Single-channel Current Measurements Research Articles

Evidence of the different effect of mercury and cadmium on the hIAPP aggregation process

hIAPP is a hormone consisting of 37 aminoacids that shows a strong tendency to self-assemble into β-sheet-rich aggregates, which evolve to form insoluble aggregates that seem to be associated with β-cell degeneration in Type 2 Diabetes Mellitus. Numerous factors, intrinsic and extrinsic to the peptide molecule, appear to influence the hIAPP aggregation process. Different metal ions are able to interact with the hIAPP molecule, modulating its secondary structure and subsequently the peptide's capacity to aggregate. In this study, the effect of Hg2+ and Cd2+ on the hIAPP aggregation process was evaluated using direct and indirect methods. The kinetics and morphology of amyloid aggregate formation were respectively evaluated with Thioflavin T assays and electron microscopy, while the ability of the peptide to incorporate into POPC PLMs and form ion channels was monitored by single-channel current measurements. Hg2+ and Cd2+ each seem to modulate the peptide's ability to aggregate in a different way, suggesting a different mechanism of hIAPP toxicity.

α4β2 Nicotinic Acetylcholine Receptors: RELATIONSHIPS BETWEEN SUBUNIT STOICHIOMETRY AND FUNCTION AT THE SINGLE CHANNEL LEVEL

Acetylcholine receptors comprising α4 and β2 subunits are the most abundant class of nicotinic acetylcholine receptor in the brain. They contribute to cognition, reward, mood, and nociception and are implicated in a range of neurological disorders. Previous measurements of whole-cell macroscopic currents showed that α4 and β2 subunits assemble in two predominant pentameric stoichiometries, which differ in their sensitivity to agonists, antagonists, and allosteric modulators. Here we compare agonist-elicited single channel currents from receptors assembled with an excess of either the α4 or β2 subunit, forming receptor populations biased toward one or the other stoichiometry, with currents from receptors composed of five concatemeric subunits in which the subunit stoichiometry is predetermined. Our results associate each subunit stoichiometry with a unique single channel conductance, mean open channel lifetime, and sensitivity to the allosteric potentiator 3-[3-(3-pyridinyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS-9283). Receptors with the composition (α4β2)2α4 exhibit high single channel conductance, brief mean open lifetime, and strong potentiation by NS-9283, whereas receptors with the composition (α4β2)2β2 exhibit low single channel conductance and long mean open lifetime and are not potentiated by NS-9283. Thus single channel current measurements reveal bases for the distinct functional and pharmacological properties endowed by different stoichiometries of α4 and β2 subunits and establish pentameric concatemers as a means to delineate interactions between subunits that confer these properties.

Correlated trapping of sugar molecules by the trimeric protein channel chitoporin

The protein channel chitoporin (ChiP), which is used by marine bacteria to translocate selected sugar molecules through the outer cell membrane, is studied via single channel current measurements in water and heavy water sugar solutions. The dynamic trapping and escape probabilities of sugar molecules from different monomers in the trimeric channel are characterized, including their dependence on channel orientation and sensitivity to a deuterium isotope effect. A detailed analysis of stochastic current fluctuations reveals that the trapping properties of chitoporin exhibit memory effects: the rate of trapping transitions depends on the previous sequence of transitions; and intermonomer correlations: the average trapping rate of an unblocked monomer is larger when its neighboring monomers are blocked. The latter, likely resulting from rapid re-trapping of recently escaped sugar molecules, is considered as a possible design strategy to enhance sugar transport.

