Changes In Ionic Selectivity Research Articles

The Ionic Selectivity of Lysenin Channels in Open and Sub-Conducting States.

The electrochemical gradients established across cell membranes are paramount for the execution of biological functions. Besides ion channels, other transporters, such as exogenous pore-forming toxins, may present ionic selectivity upon reconstitution in natural and artificial lipid membranes and contribute to the electrochemical gradients. In this context, we utilized electrophysiology approaches to assess the ionic selectivity of the pore-forming toxin lysenin reconstituted in planar bilayer lipid membranes. The membrane voltages were determined from the reversal potentials recorded upon channel exposure to asymmetrical ionic conditions, and the permeability ratios were calculated from the fit with the Goldman–Hodgkin–Katz equation. Our work shows that lysenin channels are ion-selective and the determined permeability coefficients are cation and anion-species dependent. We also exploited the unique property of lysenin channels to transition to a stable sub-conducting state upon exposure to calcium ions and assessed their subsequent change in ionic selectivity. The observed loss of selectivity was implemented in an electrical model describing the dependency of reversal potentials on calcium concentration. In conclusion, our work demonstrates that this pore-forming toxin presents ionic selectivity but this is adjusted by the particular conduction state of the channels.

Topography of the TH5 Segment in the Diphtheria Toxin T-Domain Channel.

The translocation domain (T-domain) of diphtheria toxin contains 10 α helices in the aqueous crystal structure. Upon exposure to a planar lipid bilayer under acidic conditions, it inserts to form a channel and transport the attached amino-terminal catalytic domain across the membrane. The TH5, TH8, and TH9 helices form transmembrane segments in the open-channel state, with TH1-TH4 translocated across the membrane. The TH6-TH7 segment also inserts to form a constriction that occupies only a small portion of the total channel length. Here, we have examined the TH5 segment in more detail, using the substituted-cysteine accessibility method. We constructed a series of 23 mutant T-domains with single cysteine residues at positions in and near TH5, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents added to the solutions on either side of the membrane. For 15 of the mutants, the reagent caused a decrease in single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. We also found that reaction caused large changes in ionic selectivity for some mutant channels. We determined whether reaction occurred in the open state or in the brief flicker-closed state of the channel. Finally, we compared the reaction rates from either side of the membrane. Our experiments are consistent with the hypotheses that the TH5 helix has a transmembrane orientation and remains helical in the open-channel state; they also indicate that the middle of the helix is aligned with the constriction in the channel.

TRPV1 Ionic Selectivity Dynamics are Modulated by Intracellular ATP

It was recently shown that TRPV1, a chemically- and thermally-activated cation channel highly expressed in sensory neurons, undergoes time- and agonist-dependent changes in ionic selectivity. More specifically, permeability to large cations such as N-methyl-D-glucamine (NMDG) increases during prolonged activation by capsaicin and resiniferatoxin. These changes in ionic selectivity are dose-dependent, being evoked at high, but not low, concentrations of agonist. Furthermore, protein kinase C-mediated phosphorylation of TRPV1 results in a leftward shift of the dose-response curve, inducing changes in ionic selectivity at low agonist concentrations. In addition to phosphorylation, the activity of TRPV1 is regulated by a number of mechanisms, including the binding of nucleotides and the local concentration of phospholipids. We have consequently expanded this study to investigate whether these other factors known to regulate channel activity can also modulate ionic selectivity.Whole-cell voltage-clamp recordings were taken from HEK293 cells stably expressing the wild-type rat TRPV1 channel, and capsaicin-evoked changes in NMDG permeability were monitored in the presence of intracellular ATP, or following the acute production or depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) at the plasma membrane. Neither the inclusion of ATP in the patch pipette nor an acute alteration in PIP2 levels resulted in a change to the increase in NMDG permeability evoked by micromolar concentrations of capsaicin. ATP did however induce changes in ionic selectivity at nanomolar concentrations of agonist, shifting the dose-response curve to the left, in a manner similar to that previously reported for PKC-mediated phosphorylation. With repeated agonist application, changes in NMDG permeability exhibit tachyphylaxis. Preliminary data suggest that increasing PIP2 levels in the vicinity of TRPV1 may inhibit this tachyphylaxis. Thus, dynamic ionic selectivity in TRPV1 can be differentially regulated by a number of intracellular factors.

