Single Ion Channel Currents Research Articles

Brownian Aging as One of the Mechanistic Components That Shape the Single-Channel Ionic Currents through Biological and Synthetic Membranes.

Semipermeable membranes enable the separation of a given system from its environment. In biological terms, they are responsible for cells' identity. In turn, the functioning of ion channels is crucial for the control of ionic fluxes across the membranes and, consequently, for the exchange of chemical and electrical signals. This paper presents a model and simulations of currents through ionic nanochannels in an attempt to better understand the physical mechanism(s) of open/closed (O/C) sequences, i.e., random interruptions of ionic flows through channels observed in all known biochannels and in some synthetic nanopores. We investigate whether aging, i.e., the changes in Brownian motion characteristics with the lapse of time, may be at least one of the sources of the O/C sequences (in addition to the gating machinery in biochannels). The simulations based on the approximated nanostructure of ion channels confirm this postulation. The results also show the possibility of changing the O/C characteristics through an appropriate alteration of the channel surroundings. This observation may be valuable in technical uses of nanochannels in synthetic membranes and allow for a better understanding of the reason for the differences between the biochannels' activity in diverse biological membranes. Proposals of experimental verification of this aging O/C hypothesis are also presented.

Application of Scanning Ion Conductance Microscopy in the Studies of Podocytes Morphological Changes

High‐resolution imaging of glomerular podocytes morphology is becoming an essential and helpful tool for studying their role in renal disease progression. Electron microscopy (EM) is commonly used to analyze the effacement of the podocytes’ cell foot processes and the glomerular filtration barrier integrity loss. However, EM does not allow studying living samples and requires multiple complex procedures for sample preparation. Here, we propose and demonstrate the successful application of Scanning Ion Conductance Microscopy (SICM) for studying morphological changes of cultured podocytes and freshly isolated glomeruli.SICM provides a high‐resolution (comparable to EM) non‐optical imaging of cell topography using hopping probe microscopy principles and new dynamic reconstruction software. This technique can be used for analysis of cell membrane morphology and different functional characteristics, such as cell volume, membrane potentials, single ion‐channel currents, and other parameters. The major advantage of the current method is that it can be applied to live samples with complex morphology, such as intact glomeruli, under physiologically relevant liquid conditions.We have established and demonstrated a successful application of the SICM method in the live human glomerulus and provided a proof of principle for future dynamic analysis of membrane morphology and varied functional parameters in the living glomerulus. As a proof of principle, we applied this method for estimation the pathological structural changes in podocyte foot processes in type 2 diabetic nephropathy (T2DN) rats. Using topographical quantification of the length and irregular shape of the podocyte foot processes, we showed an absence of interdigitated foot processes on the selected region of the glomerular slit diaphragm in isolated T2DN glomeruli.In addition to the application of SICM with freshly isolated glomeruli, we used this approach to visualize and quantify foot process dynamics in cultured human podocytes during pharmacological applications. Thus, we showed that destroying the F‐actin with cytochalasin D completely stopped the migration of the podocyte filopodium through the remodeling of the actin cytoskeleton. Here, we further applied SICM to detect morphological changes in human podocytes after activation of Protease‐activated receptor‐1 (PAR1) by acute application of agonist TFLLR‐NH2. A series of experiments revealed that PAR1 activation led to retraction of the lamellipodium and a significant decrease in cell surface area (+51±46 vs. ‐15±8 µm2, 30 min after application of vehicle or TFLLR, respectively; n=5, p<0.05).In summary, SICM has numerous advantages and has excellent potential for precise quantitative analyses of the morphological changes in the glomerulus and cultured podocytes. Moreover, it opens the possibility of performing patch‐clamp recordings on the specific parts of the podocyte cell body and advances our understanding of the function and mechanism of the glomerulus filtration barrier.

Integrated microfluidic biosensing platform for simultaneous confocal microscopy and electrophysiological measurements on bilayer lipid membranes and ion channels.