Probing Single-Molecule Ion Channel Conformational Dynamics in Living Cells

Stochastic and inhomogeneous conformational changes regulate the function and dynamics of ion channels that are crucial for cell functions, neuronal signaling, and brain functions. We have developed a new combined approaches of using single ion channel patch-clamp electrical recording and single-molecule fluorescence imaging for probing ion channel conformational changes simultaneously with the electrical single channel recording. By combining real-time single-molecule fluorescence imaging measurements with real-time single-channel electric current measurements in artificail lipid bilayers and in living cell membranes, we were able to probe single ion-channel-protein conformational changes simultaneously, and thus providing an understanding the dynamics and mechanism of ion-channel proteins at the molecular level. We will focus our discussion on the new development and results of real-time imaging of the dynamics of gramicidin, colicin, and NMDA receptor ion channels in lipid bilayers and living cells. We have probed NMDA (N-Methyl-D-Aspartate) receptor ion channel in live HEK-293 cell, especially, the single ion channel open-close activity and its associated protein conformational changes simultaneously. Furthermore, we have revealed that the seemingly identical electrically off states are associated with multiple conformational states. Based on our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open-close dynamics. Our results shed light on new perspectives of the intrinsic interplay of lipid membrane dynamics, solvation dynamics, and the ion channel functions.

Mechanisms of Anion Conduction by Coupled Glutamate Transporters

Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na(+)/H(+)/K(+) transporters but also function as anion-selective channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. We have combined molecular dynamics simulations with fluorescence spectroscopy of the prokaryotic homolog GltPh and patch-clamp recordings of mammalian EAATs to determine how these transporters conduct anions. Whereas outward- and inward-facing GltPh conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Fluorescence quenching of inserted tryptophan residues indicated the entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on GltPh simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels.

Catalyst-like modulation of transition states for CFTR channel opening and closing: New stimulation strategy exploits nonequilibrium gating

Cystic fibrosis transmembrane conductance regulator (CFTR) is the chloride ion channel mutated in cystic fibrosis (CF) patients. It is an ATP-binding cassette protein, and its resulting cyclic nonequilibrium gating mechanism sets it apart from most other ion channels. The most common CF mutation (ΔF508) impairs folding of CFTR but also channel gating, reducing open probability (Po). This gating defect must be addressed to effectively treat CF. Combining single-channel and macroscopic current measurements in inside-out patches, we show here that the two effects of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on CFTR, pore block and gating stimulation, are independent, suggesting action at distinct sites. Furthermore, detailed kinetic analysis revealed that NPPB potently increases Po, also of ΔF508 CFTR, by affecting the stability of gating transition states. This finding is unexpected, because for most ion channels, which gate at equilibrium, altering transition-state stabilities has no effect on Po; rather, agonists usually stimulate by stabilizing open states. Our results highlight how for CFTR, because of its unique cyclic mechanism, gating transition states determine Po and offer strategic targets for potentiator compounds to achieve maximal efficacy.

Three Novel Pathways to Stabilize the Conductive Conformation of a Voltage-Gated K+ Channel

Voltage-gated K+ channels (Kv) are tetrameric assemblies in which 4 voltage sensors open a shared K+ selective pore located at the center of the array at activating membrane potentials. A feature of many Kv channels- indispensable for the repolarization of the membrane potential following depolarization of neuronal cells- is to shut off their flux after prolonged activation. Here we show that in KvLm, a well characterized bacterial voltage-gated channel, the premature closing of the pore is bypassed by the association of the filter gate of the pore with three distinct and novel open-conformation stabilizers: A positively charged lipid, a small-molecule anti-depressant, and a protein toxin. This analysis is based on an extensive set of single channel current measurements of purified KvLm and its sensorless pore reconstituted in symmetric and asymmetric lipid bilayer membranes. Comparative assessment of the consequences produced by the three modulators in relieving the “inactivation” establishes that stabilization of the open conformation proceeds in the absence or presence of the sensors. We propose two plausible mechanisms by which all three modulators exert their demands on the pore.This work was supported by NIH grant GM 49711.

