Incorporation Of Ion Channels Research Articles

Alignment of lyotropic liquid crystals using magnetic nanoparticles improves ionic transport through built-in peptide ion channels

Alignment of lyotropic liquid crystals using magnetic nanoparticles improves ionic transport through built-in peptide ion channels

Development of an automated system to measure ion channel currents using a surface-modified gold probe

Artificial lipid bilayer single-channel recording technique has been employed to determine the biophysical and pharmacological properties of various ion channels. However, its measurement efficiency is very low, as it requires two time-consuming processes: preparation of lipid bilayer membranes and incorporation of ion channels into the membranes. In order to address these problems, we previously developed a technique based on hydrophilically modified gold probes on which are immobilized ion channels that can be promptly incorporated into the bilayer membrane at the same time as the membrane is formed on the probes’ hydrophilic area. Here, we improved further this technique by optimizing the gold probe and developed an automated channel current measurement system. We found that use of probes with rounded tips enhanced the efficiency of channel current measurements, and introducing a hydrophobic area on the probe surface, beside the hydrophilic one, further increased measurement efficiency by boosting membrane stability. Moreover, we developed an automated measurement system using the optimized probes; it enabled us to automatically measure channel currents and analyze the effects of a blocker on channel activity. Our study will contribute to the development of high-throughput devices to identify drug candidates affecting ion channel activity.

Ion channel reconstitution in lipid bilayers.

Incorporation of ion channels in planar lipid bilayers allows detecting and measuring ion channel activity in a well-controlled system. This technique provides critical information about ion channel kinetics, ion selectivity, gating mechanism, open probability, unitary conductance, subconductance states, voltage dependence, and burst opening events, particularly at the single molecule level. Planar lipid bilayers provide a unique controllable environment that enables maintaining specific regulatory components, including lipids, ligands, inhibitors, particular ions, and proteins, as well as the temperature that can modulate activity of many ion channels. Thus, this system provides explicit details about ion channel gating mechanism and enables identifying its particular regulatory molecules or components. This chapter will describe the planar lipid bilayer method using the example of a transient receptor potential (TRP) ion channel family member. The planar lipid bilayer electrophysiological approach has proven to be useful in studying intrinsic properties of TRP channels. This method is particularly valuable for our understanding of intrinsic temperature sensitivity of thermoreceptors such as TRP channels and direct effects of TRP channels agonists, antagonists, co-factors, and other modifiers.

Advances in Artificial Cell Membrane Systems as a Platform for Reconstituting Ion Channels.

An artificial cell membrane that is composed of bilayer lipid membranes (BLMs) with transmembrane proteins incorporated within them represents a well-defined system for the analysis of membrane proteins, especially ion channel proteins that are major targets for drug design. Because the BLM system has a high compatibility with recently developed cell-free expression systems, it has attracted attention as a next-generation drug screening system. However, three issues associated with BLM systems, i. e., their instability, the need for non-volatile organic solvents and a low efficiency of ion channel incorporation, have limited their use as a drug screening platform. In this personal account, we discuss our recent approaches to address these issues based on microfabrication. We also discuss the potential for using the BLM system combined with cell-free expression systems as a drug screening system for future personalized medicine.

Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes.

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4 septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO2/Si3N4 septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.

Impairment of brain mitochondrial charybdotoxin- and ATP-insensitive BK channel activities in diabetes.

Existing evidence indicates an impairment of mitochondrial functions and alterations in potassium channel activities in diabetes. Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the gating behavior of the mitochondrial ATP- and ChTx-insensitive Ca(2+)-activated potassium channel (mitoBKCa) is altered in a streptozotocin (STZ) model of diabetes. Using ion channel incorporation of brain mitochondrial inner membrane into the bilayer lipid membrane, we provide in this work evidence for modifications of the mitoBKCa ion permeation properties with channels from vesicles preparations coming from diabetic rats characterized by a significant decrease in conductance. More importantly, the open probability of channels from diabetic rats was reduced 1.5-2.5 fold compared to control, the most significant decrease being observed at depolarizing potentials. Because BKCa β4 subunit has been documented to left shift the BKCa channel voltage dependence curve in high Ca(2+) conditions, a Western blot analysis was undertaken where the expression of mitoBKCa α and β4 subunits was estimated using of anti-α and β4 subunit antibodies. Our results indicated a significant decrease in mitoBKCa β4 subunit expression coupled to a decrease in the expression of α subunit, an observation compatible with the observed decrease in Ca(2+) sensitivity. Our results thus demonstrate a modification in the mitoBKCa channel gating properties in membrane preparations coming from STZ model of diabetic rats, an effect potentially linked to a change in mitoBKCa β4 and α subunits expression and/or to an increase in reactive oxygen species production in high glucose conditions.

Endoplasmic reticulum membrane potassium channel dysfunction in high fat diet induced stress in rat hepatocytes.