Combining high-resolution imaging and electrophysiological recordings is key for various types of experimentation on lipid bilayers and ion channels. Here, we propose an integrated biosensing platform consisting of a microfluidic cartridge and a dedicated chip-holder to conduct such dual measurements on suspended lipid bilayers, in a user-friendly manner. To illustrate the potential of the integrated platform, we characterize lipid bilayers in terms of thickness and fluidity while simultaneously monitoring single ion channel currents. For that purpose, POPC lipid bilayers are supplemented with a fluorescently-tagged phospholipid (NBD-PE, 1% mol) for Fluorescence Recovery After Photobleaching (FRAP) measurements and a model ion channel (gramicidin, 1 nM). These combined measurements reveal that NBD-PE has no effect on the lipid bilayer thickness while gramicidin induces thinning of the membrane. Furthermore, the presence of gramicidin does not alter the lipid bilayer fluidity. Surprisingly, in lipid bilayers supplemented with both probes, a reduction in gramicidin open probability and lifetime is observed compared to lipid bilayers with gramicidin only, suggesting an influence of NBD-PE on the gramicidin ion function. Altogether, our proposed microfluidic biosensing platform in combination with the herein presented multi-parametric measurement scheme paves the way to explore the interdependent relationship between lipid bilayer properties and ion channel function.

Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording.

Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 μV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation.

Charging the quantum capacitance of graphene with a single biological ion channel.

The interaction of cell and organelle membranes (lipid bilayers) with nanoelectronics can enable new technologies to sense and measure electrophysiology in qualitatively new ways. To date, a variety of sensing devices have been demonstrated to measure membrane currents through macroscopic numbers of ion channels. However, nanoelectronic based sensing of single ion channel currents has been a challenge. Here, we report graphene-based field-effect transistors combined with supported lipid bilayers as a platform for measuring, for the first time, individual ion channel activity. We show that the supported lipid bilayers uniformly coat the single layer graphene surface, acting as a biomimetic barrier that insulates (both electrically and chemically) the graphene from the electrolyte environment. Upon introduction of pore-forming membrane proteins such as alamethicin and gramicidin A, current pulses are observed through the lipid bilayers from the graphene to the electrolyte, which charge the quantum capacitance of the graphene. This approach combines nanotechnology with electrophysiology to demonstrate qualitatively new ways of measuring ion channel currents.

Charging the Quantum Capacitance of Graphene with a Single Biological Ion Channel

The interaction of cell and organelle membranes (lipid bilayers) with nanoelectronics can enable new technologies to sense and measure electrophysiology in qualitatively new ways. To date, a variety of sensing devices have been demonstrated to measure membrane currents through macroscopic numbers of ion channels. However, nanoelectronic based sensing of single ion channel currents has been a challenge. Here, we report graphene-based field-effect transistors combined with supported lipid bilayers as a platform for measuring, for the first time, individual ion channel activity. We show that the supported lipid bilayers uniformly coat the single layer graphene surface, acting as a biomimetic barrier that insulates (both electrically and chemically) the graphene from the electrolyte environment. Upon introduction of pore-forming membrane proteins such as alamethicin and gramicidin A, current pulses are observed through the lipid bilayers from the graphene to the electrolyte, which charge the quantum capacitan...

Local acute serotonergic modulation on the excitability of single lateral amygdala

Differentiated murine neuroblastoma cells (NIE-115) have receptors for 5-hydroxytyptamine (5-HT), the activation of which either depolarizes or hyperpolarizes these cells with associated increase in input conductance. Ionophoresis of 5-HT on to NIE-115 cells elicits brief depolarization, but the 5-HT receptors rapidly desensitive during repeated application of 5-HT. The reversal potential of 5-HT-induced depolarization is ca. 0 mV. With the use of the improved patch clamp technique it is possible to record single ion channel currents activated by 5-HT from intact cell as well as isolated membrane patches. These currents are invariably inward at the resting potential (∼ 50mV). The channel has a conductance of∼ 140pS and reversal potential of∼ 0mV in normal saline. The channels recorded from the intact cells reveal more than one kinetically distinguishable open and closed states. The kinetic properties of 5-HT channels recorded from excised patches of membrane (as outside-out) are different from those recorded from the intact cell. Possible explanations for such differences are presented.

Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells

Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

Experimental and simulation study of the effect of pipette roughness on giga-seal formation in patch clamping

The effect of pipette tip roughness on giga-seal formation of patch clamp recording has been studied through FEA simulation and patch clamp experiments. FEA simulation results show that the membrane cannot fill up all of the peaks and valleys of a rough pipette tip. As a result in three dimensions the seal between inside and outside is compromised by channels in the order of several nanometres. These channels increase the leakage current between two electrodes, increase the noise and decrease the seal resistance. In contrast focused-ion-beam polished pipettes have very flat tips. Single ion channel currents recorded by FIB polished pipettes show significantly smaller leakage current and noise than the currents recorded by conventional pipettes. Results of FEA simulation are consistent with the results of patch clamp experiments.