Single-channel electrophysiology of cell-free expressed ion channels by direct incorporation in lipid bilayers

Single-channel electrophysiology with lipid bilayer systems requires ion channel expression, purification from cell culture, and reconstitution in proteoliposomes for delivery to a planar bilayer. Here we demonstrate that single-channel current measurements of the potassium channels KcsA and hERGS5-S6 can be obtained by direct insertion in interdroplet lipid bilayers from microliters of a cell-free expression medium.

Cyclodextrin-Scaffolded Alamethicin with Remarkably Efficient Membrane Permeabilizing Properties and Membrane Current Conductance

Bacterial resistance to classical antibiotics is a serious medical problem, which continues to grow. Small antimicrobial peptides represent a potential solution and are increasingly being developed as novel therapeutic agents. Many of these peptides owe their antibacterial activity to the formation of trans-membrane ion-channels resulting in cell lysis. However, to further develop the field of peptide antibiotics, a thorough understanding of their mechanism of action is needed. Alamethicin belongs to a class of peptides called peptaibols and represents one of these antimicrobial peptides. To examine the dynamics of assembly and to facilitate a thorough structural evaluation of the alamethicin ion-channels, we have applied click chemistry for the synthesis of templated alamethicin multimers covalently attached to cyclodextrin-scaffolds. Using oriented circular dichroism, calcein release assays, and single-channel current measurements, the α-helices of the templated multimers were demonstrated to insert into lipid bilayers forming highly efficient and remarkably stable ion-channels.

Characterization of Gating Mechanisms in Prokaryotic Pentameric Ligand-Gated Ion Channels

Pentamericligand-gated ion channels (pLGIC) mediate fast neurotransmission in the central and peripheral nervous systems and thereby regulate excitability, neurotransmitter release and muscle contraction. Upon activation, these channels rapidly switch between closed, open and desensitized conformations. The recent crystal structures of the prokaryotic pLGIC homologs from E. chrysanthemi (ELIC) and G. violaceus (GLIC) in presumably the closed and open channel conformations, respectively have provided excellent models for an atomic-level understanding of the channel structure and its role in selective ion-conduction and drug binding. However, it is still unclear as to what functional states of the pLGIC gating-scheme do these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. Here we have carried out an extensive characterization of GLIC function by both macroscopic and single-channel current measurements in reconstituted proteoliposomes. We find that GLIC channels show rapid activation (milliseconds timescale) upon jumps to acidic pH followed by a time-dependent loss of conductance (seconds timescale) due to desensitization. We have investigated various aspects of channel activation and desensitization and determined how these gating events are modulated by pH, voltage, permeant ions, pore-blockers and membrane lipid compositions. Analyzed in the framework of recent structures, these results uncover several important features of gating mechanisms in GLIC and how they relate to their eukaryotic counterparts.

The Effect of Anions on the Human P2X7 Receptor

P2X7 receptors (P2X7Rs) are nonselective cation channels that are opened by the binding of extracellular ATP and are involved in the modulation of epithelial secretion, inflammation and nociception. Here, we investigated the effect of extracellular anions on channel gating and permeation of human P2X7Rs (hP2X7Rs) expressed in Xenopus laevis oocytes. Two-microelectrode voltage-clamp recordings showed that ATP-induced hP2X7R-mediated currents increased when extracellular chloride was substituted by the organic anions glutamate or aspartate and decreased when chloride was replaced by the inorganic anions nitrate, sulfate or iodide. ATP concentration-response comparisons revealed that substitution of chloride by glutamate decreased agonist efficacy, while substitution by iodide increased agonist efficacy at high ATP concentrations. Meanwhile, the ATP potency remained unchanged. Activation of the hP2X7R at low ATP concentrations via the high-affinity ATP effector site was not affected by the replacement of chloride by glutamate or iodide. To analyze the anion effect on the hP2X7R at the single-molecule level, we performed single-channel current measurements using the patch-clamp technique in the outside-out configuration. Chloride substitution did not affect the single-channel conductance, but the probability that the P2X7R channel was open increased when chloride was replaced by glutamate and decreased when chloride was replaced by iodide. This effect was due to an influence of the anions on the mean closed times of the hP2X7R channel. We conclude that hP2X7R channels are not anion-permeable in physiological Na+-based media and that external anions allosterically affect ion channel opening in the fully ATP4--liganded P2X7R through an extracellular anion binding site.This work was supported by Deutsche Forschungsgemeinschaft Grants Ma1581/15-1 (to F.M.) and Schm536/9-1 (to G. S.) and by the Medical Faculty of the Martin-Luther-University Roux Program Grant 22/18 (to F. M.).

