John M. Tang

Single-channel recordings of apical membrane chloride conductance in A6 epithelial cells.

SummaryThe apical membrane of epithelial cells from the A6 cell line grown on impermeable substrata was studied using the patch-clamp technique. We defined the apical membrane as that membrane in contact with the growth medium. In about 50% of the patches, channels with single-unit conductances of 360±45 pS in symmetrical 105mm NaCl solutions, and characteristic voltage-dependent inactivation were observed. Using excised membrane patches and varying the ionic composition of the bathing medium, we determined that the channels were anion selective, with a permeability ratio for Cl− over Na+ of about 9∶1, calculated from the reversal potential using the constantfield equation. The channel was most active at membrane potentials between ±20 mV and inactivated, usually within a few seconds, at higher potentials of either polarity. Reactivation from this inactivation was slow, sometimes requiring minutes. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to one or more submaximal conductance levels. The channel was inhibited by the disulfonic stilbene SITS in a manner characteristic of reversible open-channel blockers.

A cation channel in frog lens epithelia responsive to pressure and calcium

SummaryPatch-clamp recording from the apical surface of the epithelium of frog lens reveals a cation-selective channel after pressure (about ±30 mm Hg) is applied to the pipette. The open state of this channel has a conductance of some 50 pS near the resting potential (−56.1±2.3 mV) when 107mm NaCl and 10 HEPES (pH 7.3) is outside the channel. The probability of the channel being open depends strongly on pressure but the current-voltage relation of the open state does not. With minimal Ca2+ (55±2 μm) outside the channel, the current-voltage relation is nonlinear even in symmetrical salt solutions, allowing more current to flow into the cell than out. The channel, in minimal Ca2+ solution, is selective among the monovalent cations in the following sequence K+>Rb+>Cs+>Na+>Li+. The conductance depends monotonically on the mole fraction of K+ when the other ion present is Li+ or Na+. The single-channel current is a saturating function of [K+] when K+ is the permeant ion, for [K+]≤214mm. When [Ca2+]=2mm, the currentvoltage relation is linearized and the channel cannot distinguish Na+ and K+.

Immunoglobulin G-induced single ionic channels in human alveolar macrophage membranes.

While it is well known that the engagement of IgG Fc receptors on the macrophage surface triggers a number of cellular responses, including particle ingestion, secretion, and respiratory burst activity, the mechanism of signal transmission following ligand binding remains poorly understood. To acquire more data in this area, we studied the electrical properties of the macrophage membrane and its response to oligomeric immunoglobulin G (IgG) using the patch-clamp technique on human alveolar macrophages that were obtained by bronchoalveolar lavage and maintained in short-term tissue culture. The results showed that cell resting potentials, as determined from whole-cell tight seal recordings, increased from -15 mV on the day of plating to -56 mV after the first day in culture and remained stable at this hyperpolarized level. Macrophages revealed an input resistance of 3.3 G omega, independent of age in culture. Extracellular application of heat-aggregated human IgG to cells voltage-clamped at -70 mV resulted in peak inward currents of approximately 470 pA. We identified an IgG-dependent, nonselective channel in both cell-attached and isolated membrane patches, with a unitary conductance of approximately 350 pS and a predominant subconductance level of 235 pS in symmetrical NaCl solutions. Single channel open times were observed to be in the range of seconds and, in addition, were dependent upon membrane voltage. Channel opening involved transitions between a number of kinetic states and subconductance levels. Channel events recorded in cell-attached patches showed characteristic exponential relaxations, which implied a variation in membrane potential as a result of a single ion channel opening. These data suggest that the IgG-dependent nonselective cation channel that we have characterized may provide the link between Fc receptor engagement and subsequent cellular activation.

Simulating Ion Permeation Through the ompF Porin Ion Channel Using Three-Dimensional Drift-Diffusion Theory

Ionic channels, natural nanotubes found in biological cells, are interesting to the electronics community because they display a range of device-like functions. The purpose of this paper is to illustrate how the solution methodology, developed for 3-D drift-diffusion models of semiconductor devices, can be applied to ion permeation in ionic channels. For this study we select the ompF porin channel, found in the membrane of the E. coli bacterium. The self-consistent 3-D model is based on the simultaneous solution of Poissons equation, which captures Coulomb interactions, and a current continuity equation for each ion species, describing permeation down an electrochemical gradient. Water is treated as a uniform background medium with a specific dielectric constant. For demonstration, a simple model is assumed for the mobility/diffusivity of each ionic species and we compute the current-voltage relations for ompF porin in a wide range of conditions. Agreement with experimental measurements is surprisingly good given that the model uses the ion diffusivity as the only calibrated parameter.