The unliganded long isoform of estrogen receptor beta stimulates brain ryanodine receptor single channel activity alongside with cytosolic Ca2+

Ca2+ release from intracellular stores mediated by endoplasmic reticulum membrane ryanodine receptors (RyR) plays a key role in activating and synchronizing downstream Ca2+-dependent mechanisms, in different cells varying from apoptosis to nuclear transcription and development of defensive responses. Recently discovered, atypical “nongenomic” effects mediated by estrogen receptors (ER) include rapid Ca2+ release upon estrogen exposure in conditions implicitly suggesting involvement of RyRs. In the present study, we report various levels of colocalization between RyR type 2 (RyR2) and ER type β (ERβ) in the neuronal cell line HT-22, indicating a possible functional interaction. Electrophysiological analyses revealed a significant increase in single-channel ionic currents generated by mouse brain RyRs after application of the soluble monomer of the long form ERβ (ERβ 1). The effect was due to a strong increase in open probability of RyR higher open channel sublevels at cytosolic [Ca2+] concentrations of 100 nM, suggesting a synergistic action of ERβ1 and Ca2+ in RyR activation, and a potential contribution to Ca2+-induced Ca2+ release rather than to basal intracellular Ca2+ concentration level at rest. This RyR/ERβ interaction has potential effects on cellular physiology, including roles of shorter ERβ isoforms and modulation of the RyR/ERβ complexes by exogenous estrogens.

Everything You Always Wanted to Know about Sachs' Seals

Everything You Always Wanted to Know about Sachs' Seals

Alternative splicing in the C‐terminus of CaV2.2 controls expression and gating of N‐type calcium channels

N-type Ca(V)2.2 calcium channels localize to presynaptic nerve terminals of nociceptors where they control neurotransmitter release. Nociceptive neurons express a unique set of ion channels and receptors important for optimizing their role in transmission of noxious stimuli. Included among these is a structurally and functionally distinct N-type calcium channel splice isoform, Ca(V)2.2e[37a], expressed in a subset of nociceptors and with limited expression in other parts of the nervous system. Ca(V)2.2[e37a] arises from the mutually exclusive replacement of e37a for e37b in the C-terminus of Ca(V)2.2 mRNA. N-type current densities in nociceptors that express a combination of Ca(V)2.2e[37a] and Ca(V)2.2e[37b] mRNAs are significantly larger compared to cells that express only Ca(V)2.2e[37b]. Here we show that e37a supports increased expression of functional N-type channels and an increase in channel open time as compared to Ca(V)2.2 channels that contain e37b. To understand how e37a affects N-type currents we compared macroscopic and single-channel ionic currents as well as gating currents in tsA201 cells expressing Ca(V)2.2e[37a] and Ca(V)2.2e[37b]. When activated, Ca(V)2.2e[37a] channels remain open for longer and are expressed at higher density than Ca(V)2.2e[37b] channels. These unique features of the Ca(V)2.2e[37a] isoform combine to augment substantially the amount of calcium that enters cells in response to action potentials. Our studies of the e37a/e37b splice site reveal a multifunctional domain in the C-terminus of Ca(V)2.2 that regulates the overall activity of N-type calcium channels in nociceptors.

Comparison between low-level 50 Hz and 900 MHz electromagnetic stimulation on single channel ionic currents and on firing frequency in dorsal root ganglion isolated neurons

Alteration of membrane surface charges represents one of the most interesting effects of the electromagnetic exposure on biological structures. Some evidence exists in the case of extremely low frequency whereas the same effect in the radiofrequency range has not been detected. Changes in transmembrane voltages are probably responsible for the mobilization of intracellular calcium described in some previous studies but not confirmed in others. These controversial results may be due to the cell type under examination and/or to the permeability properties of the membranes. According to such a hypothesis, calcium oscillations would be a secondary effect due to the induced change in the membrane voltage and thus dependent on the characteristics of ionic channels present in a particular preparation. Calcium increases could suggest more than one mechanism to explain the biological effects of exposure due to the fact that all the cellular pathways using calcium ions as a second messenger could be, in theory, disturbed by the electromagnetic field exposure. In the present work, we investigate the early phase of the signal transmission in the peripheral nervous system. We present evidence that the firing rate of rat sensory neurons can be modified by 50/60 Hz magnetic field but not by low level 900 MHz fields. The action of the 50/60 Hz magnetic field is biphasic. At first, the number of action potentials increases in time. Following this early phase, the firing rate decreases more rapidly than in control conditions. The explanation can be found at the single-channel level. Dynamic action current recordings in dorsal root ganglion neurons acutely exposed to the electromagnetic field show increased functionality of calcium channels. In parallel, a calcium-activated potassium channel is able to increase its mean open time.

