Electrical characteristics of frog atrial trabeculae in the double sucrose gap.

The electrical behavior of small single frog atrial trabeculae in the double sucrose gap has been investigated. The currents injected during voltage clamp experiments did not behave as predicted from the assumption of spatial uniformity of the voltage across a Hodgkin-Huxley membrane. Much of the difference is due to the geometrical complexities of this tissue. Nonetheless, two transient inward currents have been identified, the faster of which is blocked by tetrodotoxin (TTX). The magnitude of the slower transient varies markedly between preparations but always increases in a given preparation with increase of external calcium. The fast transient current traces, at small to intermediate depolarizations, are often marred by the presence of notches and secondary peaks due most probably to the loss of space clamp conditions. In many preparations these could be removed by reducing the current magnitude through application of a partially-blocking dose of TTX. Conversely, in the preparations whose fast transient was fully blocked by TTX, notches and secondary peaks in the slow transient could by induced through increasing calcium concentration and thereby the slow current magnitude. Previously used techniques for the measurement of the reversal potential of the fast inward transient have been shown to be invalid. In so far as they can be measured, the reversal potentials of the fast and slow inward transient are in the same neighborhood, i.e. around 120 mV from rest. The true values may be quite a bit apart. The total charge flow in the capacitive transient was measured for different sized nodes and preparations. From these data and estimates of plasma membrane area per unit trabecular volume, specific membrane capacitances of around 3 muF/cm2 were calculated for small bundles. The apparent ion current densities on this basis are approximately 1/10 of those measured in axons. The capacitive current occurring in small bundles decayed as the sum of at least three exponential functions of time. On the basis of these data and the anomalously large stable node widths, we suggest a coaxial core model of the preparation with the inner elements in series with an additional large extracellular resistance.     

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).     

A new algorithm for voltage clamp by iteration: A learning control of a nonlinear neuronal system

Voltage-clamp of excitable membrane allows the measurement of membrane currents associated with electrical potential changes across the membrane. However, it has been impossible in practice to apply the conventional analog feedback voltageclamp circuits to single electrode voltage clamping in central neurons. The reason for this is that the feedback system becomes unstable because of the positive feedback required for compensation of capacitative loss through the wall of the microelectrode. Park et al. (1981) proposed a new iterative technique to solve this problem. It requires that the potential to be clamped repeats itself with little or no change. The amount of current needed to clamp the membrane potential is not determined at once, but in a step-wise, trial and error fashion in the course of a set of repetitions. Since the feedback loop is open in real time, the system has great stability, and this advantage can be exploited in single electrode preparations. The computation algorithm which calculates the current waveform based on the voltage deviation during the last trial is the central part of the iterative voltage-clamp system. In this paper, we propose a new algorithm, which has several theoretical and practical advantages over the original one proposed by Park et al. First, two parameters used in the new algorithm are predetermined by a current-clamp experiment. Second, the speed of convergence of the new algorithm is faster than that of the Park's original algorithm. This was shown by computer simulation of iterative voltage clamp of artificial membrane following Hodgkin-Huxley equations for squid axon membrane and Rall's compartment model for a neuron with dendrites. Finally, we offer proof that the new algorithm is certain to converge for the general cases of voltage-clamp experiments with active membrane properties, synaptic membranes, etc. Consequently, the new algorithm for iterative voltage clamp is very suitable for single electrode voltage clamp in the central neurons. The new algorithm has been successfully applied to voltage-clamp experiments on rubrospinal neurons of cats (Tsukahara, Murakami, Kawato, Oda, and Etoh, in preparation).     

Fast activation of cardiac Ca++ channel gating charge by the dihydropyridine agonist, BAY K 8644.

Nonlinear charge movement (gating current) was studied by the whole-cell patch clamp method using cultured 17-d-old embryonic chick heart cells. Na+ and Ca++ currents were blocked by the addition of 10 microM TTX and 3 mM CoCl2; Cs+ replaced K+ both intra- and extracellularly. Linear capacitive and leakage currents were subtracted by a P/5 procedure. The small size (15 microns in diameter) and the lack of an organized internal membrane system in these myocytes permits a rapid voltage clamp of the surface membrane. Ca++ channel gating currents were activated positive to -60 mV; the rising phase was not distorted due to the system response time. The addition of BAY K 8644 (10(-6) M) caused a shortening of the time to peak of the Ca++ gating current, and a negative shift in the isochronal Qon vs. Vm curve. Qmax was unchanged by BAY K 8644. The voltage-dependent shift produced by BAY K 8644 is similar to that produced by isoproterenol (Josephson, I.R., and N. Sperelakis. 1990. Biophys. J. 57:305a. [Abstr.]). The results suggest that the binding of BAY K 8466 to one or more of the Ca++ channel subunits alters the kinetics and shifts the voltage dependence of gating. These changes in the gating currents can explain the parallel changes in the macroscopic Ca++ currents.     