The effect of anions on the human P2X7 receptor

P2X7 receptors (P2X7Rs) are nonselective cation channels that are opened by the binding of extracellular ATP and are involved in the modulation of epithelial secretion, inflammation and nociception. Here, we investigated the effect of extracellular anions on channel gating and permeation of human P2X7Rs (hP2X7Rs) expressed in Xenopus laevis oocytes. Two-microelectrode voltage-clamp recordings showed that ATP-induced hP2X7R-mediated currents increased when extracellular chloride was substituted by the organic anions glutamate or aspartate and decreased when chloride was replaced by the inorganic anions nitrate, sulfate or iodide. ATP concentration–response comparisons revealed that substitution of chloride by glutamate decreased agonist efficacy, while substitution by iodide increased agonist efficacy at high ATP concentrations. Meanwhile, the ATP potency remained unchanged. Activation of the hP2X7R at low ATP concentrations via the high-affinity ATP effector site was not affected by the replacement of chloride by glutamate or iodide. To analyze the anion effect on the hP2X7R at the single-molecule level, we performed single-channel current measurements using the patch-clamp technique in the outside-out configuration. Chloride substitution did not affect the single-channel conductance, but the probability that the P2X7R channel was open increased when chloride was replaced by glutamate and decreased when chloride was replaced by iodide. This effect was due to an influence of the anions on the mean closed times of the hP2X7R channel. We conclude that hP2X7R channels are not anion-permeable in physiological Na+-based media and that external anions allosterically affect ion channel opening in the fully ATP4-liganded P2X7R through an extracellular anion binding site.

Single Channel Measurements of N-Acetylneuraminic Acid-Inducible Channel (NANC) in E. coli

Escherichia coli can use N-acetylneuraminic acid (Neu5Ac) as its sole carbon source even if the general outer membrane proteins OmpF and OmpC are not expressed: NanC - a monomeric outer membrane channel - allows Neu5Ac to move into the bacterial periplasm. Recently, a high resolution structure of NanC in two different crystal forms was reported by Wirth et al., J.Mol.Biol., (2009) 394:718 (PDB codes: 2WJQ and 2WJR). Our goal is to determine appropriate ‘baseline’ ionic conditions to study the transport of Neu5Ac through NanC using single channels in lipid bilayers. Measurements of single channel currents showed that NanC has two modes of time dependent behavior (‘gating’). In the many situations we have tested, the modes are not induced or changed by surrounding ionic conditions or voltage. Single channels of NanC at pH 7.0 have: (1) a large conductance (around 100 pS to 800 pS in 100 mM KCl to 3M KCl) that varies with the polarity of the applied voltage; (2) anion over cation selectivity (Vreversal around +16 mV in 250 mM KCl ‖ 1 M KCl); (3) voltage-dependent gating (channel closures above ±200 mV). Single channel conductance of NanC decreases about 50% when HEPES concentration is increased from 100 µM to 100 mM in 250 mM KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure (PDB code: 2WJR). Studying alternative buffers, we found that phosphate interferes with the channel conductance, whereas TRIS could not be used because it reacts with Ag/AgCl electrodes producing artifacts even in the presence of Agar-KCl bridges. Our further studies of NanC will use no pH buffers, but low concentration (250 mM) salt solutions adjusted to neutral pH 7.0.

