boundary condition.

Channel activity of deamidated isoforms of prion protein fragment 106-126 in planar lipid bilayers.

Using the lipid bilayer technique, we have found that age‐related derivatives, PrP[106–126] (L‐Asp108) and PrP[106–126] (L‐iso‐Asp108), of the prion protein fragment 106–126 (PrP[106–126] (Asn108)) form heterogeneous ion channels. The deamidated isoforms, PrP[106–126] (L‐Asp108) and PrP[106–126] (L‐iso‐Asp108), showed no enhanced propensity to form heterogeneous channels compared with PrP[106–126] (Asn108). One of the PrP[106–126] (L‐Asp108)‐ and PrP[106–126] (L‐iso‐Asp108)‐formed channels had three kinetic modes. The current–voltage (I–V) relationship of this channel, which had a reversal potential, Erev, between –40 and –10 mV close to the equilibrium potential for K+ (EK –35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (gmax) was 62.5 pS at positive potentials between 0 and 140 mV. The probability (Po) and the frequency (Fo) of the channel being open had inverted and bell‐shaped curves, respectively, with a peak at membrane potential (Vm) between –80 and +80 mV. The mean open and closed times (To and Tc) had inverted bell‐shaped curves. The biophysical properties of PrP[106–126] (L‐Asp108)‐ and PrP[106–126] (L‐iso‐Asp108)‐formed channels and their response to Cu2+ were similar to those of channels formed with PrP[106–126] (Asn108). Cu2+ shifted the kinetics of the channel from being in the open state to a “burst state” in which rapid channel activities were separated by long durations of inactivity. The action of Cu2+ on the open channel activity was both time‐dependent and voltage‐dependent. The fact that Cu2+ induced changes in the kinetics of this channel with no changes in the conductance of the channel indicated that Cu2+ binds at the mouth of the channel. Consistently with the hydrophilic and structural properties of PrP[106–126], the Cu2+‐induced changes in the kinetic parameters of this channel suggest that the Cu2+ binding site could be located at M109 and H111 of this prion fragment. J. Neurosci. Res. 66:214–220, 2001.

Inhibition of Bubble Coalescence by Osmolytes: Sucrose, Other Sugars, and Urea

We report on bubble coalescence inhibition by non-surface-active, nonelectrolytes urea and sucrose, and other small sugars, in aqueous solution. Urea has no effect on bubble stability up to high concentrations>1 M, while sucrose inhibits coalescence in the range 0.01-0.3 M, similar to inhibiting electrolytes. Urea and sucrose both increase bubble coalescence inhibition in inhibiting and noninhibiting electrolytes in a cooperative manner, but urea decreases the efficacy of sucrose in mixed solutions. Several mono- and disaccharides also inhibit bubble coalescence at approximately 0.1 M, and the sugars vary in effectiveness. Disaccharides are more effective than the sum of their individual monosaccharide constituents, and sugars with very similar structures (for instance, diastereomers galactose and mannose) can show large differences in coalescence inhibition and hence thin film stability. We conclude that solute charge is not required for bubble coalescence inhibition, which indicates that the mechanism is not one of electrostatic surface repulsion and instead an effect on dynamic film thinning other than Gibbs-Marangoni elasticity is implicated. Solute structure is important in determining coalescence.

Ion Specific Electrolyte Effects on Thin Film Drainage in Nonaqueous Solvents Propylene Carbonate and Formamide

Electrolytes have been found to stabilize thin films in nonaqueous solvents propylene carbonate and formamide, in the absence of surfactant. The thin film balance microinterferometry technique has been used to measure film lifetimes, drainage kinetics, and rupture thicknesses for thin films between air-nonaqueous solution interfaces. Electrolytes that were previously found to inhibit bubble coalescence in bulk bubble column measurements also increase the lifetimes of individual thin films across a similar concentration range (from 0 to 0.3 M). We report that increasing the concentration of inhibiting electrolyte stabilizes the thin liquid film in two ways: the rate of film drainage decreases, and the film reaches a lower thickness before rupturing. In contrast, noninhibiting electrolyte shows little to no effect on film stability. We have here demonstrated that both drainage and rupture processes are affected by the addition of electrolyte and the effect on the thin film is thus ion specific.