Voltage clamping with single microelectrodes: comparison of the discontinuous mode and continuous mode using the Axoclamp 2A amplifier

The voltage clamp technique is a powerful method for studying the physiology of excitable membrane. This technique has made possible the determination of ionic responses generated by activation of either receptor-mediated or voltage-dependent processes. The development of the whole-cell, 'tight-seal' voltage clamp method has allowed the analysis and examination of membrane physiology at the single cell level. The method allows the characterization of voltage-dependent ionic conductances both at the macroscopic (whole-cell) and at the microscopic (unitary conductance or single channel) level in cells less than 10 micron in diameter, a feat difficult to achieve with 'conventional' fine-tipped micropipettes. In this paper, several methologies used for culturing neuronal and non-neuronal cells in the laboratory are described. A comparison between the two modes of voltage clamp using blunt-tipped 'patch'-microelectrodes, the switching (discontinuous) and the non-switching (continuous) modes, of the Axoclamp-2A amplifier is made. Some results on membrane currents obtained from neuronal and non-neuronal cells using the single electrode whole-cell 'tight-seal' voltage clamp is illustrated. The possible existence of two inactivating K+ currents, one dependent on Ca++ the other is not, is discussed.     

Neuron–transistor coupling: interpretation of individual extracellular recorded signals

The electrical coupling of randomly migrating neurons from rat explant brain-stem slice cultures to the gates of non-metallized field-effect transistors (FETs) has been investigated. The objective of our work is the precise interpretation of extracellular recorded signal shapes in comparison to the usual patch-clamp protocols to evaluate the possible use of the extracellular recording technique in electrophysiology. The neurons from our explant cultures exhibited strong voltage-gated potassium currents through the plasma membrane. With an improved noise level of the FET set-up, it was possible to record individual extracellular responses without any signal averaging. Cells were attached by patch-clamp pipettes in voltage-clamp mode and stimulated by voltage step pulses. The point contact model, which is the basic model used to describe electrical contact between cell and transistor, has been implemented in the electrical simulation program PSpice. Voltage and current recordings and compensation values from the patch-clamp measurement have been used as input data for the simulation circuit. Extracellular responses were identified as composed of capacitive current and active potassium current inputs into the adhesion region between the cell and transistor gate. We evaluated the extracellular signal shapes by comparing the capacitive and the slower potassium signal amplitudes. Differences in amplitudes were found, which were interpreted in previous work as enhanced conductance of the attached membrane compared to the average value of the cellular membrane. Our results suggest rather that additional effects like electrodiffusion, ion sensitivity of the sensors or more detailed electronic models for the small cleft between the cell and transistor should be included in the coupling model.     

A nonlinear electrostatic potential change in the T-system of skeletal muscle detected under passive recording conditions using potentiometric dyes

Voltage-sensing dyes were used to examine the electrical behavior of the T-system under passive recording conditions similar to those commonly used to detect charge movement. These conditions are designed to eliminate all ionic currents and render the T-system potential linear with respect to the command potential applied at the surface membrane. However, we found an unexpected nonlinearity in the relationship between the dye signal from the T-system and the applied clamp potential. An additional voltage- and time-dependent optical signal appears over the same depolarizing range of potentials where change movement and mechanical activation occur. This nonlinearity is not associated with unblocked ionic currents and cannot be attributed to lack of voltage clamp control of the T-system, which appears to be good under these conditions. We propose that a local electrostatic potential change occurs in the T-system upon depolarization. An electrostatic potential would not be expected to extend beyond molecular distances of the membrane and therefore would be sensed by a charged dye in the membrane but not by the voltage clamp, which responds solely to the potential of the bulk solution. Results obtained with different dyes suggest that the location of the phenomena giving rise to the extra absorbance change is either intramembrane or at the inner surface of the T-system membrane.     

Voltage-activated Ca2+ currents in insulin-secreting cells

Membrane voltage and voltage-clamped membrane currents have been investigated with the whole-cell patch clamp method in the insulin-secreting cell line RINm5F. The mean resting membrane potential of RINm5F cells was found to be -52 mV. Overshooting spike potentials could be evoked by depolarising voltage steps in the absence of a secretagogue. Inward membrane currents evoked by depolarising voltage steps were dependent upon extracellular Ca2+ and blocked by Co2+, nifedipine and verapamil. Outward membrane currents which were evoked by depolarising voltage steps to positive membrane potentials were reduced when Ca2+ entry was prevented. It is concluded that the voltage-activated Ca2+ currents underlie the voltage-activated spike potentials recorded from insulin-secreting cells.     