Probing Single-Molecule Ion Channel Dynamics by Combined Patch-Clamp Single-Molecule Fret Imaging Microscopy

Stochastic and inhomogeneous conformational changes regulate the function and dynamics of ion channels. Such complexity makes it difficult, if not impossible, to characterize ion channel dynamics using conventional electrical recording alone since that the measurement does not specifically interrogate the associated conformational changes but rather the consequences of the conformational changes. Recently, new technology developments on single-molecule spectroscopy, and especially, the combined approaches of using single ion channel patch-clamp electrical recording and single-molecule fluorescence imaging have provided us the capability of probing ion channel conformational changes simultaneously with the electrical single channel recording. By combining real-time single-molecule fluorescence imaging measurements with real-time single-channel electric current measurements in artificail lipid bilayers and in living cell membranes, we were able to probe single ion-channel-protein conformational changes simultaneously, and thus providing an understanding the dynamics and mechanism of ion-channel proteins at the molecular level. The function-regulating and site-specific conformational changes of ion channels are now measurable under physiological conditions in real-time, one molecule at a time. We will focus our discussion on the new development of real-time imaging of the dynamics of individual ion channels using a novel combination of single-molecule fluorescence spectroscopy and single-channel current recordings. We will then discuss a specific example of single-molecule gramicidin ion channel dynamics studied by the new approach and the future prospects.

Pannexin 1 Constitutes the Large Conductance Cation Channel of Cardiac Myocytes

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca(2+) release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.

Impact of distant charge reversals within a robust beta-barrel protein pore.

Among all beta-barrel pores, staphylococcal alpha-hemolysin (alphaHL), a heptameric transmembrane protein of known high-resolution crystal structure, features a high stability in planar lipid bilayers under a wide range of harsh experimental conditions. Here, we employed single-channel electrical recordings and standard protein engineering to explore the impact of two distant charge reversals within the interior of the beta-barrel part of the pore. The charge reversals were replacements of lysines with aspartic acids. A charge reversal within the structurally stiff region of the beta barrel near the pore constriction reduced the open-state current of the pore, but produced a quiet pore, showing current fluctuation-free channel behavior. In contrast, a charge reversal on the trans entrance, within the structurally flexible glycine-rich turn of the beta barrel, increased the open-state current and produced gating activity of the pore in the form of large-amplitude and frequent current fluctuations. Remarkably, cumulative insertion of the two distant charge reversals resulted in a large-amplitude permanent blockade of the beta barrel, as judged by both single-channel and macroscopic current measurements. The results from this work suggest that these distant charge reversals are energetically coupled, producing different impacts on the ionic transport, the unitary conductance and the open-state probability of the pore.

Effect of Altered Ion Occupancy on Permeation and Inactivation Gating in K+ Channel

The selectivity filter of a K+ channel is comprised of four equally-spaced K+-binding sites. In the first three sites the main-chain carbonyl atoms provide the octa-coordination for K+ ions. In the fourth site both main-chain carbonyl oxygen atoms and threonine side-chain hydroxyl oxygen atoms contribute to K+ coordination. This threonine is highly conserved among K+ channels with the only alternative being a serine substitution. Surprisingly, this conservative mutation, which preserves the hydroxyl moiety and the side chain polarity, has been shown to produce profound changes in permeation and gating in Shaker. Indeed, mutant T442S shows a dramatic destabilization of inactivation along with an increased conductance for Rb+ over K+. Although these studies provided early insights into the role of this position in ion coordination, the structural basis for altered single-channel conductances and gating kinetics remain unclear. KcsA has served as an archetypical K+ pore providing molecular insights into understanding selectivity, ion-permeation, gating pore-blocking and C-type inactivation. Equivalent mutation in KcsA (T75S) shows remarkably similar effects as seen in Shaker, including effects on inactivation, single-channel conductance, selectivity and rectification. Combining single-channel and macroscopic current measurements along with X-ray crystallography under different ionic conditions we now provide atomistic details that underlie these divergent channel properties and the role of Thr75 in channel function.