In a previous study we reported the presence of a large conductance K(+) channel in the membrane of endoplasmic reticulum (ER) from rat hepatocytes. The channel open probability (Po) appeared voltage dependent and reached to a minimum 0.2 at +50 mV. Channel activity in this case was found to be totally inhibited at ATP concentration 2.5 mM, glibenclamide 100 µM and tolbutamide 400 µM. Existing evidence indicates an impairment of endoplasmic reticulum functions in ER stress condition. Because ER potassium channels have been involved in several ER functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the ER potassium channel function is altered in a high fat diet model of ER stress. Male Wistar rats were made ER stress for 2 weeks with a high fat diet. Ion channel incorporation of ER stress model into the bilayer lipid membrane allowed the characterization of K(+) channel. Our results indicate that the channel Po was significantly increased at voltages above +30 mV. Interestingly, addition of ATP 7.5 mM, glibenclamide 400 µM and tolbutamide 2400 µM totally inhibited the channel activities, 3-fold, 4-fold and 6-fold higher than that in the control groups, respectively. Our results thus demonstrate a modification in the ER K(+) channel gating properties and decreased sensitivity to drugs in membrane preparations coming from ER high fat model of ER stress, an effect potentially linked to a change in ER K(+) channel subunits in ER stress condition. Our results may provide new insights into the cellular mechanisms underlying ER dysfunctions in ER stress.

Gating behavior of endoplasmic reticulum potassium channels of rat hepatocytes in diabetes.

Background: Defects in endoplasmic reticulum homeostasis are common occurrences in different diseases, such as diabetes, in which the function of endoplasmic reticulum is disrupted. It is now well established that ion channels of endoplasmic reticulum membrane have a critical role in endoplasmic reticulum luminal homeostasis. Our previous studies showed the presence of an ATP-sensitive cationic channel in endoplasmic reticulum. Therefore, in this study, we examined and compared the activities of this channel in control and diabetic rats using single-channel recording techniques. Method: Male Wistar rats were made diabetic for 2 weeks with a single dose injection of streptozotocin (45 mg/kg). Ion channel incorporation of rough endoplasmic reticulum of diabetic hepatocytes into the bilayer lipid membrane allowed the characterization of K+ channel. Results: Ion channel incorporation of rough endoplasmic reticulum vesicles into the bilayer lipid revealed that the channel current-voltage (I-V) relation with a mean slope conductance of 520 ± 19 pS was unaffected in diabetes. Interestingly, the channel Po-voltage relation was significantly lower in diabetic rats at voltages above +30 mV. Conclusion: We concluded that the endoplasmic reticulum cationic channel is involved in diabetes. Also, this finding could be considered as a goal for further therapeutic plans.

Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.

Probing Simultaneously Membrane Dynamics and Protein Activity in Suspended Bilayers in a Microfluidic Format

Membrane dynamics affect the structure and function of ion channels, a point that deserves more attention while studying membrane proteins. One important factor in the local lipidic environment of the ion channels, is the membrane fluidity which is directly connected to the free diffusion and packing of the phospholipids. Typically, FRAP is utilized to investigate this parameter in simplified cell membrane models, and preferably supported bilayer lipid membranes (BLMs), which are more stable than their free-standing counterparts. However, supported membranes are less suitable for the incorporation of ion channels and electrophysiological monitoring of their activity than suspended ones.

1P287 Simple method for lipid bilayer formation with simultaneous incorporation of ion channels using gold electrode(26. Measurements,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

1P287 Simple method for lipid bilayer formation with simultaneous incorporation of ion channels using gold electrode(26. Measurements,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

Storable droplet interface lipid bilayers for cell-free ion channel studies

An artificially created lipid bilayer is an important platform in studying ion channels and engineered biosensor applications. However, a lipid bilayer created using conventional techniques is fragile and short-lived, and the measurement of ion channels requires expertise and laborious procedures, precluding practical applications. Here, we demonstrate a storable droplet lipid bilayer precursor frozen with ion channels, resulting in a droplet interface bilayer upon thawing. A small vial with an aqueous droplet in organic solution was flash frozen in -80 °C methanol immediately after an aqueous droplet was introduced into the organic solution and gravity draws the droplet down to the interface upon thawing. A lipid bilayer created along the interface using this method had giga-ohm resistance and typical specific capacitance values. The noise level of this system is favorably comparable to the conventional system. The subsequent incorporation of ion channels, alpha-hemolysin and gramicidin A, showed typical conductance values consistent with those in previous literatures. This novel system to create a lipid bilayer as a whole can be automated from its manufacture to use and indefinitely stored when frozen. As a result, ion channel measurements can be carried out in any place, increasing the accessibility of ion channel studies as well as a number of applications, such as biosensors, ion channel drug screening, and biophysical studies.

Single molecule measurements within individual membrane-bound ion channels using a polymer-based bilayer lipid membrane chip

The measurement of single poly(ethylene glycol) (PEG) molecules interacting with individual bilayer lipid membrane-bound ion channels is presented. Measurements were performed within a polymer microfluidic system including an open-well bilayer lipid membrane formation site, integrated Ag/AgCl reference electrodes for on-chip electrical measurements, and multiple microchannels for independent ion channel and analyte delivery. Details of chip fabrication, bilayer membrane formation, and alpha-hemolysin ion channel incorporation are discussed, and measurements of interactions between the membrane-bound ion channels and single PEG molecules are presented.