A patch clamp study on the electro-permeabilization of higher plant cells: Supra-physiological voltages induce a high-conductance, K+ selective state of the plasma membrane

Permeabilization of biological membranes by pulsed electric fields ("electroporation") is frequently used as a tool in biotechnology. However, the electrical properties of cellular membranes at supra-physiological voltages are still a topic of intensive research efforts. Here, the patch clamp technique in the whole cell and the outside out configuration was employed to monitor current-voltage relations of protoplasts derived from the tobacco culture cell line "Bright yellow-2". Cells were exposed to a sequence of voltage pulses including supra-physiological voltages. A transition from a low-conductance (~0.1 nS/pF) to a high-conductance state (~5 nS/pF) was observed when the membrane was either hyperpolarized or depolarized beyond threshold values of around -250 to -300 mV and +200 to +250 mV, respectively. Current-voltage curves obtained with ramp protocols revealed that the electro-permeabilized membrane was 5-10 times more permeable to K+ than to gluconate. The K+ channel blocker tetraethylammonium (25 mM) did not affect currents elicited by 10 ms-pulses, suggesting that the electro-permeabilization was not caused by a non-physiological activation of K+ channels. Supra-physiological voltage pulses even reduced "regular" K+ channel activity, probably due to an increase of cytosolic Ca2+ that is known to inhibit outward-rectifying K+ channels in Bright yellow-2 cells. Our data are consistent with a reversible formation of aqueous membrane pores at supra-physiological voltages.     

Electrophysiological properties of macrophages

Electrophysiological studies indicate that the macrophage can display at least two different K conductances, a Ca-mediated K conductance and an inward rectifying K conductance, as well as an electrogenic Na+-K+ pump. Spontaneous hyperpolarizations associated with a Ca-mediated K permeability have been noted in all types of macrophages studied. Similar membrane hyperpolarizations can be elicited by a variety of stimuli that presumably increase intracellular calcium. These include mechanical and electrical stimulation as well as exposure to endotoxin-activated serum, chemotactic peptides, and the Ca ionophore A23187. Recent patch clamp studies on macrophages demonstrated channel activity that probably corresponds to currents through the inward rectifying K conductance previously described with current clamp techniques. With the advent of the patch clamp, this and other conductances can be effectively examined by using both whole-cell voltage clamp and patch recordings in a variety of different macrophages, including small freshly isolated cells.     

The N-Shaped Current-Potential Characteristic in Frog Skin: II. Kinetic Behavior during Ramp Voltage Clamp

Previous step voltage-clamp measurements on frog skin showed the presence of an N-shaped current-potential (I-V) relation in excitable skin. However, the collection and reconstruction of I-V data using discrete step changes of skin potential was tedious because of the long refractory period (up to 1 min) in frog skin. A direct and rapid (5 msec) method for recording the N-shaped I-V characteristic in real time is presented. Ramp functions are used as the command to the clamp system instead of a step function. Consequently the skin potential is forced to change in a linear manner (as commanded) and the skin current can be recorded as a continuous function of the controlled change of skin potential. With the ramp clamp, a low-resistance membrane state ( 10 Omega . cm(2)) resembling a breakdown phenomenon was observed at high skin potential ( 300 mv). Entry into the low resistance state resulted in a collapse of the N-shaped I-V relation to a nearly linear function. The utility of the ramp measurement is demonstrated by predicting (1) that the maximum rate of rise of the spike occurs at a voltage corresponding to the valley (local minimum) in the N-shaped I-V curve, (2) that the rate of rise of the spike increases with increasing clamp currents, (3) the voltage peak of the spike, and (4) the time course of the rising phase of the spike.     

Properties of internally perfused, voltage-clamped, isolated nerve cell bodies

The membrane properties of isolated neurons from Helix aspersa were examined by using a new suction pipette method. The method combines internal perfusion with voltage clamp of nerve cell bodies separated from their axons. Pretreatment with enzymes such as trypsin that alter membrane function is not required. A platinized platinum wire which ruptures the soma membrane allows low resistance access directly to the cell's interior improving the time resolution under voltage clamp by two orders of magnitude. The shunt resistance of the suction pipette was 10-50 times the neuronal membrane resistance, and the series resistance of the system, which was largely due to the tip diameter, was about 10(5) omega. However, the peak clamp currents were only about 20 nA for a 60-mV voltage step so that measurements of membrane voltage were accurate to within at least 3%. Spatial control of voltage was achieved only after somal separation, and nerve cell bodies isolated in this way do not generate all-or-none action potentials. Measurements of membrane potential, membrane resistance, and membrane time constant are equivalent to those obtained using intracellular micropipettes, the customary method. With the axon attached, comparable all-or-none action potentials were also measured by either method. Complete exchange of Cs+ for K+ was accomplished by internal perfusion and allowed K+ currents to be blocked. Na+ currents could then be blocked by TTX or suppressed by Tris-substituted snail Ringer solution. Ca2+ currents could be blocked using Ni2+ and other divalent cations as well as organic Ca2+ blockers. The most favorable intracellular anion was aspartate-, and the sequence of favorability was inverted from that found in squid axon.     