Fe65 Interacts with P2X2 Subunits at Excitatory Synapses and Modulates Receptor Function

Ionotropic receptors in the neuronal plasma membrane are organized in macromolecular complexes, which assure their proper localization and regulate signal transduction. P2X receptors, the ionotropic receptors activated by extracellular ATP, have been shown to influence synaptic transmission. Using a yeast two-hybrid approach with the P2X(2) subunit C-terminal domain as bait we isolated the beta-amyloid precursor protein-binding proteins Fe65 and Fe65-like 1 as the first identified proteins interacting with neuronal P2X receptors. We confirmed the direct interaction of Fe65 and the P2X(2) C-terminal domain by glutathione S-transferase pull-down experiments. No interaction was observed between Fe65 and the naturally occurring P2X(2) splice variant P2X(2(b)), indicating that alternative splicing can regulate the receptor complex assembly. We generated two antibodies to Fe65 to determine its subcellular localization using postembedding immunogold labeling electron microscopy. We found labeling for Fe65 at the pre- and postsynaptic specialization of CA1 hippocampal pyramidal cell/Schaffer collateral synapses. By double immunogold labeling, we determined that Fe65 colocalizes with P2X(2) subunits at the postsynaptic specialization of excitatory synapses. Moreover, P2X(2) and Fe65 could be coimmunoprecipitated from brain membrane extracts, demonstrating that the interaction occurs in vivo. The assembly with Fe65 regulates the functional properties of P2X(2) receptors. Thus, the time- and activation-dependent change in ionic selectivity of P2X(2) receptors was inhibited by coexpression of Fe65, suggesting a novel role for Fe65 in regulating P2X receptor function and ATP-mediated synaptic transmission.

Contribution of Transmembrane Regions to ATP-gated P2X2 Channel Permeability Dynamics

ATP-gated P2X(2) channels undergo activation-dependent permeability increases as they proceed from the selective I(1) state to the I(2) state that is readily permeable to organic cations. There are two main models about how permeability changes may occur. The first proposes that permeability change-competent P2X channels are clustered or redistribute to form such regions in response to ATP. The second proposes that permeability changes occur because of an intrinsic conformational change in P2X channels. In the present study we experimentally tested these views with total internal reflection fluorescence microscopy, electrophysiology, and mutational perturbation analysis. We found no evidence for clusters of P2X(2) channels within the plasma membrane or for cluster formation in response to ATP, suggesting that channel clustering is not an obligatory requirement for permeability changes. We next sought to identify determinants of putative intrinsic conformational changes in P2X(2) channels by mapping the transmembrane domain regions involved in the transition from the relatively selective I(1) state to the dilated I(2) state. Initial channel opening to the I(1) state was only weakly affected by Ala substitutions, whereas dramatic effects were observed for the higher permeability I(2) state. Ten residues appeared to perturb only the I(1)-I(2) transition (Phe(31), Arg(34), Gln(37), Lys(53), Ile(328), Ile(332), Ser(340), Gly(342), Trp(350), Leu(352)). The data favor the hypothesis that permeability changes occur because of permissive motions at the interface between first and second transmembrane domains of neighboring subunits in pre-existing P2X(2) channels.

A glucocorticoid-induced Na+ conductance in human airway epithelial cells identified by perforated patch recording.

The perforated patch recording technique was used to investigate the effects of dexamethasone (0.2 microm, 24-30 h), a synthetic glucocorticoid, on membrane conductance in the human airway epithelial cell line H441. Under zero current clamp conditions this hormone induced amiloride-sensitive depolarization of the membrane potential (V(m)). Lowering external Na(+) to 10 mm by replacing Na(+) with N-methyl-d-glucammonium (NMDG(+)) also hyperpolarized the dexamethasome-treated cells, whilst replacing Na(+) with Li(+) caused a small depolarization. Although V(m) was insensitive to amiloride in control cells, NMDG(+) substitution caused a small hyperpolarization and so an amiloride-insensitive cation conductance is present. Replacing Na(+) with Li(+) had no effect on V(m) in such cells. Voltage clamp studies of dexamethasone-treated cells showed that the amiloride-sensitive component of the membrane current reversed at a potential close to the Na(+) equilibrium potential (E(Na)), and replacing Na(+) with K(+) caused a leftward shift in reversal potential (V(Rev)) that correlated with the corresponding shift in E(Na). Lowering [Na(+)](o) to 10 mm, the concentration in the pipette solution, by substitution with NMDG(+) shifted V(Rev) to 0 mV, whilst replacing Na(+) with Li(+) caused a rightward shift. Exposing dexamethasone-treated cells to a cocktail of cAMP-activating compounds (20 min) caused a approximately 2-fold increase in amiloride-sensitive conductance that was associated with no discernible change in ionic selectivity and an 18 mV depolarization. Dexamethasone thus induces the expression of a selective Na(+) conductance with a substantial permeability to Li(+) that is subject to acute regulation via cAMP. These data thus suggest that selective Na(+) channels underlie cAMP-regulated Na(+) transport in airway epithelia.

