Planar bilayer membranes made from phospholipid monolayers form by a thinning process.

We investigated the manner in which planar phospholipid membranes form when monolayers are sequentially raised. Simultaneous electrical and optical recordings showed that initially a thick film forms, and the capacitance of the film increases with the same time course as the observed thinning. The diameter of fully thinned membranes varies from membrane to membrane and a torus is readily observed. The frequency-dependent admittance of the membrane was measured using a wide-bandwidth voltage clamp whose frequency response is essentially independent of capacitative load. The membrane capacitance dominates the total admittance and the membrane dielectric is not lossy. The specific capacitance of membranes of several mixtures was measured. A schematic diagram of the formation of these membranes is presented.     

IR Laser-Induced Perturbations of the Voltage-Dependent Solute Carrier Protein SLC26a5

Alterations in membrane capacitance can arise from linear and nonlinear sources. For example, changes in membrane surface area or dielectric properties can modify capacitance linearly, whereas sensor residues of voltage-dependent proteins can modify capacitance nonlinearly. Here, we examined the effects of fast temperature jumps induced by an infrared (IR) laser in control and prestin (SLC26a5)-transfected human embryonic kidney (HEK) cells under whole-cell voltage clamp. Prestin’s voltage sensor imparts a characteristic bell-shaped, voltage-dependent nonlinear capacitance (NLC). Temperature jumps in control HEK cells cause a monophasic increase in membrane capacitance (C_m) regardless of holding voltage due to double-layer effects. Prestin-transfected HEK cells, however, additionally show a biphasic increase/decrease in C_m with a reversal potential corresponding to the voltage at peak NLC of prestin (V_h), attributable to a rapid temperature-following shift in V_h, with shift rates up to 14 V/s over the course of a 5 ms IR pulse. Treatment with salicylate, a known inhibitor of NLC, reestablishes control cell behavior. A simple kinetic model recapitulates our biophysical observations. These results verify a voltage-dependent protein’s ability to respond to fast temperature perturbations on a par with double-layer susceptibility. This likely arises from prestin’s unique ability to move sensor charge at kilohertz rates, which is required for the outer hair cells’ role as a cochlear amplifier.     

IR Laser-Induced Perturbations of the Voltage-Dependent Solute Carrier Protein SLC26a5

Alterations in membrane capacitance can arise from linear and nonlinear sources. For example, changes in membrane surface area or dielectric properties can modify capacitance linearly, whereas sensor residues of voltage-dependent proteins can modify capacitance nonlinearly. Here, we examined the effects of fast temperature jumps induced by an infrared (IR) laser in control and prestin (SLC26a5)-transfected human embryonic kidney (HEK) cells under whole-cell voltage clamp. Prestin’s voltage sensor imparts a characteristic bell-shaped, voltage-dependent nonlinear capacitance (NLC). Temperature jumps in control HEK cells cause a monophasic increase in membrane capacitance (C_m) regardless of holding voltage due to double-layer effects. Prestin-transfected HEK cells, however, additionally show a biphasic increase/decrease in C_m with a reversal potential corresponding to the voltage at peak NLC of prestin (V_h), attributable to a rapid temperature-following shift in V_h, with shift rates up to 14 V/s over the course of a 5 ms IR pulse. Treatment with salicylate, a known inhibitor of NLC, reestablishes control cell behavior. A simple kinetic model recapitulates our biophysical observations. These results verify a voltage-dependent protein’s ability to respond to fast temperature perturbations on a par with double-layer susceptibility. This likely arises from prestin’s unique ability to move sensor charge at kilohertz rates, which is required for the outer hair cells’ role as a cochlear amplifier.     

电压钳控制电流钳的设计

研究证明,传统膜片钳放大器在电流钳模式下记录到的快速电压信号会存在失真,且造成失真的根本原因是由于膜片钳的探头电路设计.为了克服这些缺陷重新设计了一种探头,新探头电路不仅能像传统的电压跟随器一样测量瞬态电压,而且适用于传统的电压钳工作模式.此外,一种命名为电压钳控制的电流钳技术被应用来改进传统的膜片钳放大器.用可变的低通滤波器来调整电压钳模块的响应速度,从而在实现膜电位钳位的同时准确记录快速电压信号.桥平衡电路用来消除命令电流流过串联电阻时带来的电压误差.而传统膜片钳中的快电容补偿环节则被改进用来补偿电极分布电容和探头放大器输入电容并提高电流钳模式下系统的响应速度.细胞模型实验结果表明,改进后的膜片钳放大器能够满足电生理研究中快速电位变化测量的需要.     