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.     

Effects on membrane capacitance of steroids with antagonist properties at GABAA receptors

We investigated the electrophysiological signature of neuroactive steroid interactions with the plasma membrane. We found that charged, sulfated neuroactive steroids, those that exhibit noncompetitive antagonism of GABA_A receptors, altered capacitive charge movement in response to voltage pulses in cells lacking GABA receptors. Uncharged steroids, some of which are potent enhancers of GABA_A receptor activity, produced no alteration in membrane capacitance. We hypothesized that the charge movements might result from physical translocation of the charged steroid through the transmembrane voltage, as has been observed previously with several hydrophobic anions. However, the charge movements and relaxation time constants of capacitive currents did not exhibit the Boltzmann-type voltage dependence predicted by a single barrier model. Further, a fluorescently tagged analog of a sulfated neurosteroid altered membrane capacitance similar to the parent compound but produced no voltage-dependent fluorescence change, a result inconsistent with a strong change in the polar environment of the fluorophore during depolarization. These findings suggest that negatively charged sulfated steroids alter the plasma membrane capacitance without physical movement of the molecule through the electric field.     

游离心肌细胞晚钠通道爆发型开放模式的电位依赖性

应用膜片箝技术记录豚鼠游离心室肌细胞钠通道电流,发现除极可引起晚钠电流爆发型开放,而复极可终止爆发型活动。爆发型模式的通道电流不仅有浓度依赖性和电位依赖性,其开放时间常数也随箝制电位变正而增大。多步阶梯式除极和斜线除极的实验结果首次表明电位变化愈迅速、除极步数愈多,爆发型出现的机率也就愈高。     

An improved vaseline gap voltage clamp for skeletal muscle fibers

A Vaseline gap potentiometric recording and voltage clamp method is developed for frog skeletal muscle fibers. The method is based on the Frankenhaeuser-Dodge voltage clamp for myelinated nerve with modifications to improve the frequency response, to compensate for external series resistance, and to compensate for the complex impedance of the current-passing pathway. Fragments of single muscle fibers are plucked from the semitendinosus muscle and mounted while depolarized by a solution like CsF. After Vaseline seals are formed between fluid pools, the fiber ends are cut once again, the central region is rinsed with Ringer solution, and the feedback amplifiers are turned on. Errors in the potential and current records are assessed by direct measurements with microelectrodes. The passive properties of the preparation are simulated by the "disk" equivalent circuit for the transverse tubular system and the derived parameters are similar to previous measurements with microelectrodes. Action potentials at 5 degrees C are long because of the absence of delayed rectification. Their shape is approximately simulated by solving the disk model with sodium permeability in the surface and tubular membranes. Voltage clamp currents consist primarily of capacity currents and sodium currents. The peak inward sodium current density at 5 degrees C is 3.7 mA/cm2. At 5 degrees C the sodium currents are smoothly graded with increasing depolarization and free of notches suggesting good control of the surface membrane. At higher temperatures a small, late extra inward current appears for small depolarizations that has the properties expected for excitation in the transverse tubular system. Comparison of recorded currents with simulations shows that while the transverse tubular system has regenerative sodium currents, they are too small to make important errors in the total current recorded at the surface under voltage clamp at low temperature. The tubules are definitely not under voltage clamp control.     

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.     

Effects of hypothalamic peptides on electrical activity and membrane currents of 'patch perforated' clamped GH3 anterior pituitary cells.

'Perforated-patch' recordings of rat anterior pituitary GH3 cells allow long and stable monitoring of electrical activity and membrane currents. Under current clamp conditions, the biphasic effect of thryotropin releasing hormone (TRH) consisting of a transient hyperpolarization followed by a longer phase of increased action potential frequency is fully preserved. Somatostatin suppresses action potential activity and antagonizes the second phase of enhanced spiking caused by TRH. Voltage clamp records of isolated currents indicate that TRH affects calcium-dependent potassium currents, but does not alter either voltage-dependent potassium or calcium currents at times and concentrations at which the electrical activity is increased.