The Link between Ion Permeation and Inactivation Gating of Kv4 Potassium Channels

Kv4 potassium channels undergo rapid inactivation but do not seem to exhibit the classical N-type and C-type mechanisms present in other Kv channels. We have previously hypothesized that Kv4 channels preferentially inactivate from the preopen closed state, which involves regions of the channel that contribute to the internal vestibule of the pore. To further test this hypothesis, we have examined the effects of permeant ions on gating of three Kv4 channels (Kv4.1, Kv4.2, and Kv4.3) expressed in Xenopus oocytes. Rb + is an excellent tool for this purpose because its prolonged residency time in the pore delays K + channel closing. The data showed that, only when Rb + carried the current, both channel closing and the development of macroscopic inactivation are slowed (1.5- to 4-fold, relative to the K + current). Furthermore, macroscopic Rb + currents were larger than K + currents (1.2- to 3-fold) as the result of a more stable open state, which increases the maximum open probability. These results demonstrate that pore occupancy can influence inactivation gating in a manner that depends on how channel closing impacts inactivation from the preopen closed state. By examining possible changes in ionic selectivity and the influence of elevating the external K + concentration, additional experiments did not support the presence of C-type inactivation in Kv4 channels.

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.

The K+ channel signature sequence of murine Kir2.1: mutations that affect microscopic gating but not ionic selectivity.

1. We have studied the effects on ionic selectivity and gating of Kir2.1 of replacing Tyr (Y) in the GYG signature sequence with Phe (Y145F), Leu (Y145L), Met (Y145M), Ala (Y145A) or Val (Y145V). 2. The mutant Y145F showed no changes in ionic selectivity (as indicated by the permeability coefficient ratios PNa/PK or PRb/PK), indicating that a hydrogen bond between Tyr and other residues is not essential for K+ selectivity. Y145L, Y145M, Y145A and Y145V did not express as monomers. 3. None of the channels made from covalently linked tandem dimers with wild-type and mutant subunits (WT-mutant) had altered ionic selectivity (PNa/PK or PRb/PK), indicating that 4-fold symmetry is not required. 4. Macroscopic currents activated under hyperpolarization and the time constants for activation were reduced e-fold per 23 mV hyperpolarization in wild-type. This gating, believed to be due to the release of polyamines from the pore, was little affected by mutation of Y14. There was similarly little effect on the relationship between chord conductance (gK) and membrane potential. 5. Unitary conductance (140 mM [K+]o) was also little affected by mutation and was reduced only in channels formed from WT-Y145M, from 22.7 +/- 0.4 pS (n = 5) in wild-type to 17.1 +/- 0.5 pS (n = 4) in WT-Y145M. 6. Steady-state recording of unitary currents showed that channel open times were affected by the residue that replaced Tyr in GYG. Channel openings were particularly brief in WT-Y145V, where the mean open time was reduced from 102 ms at -120 mV in wild-type to 6 ms in WT-Y145V. 7. Thus in Kir2.1, GFG can act as a K+ selectivity filter, as can G(L/M/A/V)G, at least in dimers also containing GYG. Channel open time duration depended on the residue at position 145, consistent with the H5 region helping to determine the dwell time of the channel in the open state.

Dynamic selectivity filters in ion channels.

This work was supported by a Wellcome Trust (UK) International Prize Travelling Fellowship and the NIH (NS-11756).