Genetic alterations of the BRI2 gene: familial British and Danish dementias.

Classic arguments sustaining the importance of amyloid in the pathogenesis of dementia are usually centered on amyloid beta (Abeta) and its role in neuronal loss characteristic of Alzheimer disease, the most common form of human cerebral amyloidosis. Two non-Abeta cerebral amyloidoses, familial British and Danish dementias, share many aspects of Alzheimer disease, including the presence of neurofibrillary tangles, parenchymal pre-amyloid and amyloid deposits, cerebral amyloid angiopathy, and a widespread inflammatory response. Both early-onset conditions are linked to specific mutations in the BRI2 gene, causing the generation of longer-than-normal protein products and the release of 2 de novo created peptides ABri and ADan, the main components of amyloid fibrils in these inherited dementias. Although the molecular mechanisms and signal transduction pathways elicited by the amyloid deposits and their relation to cognitive impairment remain to be clarified, new evidence indicates that, independent of the differences in their primary structures, Abeta, ABri, and ADan subunits are able to form morphologically compatible ion-channel-like structures and elicit single ion-channel currents in reconstituted lipid membranes. These findings reaffirm the notion that non-Abeta amyloidosis constitute suitable alternative models to study the role of amyloid deposition in the mechanism of neuronal cell death.

Estimating rate constants from single ion channel currents when the initial distribution is known

Single ion channel currents can be analysed by hidden or aggregated Markov models. A classical result from Fredkin et al. (Proceedings of the Berkeley conference in honor of Jerzy Neyman and Jack Kiefer, vol I, pp 269-289, 1985) states that the maximum number of identifiable parameters is bounded by 2n(o)n(c), where n(o) and n(c) denote the number of open and closed states, respectively. We show that this bound can be overcome when the probabilities of the initial distribution are known and the data consist of several sweeps.

Ionic currents in isolated and in situ squid Schwann cells.

Ionic currents from Schwann cells isolated enzymatically from the giant axons of the squids Loligo forbesi, Loligo vulgaris and Loligo bleekeri were compared with those obtained in situ. Macroscopic and single channel ionic currents were recorded using whole-cell voltage and patch clamp. In the whole-cell configuration, depolarisation from negative holding potentials evoked two voltage-dependent currents, an inward current and a delayed outward current. The outward current resembled an outwardly rectifying K+ current and was activated at -40 mV after a latent period of 5-20 ms following a step depolarisation. The current was reduced by externally applied nifedipine, Co2+ or quinine, was not blocked by addition of apamin or charibdotoxin and was insensitive to externally applied L-glutamate or acetylcholine. The voltage-gated inward current was activated at -40 mV and was identified as an L-type calcium current sensitive to externally applied nifedipine. Schwann cells were impaled in situ in split-open axons and voltage clamped using discontinuous single electrode voltage clamp. Voltage dependent outward currents were recorded that were kinetically identical to those seen in isolated cells and that had similar current-voltage relations. Single channel currents were recorded from excised inside-out patches. A single channel type was observed with a reversal potential close to the equilibrium potential for K+ (E(K)) and was therefore identified as a K+ channel. The channel conductance was 43.6 pS when both internal and external solutions contained 150 mM K+. Activity was weakly dependent on membrane voltage but sensitive to the internal Ca2+ concentration. Activity was insensitive to externally or internally applied L-glutamate or acetylcholine. The results suggest that calcium channels and calcium-activated K+ channels play an important role in the generation of the squid Schwann cell membrane potential, which may be controlled by the resting intracellular Ca2+ level.

Applying Hidden Markov Models to the Analysis of Single Ion Channel Activity

Hidden Markov models have recently been used to model single ion channel currents as recorded with the patch clamp technique from cell membranes. The estimation of hidden Markov models parameters using the forward-backward and Baum-Welch algorithms can be performed at signal to noise ratios that are too low for conventional single channel kinetic analysis; however, the application of these algorithms relies on the assumptions that the background noise be white and that the underlying state transitions occur at discrete times. To address these issues, we present an “ H-noise” algorithm that accounts for correlated background noise and the randomness of sampling relative to transitions. We also discuss three issues that arise in the practical application of the algorithm in analyzing single channel data. First, we describe a digital inverse filter that removes the effects of the analog antialiasing filter and yields a sharp frequency roll-off. This enhances the performance while reducing the computational intensity of the algorithm. Second, the data may be contaminated with baseline drifts or deterministic interferences such as 60-Hz pickup. We propose an extension of previous results to consider baseline drift. Finally, we describe the extension of the algorithm to multiple data sets.