An Ion Selectivity Filter in the Extracellular Domain of Cys-loop Receptors Reveals Determinants for Ion Conductance

Neurotransmitter binding to Cys-loop receptors promotes a prodigious transmembrane flux of several million ions/s, but to date, structural determinants of ion flux have been identified flanking the membrane-spanning region. Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO4(2-) with a ring of lysines at a position equivalent to 24 A above the lipid membrane in homologous Cys-loop receptors. Analysis of multiple sequence alignments revealed that residues equivalent to the ring of lysines are negatively charged in cation-selective receptors but are positively charged in anion-selective receptors. Charge reversal of side chains at homologous positions in the nicotinic receptor from the motor end plate decreases unitary conductance up to 80%. Selectivity filters stemming from transmembrane alpha-helices have similar pore diameters and compositions of amino acids. These findings establish that when the channel opens under a physiological electrochemical gradient, permeant ions are initially stabilized within the extracellular vestibule of Cys-loop receptors, and this stabilization is a major determinant of ion conductance.

Asymmetry of Rb+ Conduction Emerged under Bi-Ionic Conditions in Epithelial Maxi-K+ Channels

K(+) channels permit more than one ion within their conducting pathway at any given moment and show a saturating single-file behavior. The conduction of Rb(+) shows an unusual behavior, a so-called "Rb(+) anomaly," and it has been used to probe the mechanism of the ion conduction through K(+)-selective channels. Under the bi-ionic condition of K(+) and Rb(+), we carried out patch-clamp single-channel current measurements in MaxiK(+) channels from mouse submandibular acinar cells. Keeping only K(+) on one side of the membrane while varying fractional Rb(+) concentration on the opposite, we had a series of current-voltage relationships. It showed a characteristic inflection at which the ion conductance was divided into two components, one ascribed to pure K(+) conduction and the other to K(+) and Rb(+) bi-ionic conduction. By analyzing the latter, we depicted that (1) the bi-ionic conductance showed a characteristic reduction curve as the Rb(+) fractional concentration increased; (2) Rb(+) can bind the channel more tightly when it accesses from the outside than from the inside. Thus we conclude that such asymmetry of the Rb(+) binding determines the pattern of bi-ionic conductance reduction in K-selective channels.

A Quantitative Description of KcsA Gating I: Macroscopic Currents

The prokaryotic K+ channel KcsA is activated by intracellular protons and its gating is modulated by transmembrane voltage. Typically, KcsA functions have been studied under steady-state conditions, using macroscopic Rb+-flux experiments and single-channel current measurements. These studies have provided limited insights into the gating kinetics of KcsA due to its low open probability, uncertainties in the number of channels in the patch, and a very strong intrinsic kinetic variability. In this work, we have carried out a detailed analysis of KcsA gating under nonstationary conditions by examining the influence of pH and voltage on the activation, deactivation, and slow-inactivation gating events. We find that activation and deactivation gating of KcsA are predominantly modulated by pH without a significant effect of voltage. Activation gating showed sigmoidal pH dependence with a pKa of ∼4.2 and a Hill coefficient of ∼2. In the sustained presence of proton, KcsA undergoes a time-dependent decay of conductance. This inactivation process is pH independent but is modulated by voltage and the nature of permeant ion. Recovery from inactivation occurs via deactivation and also appears to be voltage dependent. We further find that inactivation in KcsA is not entirely a property of the open-conducting channel but can also occur from partially “activated” closed states. The time course of onset and recovery of the inactivation process from these pre-open closed states appears to be different from the open-state inactivation, suggesting the presence of multiple inactivated states with diverse kinetic pathways. This information has been analyzed together with a detailed study of KcsA single-channel behavior (in the accompanying paper) in the framework of a kinetic model. Taken together our data constitutes the first quantitative description of KcsA gating.