Membrane tension directly shifts voltage dependence of outer hair cell motility and associated gating charge.

The unique electromotility of the outer hair cell (OHC) is believed to promote sharpening of the passive mechanical vibration of the mammalian basilar membrane. The cell also presents a voltage-dependent capacitance, or equivalently, a nonlinear gating current, which correlates well with its mechanical activity, suggesting that membrane-bound voltage sensor-motor elements control OHC length. We report that the voltage dependence of the gating charge and motility are directly related to membrane stress induced by intracellular pressure. A tracking procedure was devised to continuously monitor the voltage at peak capacitance (VpkCm) after obtaining whole cell voltage clamp configuration. In addition, nonlinear capacitance was more fully evaluated with a stair step voltage protocol. Upon whole cell configuration, VpkCm was typically near -20 mV. Negative patch pipette pressure caused a negative shift in VpkCm, which obtained a limiting value near the normal resting potential of the OHC (approximately -70 mV) at the point of cell collapse. Positive pressure in the pipette caused a positive shift that could reach values greater than 0 mV. Measures of the mechanical activity of the OHC mirrored those of charge movement. Similar membrane-tension dependent peak shifts were observed after the cortical cytoskeletal network was disrupted by intracellular dialysis of trypsin from the patch pipette. We conclude that unlike stretch receptors, which may sense tension through elastic cytoskeletal elements, the OHC motor senses tension directly. Furthermore, since the voltage dependence of the OHC nonlinear capacitance and motility is directly regulated by intracellular turgor pressure, we speculate that modification of intracellular pressure in vivo provides a mechanism for controlling the gain of the mammalian "cochlear amplifier".     

Intracellular microelectrode measurements in small cells evaluated with the patch clamp technique.

Microelectrode penetration of small cells leads to a sustained depolarization of the resting membrane potential due to a transmembrane shunt resistance (Rs) introduced by the microelectrode. This has led to underestimation of the resting membrane potential of various cell types. However, measurement of the fast potential transient occurring within the first few milliseconds after microelectrode penetration can provide information about pre-impalement membrane electrophysiological properties. We have analyzed an equivalent circuit of a microelectrode measurement to establish the conditions under which the peak of the impalement transients (Ep) approaches the pre-impalement resting membrane potential (Em) of small cells most closely. The simulation studies showed that this is the case when the capacitance of the microelectrode is low and the membrane capacitance of the cell high. In experiments performed to assess the reliability of Ep as a measure of Em, whole-cell patch clamp measurements were performed in the current clamp mode to monitor, free from the effects of Rs, Em in cultured human monocytes. Microelectrode impalement of such patch clamped cells and measurement of Ep made it possible to detect correlation between Ep and Em and showed that for small cells such as human monocytes Ep is on average 6 mV less negative than the resting membrane potential.     

Two-microelectrode voltage clamp of Xenopus oocytes: voltage errors and compensation for local current flow.

Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The most common method for their electrophysiological investigation is the two-microelectrode voltage clamp technique. The quality of voltage clamp recordings obtained with this technique is poor when membrane currents are large and when rapid charging of the membrane is desired. Detailed mathematical modeling of the experimental setup shows that the reasons for this weak performance are the electrical properties of the oocytes and the geometry of the setup. We measured the cytosolic conductivity to be approximately 5 times lower than that of the typical bath solution, and the specific membrane capacitance to be approximately 6 times higher than that of a simple lipid bilayer. The diameter of oocytes is typically approximately 1 mm, whereas the penetration depth of the microelectrodes is limited to approximately 100 microm. This eccentric current injection, in combination with the large time constants caused by the low conductivity and the high capacitance, yields large deviations from isopotentiality that decay slowly with time constants of up to 150 micros. The inhomogeneity of the membrane potential can be greatly reduced by introducing an additional, extracellular current-passing electrode. The geometrical and electrical parameters of the setup are optimized and initial experiments show that this method should allow for faster and more uniform control of membrane potential.     