Deposition of highly resistive lipid bilayer on silicon–silicon dioxide electrode and incorporation of gramicidin studied by ac impedance spectroscopy

Highly resistive planar supported lipid membranes were deposited onto highly doped p-type silicon–silicon dioxide electrodes. Physical parameters of the substrates (e.g. dopant, doping ratio and oxide layer thickness) were optimized by a combined study using ellipsometry and ac impedance spectroscopy. Lipid bilayer was deposited by fusion of small unilamellar vesicles, and the self-assembling of the homogeneous bilayer could be monitored as a function of time. Impedance spectroscopy over a wide frequency range (from 20 kHz to 10 mHz) enables separating membrane resistance and capacitance from the background signals. Membrane resistance amounted to 1.0 MΩ cm 2, and the capacitance was around 0.7 μF cm −2. The resistance obtained here is comparable to that of the freestanding black lipid membrane. Although the area of the supported membrane (0.5 cm 2) is much larger than that of the black lipid membrane (∼0.002 cm 2), the electrical properties were stable for more than a week. Gramicidin D was inserted into the membrane from trifluoroethanol solution, and activity of the channels was checked in terms of membrane conductance and ion selectivity. Functional incorporation of ion channels into the supported membrane demonstrated here suggested that the gramicidin monomers could diffuse over the membranes to form transmembrane pores.

A novel method for incorporation of ion channels into a planar phospholipid bilayer which allows solution changes on a millisecond timescale.

We have developed a method of rapidly changing the solutions on one side of a planar phospholipid bilayer. Bilayers can be painted on glass pipettes of tip diameter > or = 50 microns. By modifying an established method for rapid exchange of solutions bathing excised membrane patches, solution changes can be made at the bilayer within 10 ms. After incorporation of channels into the bilayer, the bilayer is moved into one of two parallel streams of solution flowing from a length of double-barrelled glass theta tubing. Activation of a solenoid system rapidly moves the theta tubing so that the bilayer is in the flow of the adjacent solution. For various reasons, the single-channel gating mechanisms of many channels are studied in planar bilayer systems. The conventional bilayer technique only allows for steady-state single-channel gating to be monitored. This novel method now allows the effects of rapid changes in modulators of channels incorporated into planar phospholipid bilayers to be measured.

31] Planar lipid bilayers on patch pipettes: Bilayer formation and ion channel incorporation

This chapter describes how to make bilayers on the tip of patch-style pipettes and then incorporate channels into these bilayers. The chapter also explains the transcription of some oral lore and traditions accompanying the formation of bilayers on the tip of patch-style pipettes and the study of channels in these bilayers. Tip-dip membranes, double dip membranes, and patch membranes are bilayers, whose name refers to the method of membrane formation. Tip-dip bilayers represent the combination of two other techniques, patch damping and planar lipid bilayers. There are numerous steps in each of the methods. A better understanding of the physical processes that govern protein–lipid and lipid–lipid interactions would assist in future efforts in bilayer formation and channel reconstitution.

Incorporation of ion channels from bovine rod outer segments into planar lipid bilayers

Membranes vesicles, prepared from bovine rod outer segments were fused with planar lipid bilayers. Two different ion channels were identified by recording currents from single channels. Both types of channels were selective for sodium rather than potassium and were impermeable to chloride ions. Unit conductances were 20 and 120 pS, respectively, in 150 mM sodium chloride. The channel with the larger unit conductance was sensitive to the transmembrane potential. This channel rapidly activated within less than 10 ms after a voltage jump to a more negative membrane potential and then inactivated after several seconds. The duration of the active period and the properties of the channel depended on the amplitude of the voltage jump. The channel of smaller unit conductance did not show any voltage-dependent activation or inactivation. Both types of channels were insensitive to light in the planar bilayer system. Channels incorporated into planar bilayers on a Teflon sandwich septum or on the tip of a glass micropipette gave similar results.

An ion-channel forming protein produced by Entamoeba histolytica.

We have identified a remarkable ion-channel forming material in virulent strains of Entamoeba histolytica that may be responsible for many of the symptoms associated with amoebic dysentery. A polypeptide that we refer to as amoebapore is shed into the growth media and is also found within the amoeba in a high speed sedimentable fraction. Amoebapore has the distinctive property of spontaneously incorporating into lipid bilayers, liposomes, and cells, leading to progressive and irreversible changes in the ion conductance of the target membranes. Exposure of planar lipid bilayers to amoebapore -containing fractions under voltage clamp conditions results in an almost immediate and progressive incorporation of ion channels which continues in an irreversible manner leading to a fall in membrane impedance of up to five orders of magnitude. The ion-channel conductance is moderately cation-selective, voltage dependent, and displays a unit size of 1.6 +/- 0.2 nanoSiemens in 1 M KCl at -10 mV. In the bilayer, the amoebapore -induced conductance exhibits an in situ sensitivity to protease. Amoebapore is mainly concentrated in a fraction sedimenting at 150 000 g. It is insoluble in Triton X-100 but can be dissociated in an active state in 1% SDS. Under these conditions it has an apparent mol. wt. of 13 000 daltons.