Changes in ionic selectivity with changes in density of water in gels and cells

Gels equilibrated with aqueous solutions of impermeant solutes reached a steady state in which, in the absence of a pressure difference, the activity of water in the pores of the gel was higher than that of water in the external solution. The chemical potential of water in the gel/polymer solution slurry was higher than that in the supernatant polymer solution removed from the gel. Water in the pores of the gel decreased in density to 0.96 as increasing osmotic stress was applied. It is argued that at constant temperature and pressure water can equilibrate between two compartments of unequal osmolality only by adjusting its molar volume. Experiments showed that when gel water had a higher activity than external water it was K(+) selective; when it had a lower activity it was Na(+) selective. It is proposed that a continuous spectrum of water structures can exist in these two compartment systems from dense, reactive, weakly-bonded water which selects highly hydrated ions, to expanded, stretched, unreactive, viscous water which is strongly hydrogen bonded and selects K(+) and univalent anions. These findings are related to the state and properties of cytoplasmic water which is probably held under osmotic stress by the activity of the sodium pump.

Potential-dependent changes in ionic selectivity of batrachotoxin-modified sodium channels of frog nerve fiber

Currents through batrachotoxin-modified sodium channels in frog myelinated fibres were measured under voltage-clamp conditions. Reversal potential (Erev) of steady-state currents is shown to be about 5 mV less positive than Erev of initial (peak) currents. Control experiments with procaine and tetrodotoxin in external solutions showed that this shift of Erev during depolarizing pulse cannot be accounted for by the presence of unmodified sodium channels, unblocked potassium channels, nonlinearity of the leakage or any changes in transmembrane gradients of current-carrying cations. "Instantaneous" current measurements showed that Erev becomes less positive as amplitude and duration of preliminary depolarization increase. The results obtained are consistent with assumption that sodium-potassium selectivity of the batrachotoxin-modified channels depends on potential.

The specificity of the sodium channel for monovalent cations.

The specificity of bthe Na+ channel for monovalent cations has been analyzed in three different ways using neutrotoxins and excitable cells in culture. Influx experiments using radioactive monovalent cations have shown that all neurotoxins or combination of neurotoxins which change the gating system of the Na+ channel (veratridine, batrachotoxin, dihydrograyanotoxin, scorpion and sea‐anemone toxins) induce a considerable alteration of the permeability of the channel for monovalent cations. Theer is a much decreased ionic selectivity of the channel. Toxin‐induced changes in ionic selectivity are the same or very similar in neuroblastoma cells and in skeletal myotubes. Thefact that toxins which change the kinetics of the gating system also change channel selectivity indicates that the selectivity filter and the gating system may be coupled entities. Efflux experiments using neuroblastoma cells preloaded with 22N+, 42K+ or 86Rb+ indicate that the toxin stimulated release of these monovalent cations through the Na+ channel is not seen in the presence of cholinium in the external medium. It is seen only if the external medium contains permeant monovalent cations. The ‘chemical’ activation of the Na+ channel by neurotoxins necessitates the saturation at the external face of the channel of sites specific for permeant monovalent cations. Dose‐response curves for the effect of increasing external concentrations of Na+, K+, Rb+ or guanidinium on 22Na+ efflux indicate positive cooperativity in the stimulating effect of these cations. The best apparent affinity is observed for guanidinium, then for Na+, K+ and Rb+. Competition experiments between monovalent cations and a radiolabelled tritiated derivative of tetrodotoxin for binding to the tetrodotoxin receptor of the Na+ channel in neuroblastoma cells indicatae that monovalent cations bind cooperatively to external sites for Na+. The Hill coefficients and the dissociation constants for Na+ and guanidinium binding to the Na+ channel measured with this approach compare well to values found from 22Na+efflux experiments.

Monolayer and Interfacial Permeation

Transport across physical-chemical interfaces is considered in connection with three particular problems of biological interfaces: the structure and properties of cell membranes, the properties of the lung surfactant, and the effects of ionic currents across excitable membranes. With regard to cell membranes, studies of monolayer permeation suggest that permselectivity on the basis of size is a property of bilayer structure and probably gives rise to the observed dependence of the permeability on partition coefficients. The permeabilities of lipid and protein monolayers are consistent with the bimolecular leaflet (BML) model of the membrane and not with mosaic models. Experiments with the lung surfactant indicate that, in addition to its surface tension-lowering properties, it is unusual in its ability to form a strong two-dimensional network, which probably contributes to alveolar stability. Finally, the results of studies of interfacial ionic transference suggest a new way of accounting for the ionic fluxes in excitable membranes during an action potential without assuming ion-selective pores or carriers. In the suggested mechanism, it is possible to account for the change in ionic selectivity and the proper phasing of the fluxes, as well as other aspects of excitation in natural membranes.