Model for the dynamic responses of taste receptor cells to salty stimuli. I. Function of lipid bilayer membranes.

The dynamic response of the lipid bilayer membrane is studied theoretically using a microscopic model of the membrane. The time courses of membrane potential variations due to monovalent salt stimulation are calculated explicitly under various conditions. A set of equations describing the time evolution of membrane surface potential and diffusion potential is derived and solved numerically. It is shown that a rather simple membrane such as lipid bilayer has functions capable of reproducing the following properties of dynamic response observed in gustatory receptor potential. Initial transient depolarization does not occur under Ringer adaptation but does under water. It appears only for comparatively rapid flows of stimuli, the peak height of transient response is expressed by a power function of the flow rate, and the membrane potential gradually decreases after reaching its peak under long and strong stimulation. The dynamic responses in the present model arise from the differences between the time dependences in the surface potential phi s and the diffusion potential phi d across a membrane. Under salt stimulation phi d cannot immediately follow the variation in phi s because of the delay due to the charging up of membrane capacitance. It is suggested that lipid bilayer in the apical membrane is the most probable agency producing the initial phasic response to the stimulation.     

A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons

We report on a simple and high‐yield manufacturing process for silicon planar patch‐clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high‐quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high‐impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high‐fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole‐cell current recordings obtained from a voltage‐clamp stimulation protocol, and in current‐clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch‐clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high‐information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity. Biotechnol. Bioeng. 2010;107:593–600. © 2010 Wiley Periodicals, Inc.     

Computed Membrane Currents in Cardiac Purkinje Fibers during Voltage Clamps

Recent measurements have indicated that some of the cardiac cell electrical capacitance is in series with a resistance. The computations of currents in a voltage clamp presented below show that, in this case, there is a danger that capacitive transient currents recorded during voltage clamp experiments may be confused with currents arising through rapid active membrane conductance changes. Secondly, a voltage clamp technique aimed at avoiding capacitive transients, namely the linear or ramp clamp, has recently been introduced. An attempt has been made here to evaluate the usefulness of ramp clamps in studying membrane electrical properties, by computing ramp clamp results and considering the difficulties in reconstructing the original model from these results. It is concluded that such a reconstruction is not feasible.     

Asymmetry currents and admittance in squid axons.

The complex admittance of squid (Loligo pealei) axon was measured rapidly (within 1 s) with pseudo-random small signals and discrete Fourier transform techniques under guarded, "space-clamp" conditions and during suppression of ion conduction. Asymmetry currents were measured by paired step clam pulses of +/-70 mV from a holding potential of -97 mV and gave an apparent capacitance of 0.36 muF/cm2. However, the admittance data showed no change in capacitance at holding potentials from -97 to -67 mV and gave a decrease of 0.07 of 0.15 muF/cm2 at -37 mV. The failure to observe a capacitance increase at low membrane potentials suggests the following possibilities: (a) the asymmetry current is a displacement current that inactivates completely with time, and (b) the asymmetry current is not a displacement current and arises from large signal effects (i.e., delayed nonlinearity in ionic current) on the membrane.     

Compensation for resistance in series with excitable membranes.

Extracellular resistance in series (Rs) with excitable membranes can give rise to significant voltage errors that distort the current records in voltage-clamped membranes. Electrical methods for measurement of and compensation for such resistances are described and evaluated. Measurement of Rs by the conventional voltage jump in response to a current step is accurate but the measurement of sine-wave admittance under voltage-clamp conditions is better, having about a fivefold improvement in resolution (+/- 0.1 omega cm2) over the conventional method. Conventional feedback of the membrane current signal to correct the Rs error signal leads to instability of the voltage clamp when approximately two-thirds of the error is corrected. We describe an active electronic bridge circuit that subtracts membrane capacitance from the total membrane current and allows full, yet stable, compensation for the voltage error due to ionic currents. Furthermore, this method provides not only fast and accurate control of the membrane potential in response to a command step, but also fast recovery following an abrupt change in the membrane conductance. Marked changes in the kinetics and amplitude of ionic currents resulting from full compensation for Rs are shown for several typical potential patterns.     