Voltage-dependent ion channels formed by dodeca- and pentadecaoligopeptides with two charged terminal groups

Two voltage-dependent ion channels composed of dodeca- and pentadecaoligopeptides, NH 3 +- l-Ala- d-Leu- l-Ala- d-Val- l-Val- d-Val- l-Trp- d-Leu- l-Trp- d-Leu- l-Trp- d-Leu-CO 2 − ( 3) and NH 3 +- l-Val-Gly- l-Ala- d-Leu- l-Ala- d-Val- l-Val- d-Val- l-Trp- d-Leu- l-Trp- d-Leu- l-Trp- d-Leu- l-Trp-CO 2 − ( 4), were synthesized. Both oligopeptides have charged species asymmetrically located at the N- and C-terminals. When dodecapeptide 3 was incorporated into planar bilayer membranes and voltage was applied in the range +200 to −200 mV under symmetric 500 mM KCl conditions, stable single ion channel currents were observed. The open probability of these single ion channels was dependent on the applied voltage, and higher open probabilities were observed either at high positive or negative applied voltages, with the voltage-dependence of the open probability being almost randomly distributed. The current–voltage relationship for 3 was ohmic and channel conductances were in the range 5.2–11.2 pS under the same salt conditions. On the other hand, pentadecapeptide 4 showed multi-channel activity with voltage dependency, i.e., the magnitude of the whole currents per unit time was dependent on the applied voltage. For large applied voltage steps, large amplitude whole channel currents were observed. Based on comparisons of the behaviors of various lengths of oligopeptides, extended structures through the lipid membrane of 3 and 4 were proposed as the active forms of the ion channel species.

Hidden Markov model analysis of intermediate gating steps associated with the pore gate of shaker potassium channels.

Cooperativity among the four subunits helps give rise to the remarkable voltage sensitivity of Shaker potassium channels, whose open probability changes tenfold for a 5-mV change in membrane potential. The cooperativity in these channels is thought to arise from a concerted structural transition as the final step in opening the channel. Recordings of single-channel ionic currents from certain other channel types, as well as our previous recordings from T442S mutant Shaker channels, however, display intermediate conductance levels in addition to the fully open and closed states. These sublevels might represent stepwise, rather than concerted, transitions in the final steps of channel activation. Here, we report a similar fine structure in the closing transitions of Shaker channels lacking the mutation. Describing the deactivation time course with hidden Markov models, we find that two subconductance levels are rapidly traversed during most closing transitions of chimeric, high conductance Shaker channels. The lifetimes of these levels are voltage-dependent, with maximal values of 52 and 22 micros at -100 mV, and the voltage dependences of transitions among these states suggest that they arise from equivalent conformational changes occurring in individual subunits. At least one subconductance level is found to be traversed in normal conductance Shaker channels. We speculate that voltage-dependent conformational changes in the subunits give rise to changes in a "pore gate" associated with the selectivity filter region of the channel, producing the subconductance states. As a control for the hidden Markov analysis, we applied the same procedures to recordings of the recovery from N-type inactivation in Shaker channels. These transitions are found to be instantaneous in comparison.

Transmembrane ion channels constructed of cholic acid derivatives.

A new class of supramolecular transmembrane ion channels was prepared by linking two amphiphilic cholic acid methyl ethers through biscarbamate bonds to afford bis(7,12-dimethyl-24-carboxy-3-cholanyl)-N,N'-xylylene dicarbamate 2 and bis[7,12-dimethyl-24-(N,N,N-trimethylethanaminium-2-carboxylate)-3-cholanyl]-N,N'-xylylene dicarbamate dichloride 3. When incorporated into a planar bilayer membrane, both compounds showed stable (lasting 10 ms to 10 s) single ion channel currents. Only limited numbers of relatively small conductances were characterized for these channels (5-20 pS for 2 and 5-10 pS for 3, 10 and 17 pS for 2, and 9 pS for 3 in particular). Both channels were cation selective, and permeability ratios of potassium cation to chloride anion were 17 and 7.9 for 2 and 3, respectively, reflecting the difference in ionic species of the headgroup. Both channels 2 and 3 showed significant potassium selectivity over sodium by a factor of 3.1 and 3.2, respectively. No Li(+) currents were observed for 2, showing sharp discrimination between Na(+) or K(+).