Teflon™-coated silicon apertures for supported lipid bilayer membranes

We present a method for microfabricating apertures in a silicon substrate using well-known cleanroom technologies resulting in highly reproducible giga-seal resistance bilayer formations. Using a plasma etcher, 150μm apertures have been etched through a silicon wafer. Teflon™ has been chemically vapor deposited so that the surface resembles bulk Teflon and is hydrophobic. After fabrication, reproducible high resistance bilayers were formed and characteristic measurements of a self-inserted single OmpF porin ion channel protein were made.

Three-Dimensional Continuum Simulations of Ion Transport Through Biological Ion Channels: Effect of Charge Distribution in the Constriction Region of Porin

Drift-diffusion models are useful for studying ion transport in open protein channel systems over time scales that cannot be resolved practically by detailed molecular dynamics or quantum approaches. Water is treated as a uniform background medium with a specific dielectric constant and macroscopic current flow is resolved by assigning an appropriate mobility and diffusivity to each ionic species. The solution of Poissons equation over the entire domain provides a simple way to include external boundary conditions and image force effects at dielectric discontinuities. Here we present a 3-D drift-diffusion model of ion (K+ and Cl−) permeation through the porin channel ompF, and its mutant G119D, implemented using the computational platform PROPHET.

Perfusing patch pipettes

Publisher Summary This chapter discusses the construction of a perfusion capillary that is a slight modification of the standard patch clamp apparatus. The patch clamp technique measures current flow through individual protein molecules, thus specifying the type of channel with little ambiguity. The technique depends on the electrical and physical isolation of one compartment, the pipette lumen, from another, the surrounding bath. The isolation of the lumen of the pipette is the essential feature of the patch clamp technique; failures in isolation introduce artifacts, always excess electrical noise and sometimes distortion in the time course of currents. Measurements of the rate of perfusion show that it is complete in 1 minute, judging by the change in reversal potential with time of a channel in the patch. Noise also reduces in this setup by placing the Beem capsules of solution in an expanded polystyrene block a few centimeters thick. Thus, the perfusion technique works well and conveniently in the laboratory, and it should prove generally useful and convenient.

A calcium conducting channel akin to a calcium pump

SummaryCalcium conducting channels were studied in blebs of sarcoplasmic reticulum described by Stein & Palade (1988). The calcium channels had at least three conductance states (70 pS, 50 pS and 37 pS) and were weakly selective for calcium ions, with a permeability ratio Ca2+ to K+ of about 3.4. The open probability of the channel was strongly voltage dependent, decreasing at positive membrane voltages. 10 μm ryanodine and 5 μm ruthenium red had no effect on this channel; neither did millimolar concentrations of ATP, Mg2+, caffeine, and Ca2+, implying that the calcium conducting channels are not ryanodine receptors. Several calcium pump inhibitors—namely, vanadate, AlF4−, reactive red 120, and cyclopiazonic acid—had obvious effects on the calcium conducting channels, suggesting that the calcium conducting channel of SR membrane blebs is some form of the SR calcium pump.We thank the National Science Foundation for steadfast support.

Ion channel sensor on a silicon support

We are building a biosensor based on ion channels inserted into lipid bilayers that are suspended across an aperture in silicon. The process flow only involves conventional optical lithography and deep Si reactive ion etching to create micromachined apertures in a silicon wafer. In order to provide surface properties for lipid bilayer attachment that are similar to those of the fluorocarbon films that are currently used, we coated the silicon surface with a fluoropolymer using plasma-assisted chemical vapor deposition. When compared with the surface treatment methods using self-assembled monolayers of fluorocarbon chemicals, this novel approach towards modifying the wettability of a silicon dioxide surface provides an easy and fast method for subsequent lipid bilayer formation. Current-Voltage measurements on OmpF ion channels incorporated into these membranes show the voltage dependent gating action expected from a working porin ion channel.

Integrated platform for ion channel sensing

Conductance measurement of single ion channels and related stochastic signals is a promising technique for the development of a functional biosensor. We present results showing that silicon substrates can be used as a low noise, universal platform for recording the electrical activity of single ion channels inserted into bilayer membranes. Bilayers were suspended on polytetrafluoroethylene coated, 150mum apertures etched into silicon substrates and then oxidized with common silicon processing techniques. The overall noise of the system was reduced with a 75mum thick SU-8 layer and integrated silver/silver chloride electrodes. Phase sensitive detection of single OmpF porin ion channels using the silicon device and a lock-in amplifier were made and demonstrate the systems capability as a sensor in high noise environments