Patch voltage clamp of squid axon membrane.

A small area (patch) of the external surface of a squid axon can be "isolated" electrically from the surrounding bath by means of a pair of concentric glass pipettes. The seawater-filled inner pipette makes contact with the axon and constitutes the external access to the patch. The outer pipette is used to direct flowing sucrose solution over the area surrounding the patch of membrane underlying the inner pipette. Typically, sucrose isolated patches remain in good condition (spike amplitude greater than 90 mV) for periods of approximately one half hour. Patches of axon membrane which had previously been exposed to sucrose solution were often excitable. Membrane survival of sucrose treatment apparently arises from an outflow of ions from the axon and perhaps satellite cells into the interstitial cell space surrounding the exolemma. Estimate of the total access resistance (electrode plus series resistance) to the patch is about 100 komega (7 omega cm2). Patch capacitance ranges from 10-100 pF, which suggests areas of 10(-4) to 10(-5) cm2 and resting patch resistances of 10-100 Momega. Shunt resistance through the interstitial space exposed to sucrose solution, which isolates the patch, is typically 1-2 Momega. These parameters indicate that good potential control and response times can be achieved on a patch. Furthermore, spatial uniformity is demonstrated by measurement of an exoplasmic isopotential during voltage clamp of an axon patch. The method may be useful for other preparations in which limited membrane area is available or in special instances such as in the measurement of membrane conduction noise.     

大鼠嗜铬细胞分泌的膜电容和微碳纤电极检测

在原代培养的大鼠肾上腺嗜铬细胞上,综合运用细胞内钙测定法和全细胞膜片钳法,以检测膜电容变化为手段测定单一肾上腺嗜铬细胞的胞吐过程。－７０ｍＶ到＋２０ｍＶ去极化引起的钙电流和细胞膜电容的变化以及吹加６０ｍｍｏｌ／ＬＫＣｌ时,细胞内游离钙离子浓度［Ｃａ２＋］ｉ和细胞膜电容变化的同时检测,表明了Ｃａ２＋对细胞胞吐的控制作用。而用微碳纤电极则能检测到吹加６０ｍｍｏｌ／ＬＫＣｌ导致嗜铬细胞胞吐时儿茶酚胺的量子化释放。细胞膜电容检测和微碳纤电极检测从不同侧面动态的反映了细胞胞吐过程与Ｃａ２＋的相关性     

Fusion of membranes during fertilization. Increases of the sea urchin egg''s membrane capacitance and membrane conductance at the site of contact with the sperm

The early events of fertilization that precede and cause activation of an egg have not been fully elucidated. The earliest electrophysiological change in the sea urchin egg is a sperm-evoked increase of the egg's membrane conductance. The resulting depolarization facilitates entry of the fertilizing sperm and precludes the entry of supernumerary sperm. The sequence of the increase in the egg's membrane conductance, gamete membrane fusion, egg activation, and sperm entry, including causal relationships between these events, are not known. This study reports the use of whole egg voltage clamp and loose patch clamp to monitor simultaneously changes of membrane conductance and capacitance at the site of sperm-egg contact. Measurements were made during sperm-egg interactions where sperm entry readily proceeded or was precluded by maintaining the egg's membrane potential either at large, negative values or at positive values. Whenever the sperm evoked an increase of the egg's membrane conductance, that increase initiated abruptly, was localized to the site of sperm attachment, and was accompanied by a simultaneous abrupt increase of the membrane capacitance. This increase of capacitance indicated the establishment of electrical continuity between gametes (possibly fusion of the gametes' plasma membranes). If sperm entry was blocked by large negative membrane potentials, the capacitance cut off rapidly and simultaneously with a decrease of the membrane conductance, indicating that electrical continuity between gametes was disrupted. When sperm entry was precluded by positive membrane potentials, neither conductance nor capacitance increased, indicating that sperm entry was halted before the fusion of membranes. A second, smooth increase of capacitance was associated with the exocytosis of cortical granules near the sperm in eggs that were activated. Electrical continuity between the gametes always preceded activation of the egg, but transient electrical continuity between the gametes alone was not always sufficient to induce activation.     

Optocapacitance: physical basis and its application

The observation that membrane capacitance increases with temperature has led to the development of new methods of neuronal stimulation using light. The optocapacitive effect refers to a light-induced change in capacitance produced by the heating of the membrane through a photothermal effect. This change in capacitance manifests as a current, named optocapacitive current that depolarizes cells and therefore can be used to stimulate excitable tissues. Here, we discuss how optocapacitance arises from basic membrane properties, the characteristics of the optocapacitive current, its use for neuronal stimulation, and the challenges for its application in vivo.     

cAMP directly facilitates Ca-induced exocytosis in bovine lactotrophs

We have used the whole cell patch clamp technique on single prolactin-secreting bovine lactotrophs to measure plasma membrane capacitance (Cm), an index of membrane surface area, under voltage-clamp during cytosol dialysis with Ca and cAMP. cAMP increased the magnitude and rate of Ca-induced exocytosis (Cm increase) without affecting membrane conductance; however, cAMP had no detectable effect on Cm when intracellular Ca was low. We thus report new evidence that cAMP can facilitate Ca-induced secretion in a synergistic fashion, by acting directly on the secretory apparatus, independently of membrane conductance activation.     

Sperm factor initiates capacitance and conductance changes in mouse eggs that are more similar to fertilization than IP(3)- or Ca(2+)-induced changes

We used patch clamp electrophysiology and concurrent imaging with the Ca(2+)-sensitive dye, fura-2, to study the temporal relationship between membrane capacitance and conductance and intracellular free Ca(2+) concentration ([Ca(2+)](i)) during mouse egg fertilization. We found an approximately 2 pF step increase in egg membrane capacitance and a minor increase in conductance with no change in [Ca(2+)](i) at sperm fusion. This was followed approximately 1 min later by a rise in [Ca(2+)](i) that led to larger changes in capacitance and conductance. The most common pattern for these later capacitance changes was an initial capacitance decrease, followed by a larger increase and eventual return to the approximate starting value. There was some variation in this pattern, and sub-microM peak [Ca(2+)](i) favored capacitance decrease, while higher [Ca(2+)](i) favored capacitance increase. The magnitude of accompanying conductance increases was variable and did not correlate well with peak [Ca(2+)](i). The intracellular introduction of porcine sperm factor reproduced the postfusion capacitance and conductance changes with a similar [Ca(2+)](i) dependence. Raising [Ca(2+)](i) by the intracellular introduction of IP(3) initiated fertilization-like capacitance changes, but the conductance changes were slower to activate. Capacitance decrease could be induced when [Ca(2+)](i) was increased modestly by activation of an endogenous Ca(2+) current, with little effect on resting conductance. These results suggest that net turnover of the mouse egg surface membrane is sensitive to [Ca(2+)](i) and that sperm and the active component of sperm factor may be doing more than initiating the IP(3)-mediated release of intracellular Ca(2+).     

Mechanism of the electric response of lipid bilayers to bitter substances.

In order to clarify by what mechanism the lipid bilayer membrane changes its potential under the stimulation of bitter substances, a microscopic model for the effects of the substances on the membrane is presented and studied theoretically. It is assumed that the substances are adsorbed on the membrane and change the partition coefficients of ions between the membrane and the stimulation solution, the dipole orientation in the polar head, and the diffusion constants of ions in the membrane. It is shown, based on the comparison of the calculated results with the experimental ones, that the response arises mainly from a change in the partition coefficients. Protons play an essential role in the membrane potential variation due to the change in their partition coefficients. The present model reproduces the following observed unique properties in the response of lipid bilayers to bitter substances, which cannot be accounted for by the usual channel model for the membrane potential: 1) the response of the membrane potential appears even under the condition that there is no ion gradient across the membrane, 2) the response remains even when the salt in the stimulating solution is replaced with the salt made of an impermeable cation, and 3) the direction of the polarization of the potential is not reversed, even when the ion gradient across the bilayer is reversed.