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PubMed1961

An analysis of the membrane potential along a clamped squid axon.

A partially depolarized squid axon membrane is assumed to have a quasi-steady state negative resistance, the membrane potential is clamped at one point, and a distribution of potential along the axon is obtained from the cable equation. Nominal experimental values of -2 ohm cm(2) for the membrane and 6 ohm cm(2) for the internal and external current electrodes and the axoplasm and sea water between them are used for illustration. The potential and current may be uniform for an axon and electrode length less than 1.2 mm. For a long axon the potential varies as the cosine of the distance within 0.8 mm of the control point. Beyond this the potential variation is exponential and the entire pattern is about 5 mm long. The average current density out to 0.3 mm from the control point is within 10 per cent of the potential clamp value. These distributions are stable for control amplifications of about unity and more.

Animals↗

PubMed1962

Theoretical stability properties of a space-clamped axon.

A mathematical method for determining the stability properties of a uniform nerve membrane is developed. Two basically similar tests of stability are considered: examination of the real characteristic roots of the linearized equations and application of a modified Nyquist criterion to the linearized alternating current admittance. The method is applied to the Hodgkin-Huxley equations for the squid axon membrane at 6.3 degrees C to decide theoretically whether stable membrane behavior might be expected in a space clamp experiment. The equations are solved for step depolarizations similar to those used in voltage clamp experiments. Each solution can be represented by a trajectory in the phase space of the variables V, m, h, and n. The stability of motion of a phase point on a given trajectory, and hence the adequacy of the control of the membrane potential, is shown to be a function of the effective conductance in series with the membrane. (For a patch of membrane away from the point controlled by feedback, the effective conductance is the combined conductance of the axial current electrode, axoplasm, and an external layer of sea water, all in series.) In particular, there is a (uniquely determined) critical conductance, defined as the minimum effective series conductance consistent with stability, associated with each point on the trajectory. During a step depolarization the critical conductance goes through a maximum. The values of such maxima as a function of voltage are closely similar to the negative slopes of the peak inward current versus voltage curve. This empirical correlation may be helpfup in the prediction of stability in experimental situations.

Animals↗

PubMed1962

Sodium and potassium ion effluxes from squid axons under voltage clamp conditions.

Squid giant axons loaded with Na(24) were subjected to short duration (0.5 msec.) clamped depolarizations of about 100 mv at frequencies of 20/sec. and 60/sec. while in choline sea water. Under such conditions the early outward current was just about maximal at the time of termination of the clamping pulse. An integration of the early current versus time record gave 1.2 mucoulomb/cm(2) pulse, while a measurement of the extra Na(24) efflux resulting from repetitive pulsing gave a charge transfer of 1.4 mucoulomb/cm(2) pulse. In sodium-containing sea water and with pulses 50-75 mv more positive than E(Na) the Na(24) efflux is about 3 times the measured charge transfer. The efflux of K(42) from a previously loaded axon into normal sea water is only 50 per cent of the measured charge transfer when the membrane is held for about 5 msec. at a potential such that there is no early current, and such pulses are at 10-20/sec. The experiments appear to confirm the suggestion that the early current during bioelectric activity is sodium but provide unsatisfactory support for the identification of the delayed but sustained current solely with potassium ions. Resting Na(+) efflux is 0.6 pmole/cm(2) sec. mmole [Na](1), while the apparent K(+) efflux is about 250 pmole/cm(2) sec. and is little affected by hyperpolarization.

Animals↗

PubMed1971

Membrane current noise in lobster axon under voltage clamp.

Random fluctuations in the steady-state current of neural membrane were measured in the giant lobster axon by means of a low noise voltage-clamp system. The power density spectrum S(f) of the fluctuations was evaluated between 20 and 5120 Hz and found to be of the type 1/f. Mean values of the potassium, sodium, and leakage currents I(K), I(Na), and I(L) were also measured by usual voltage-clamp techniques. Comparisons between these two types of data recorded under a number of different experimental conditions, such as presence of tetrodotoxin (TTX), substitution of calcium by lanthanum, and changes in the external concentration of potassium, have strongly suggested that the intensity of the fluctuations is related to the magnitude of I(K).

Animals↗

PubMed1973

Voltage and current clamp transients with membrane dielectric loss.

Transient responses of a space-clamped squid axon membrane to step changes of voltage or current are often approximated by exponential functions of time, corresponding to a series resistance and a membrane capacity of 1.0 muF/cm(2). Curtis and Cole (1938, J. Gen. Physiol.21:757) found, however, that the membrane had a constant phase angle impedance z = z(1)(jomegatau)(-alpha), with a mean alpha = 0.85. (alpha = 1.0 for an ideal capacitor; alpha < 1.0 may represent dielectric loss.) This result is supported by more recently published experimental data. For comparison with experiments, we have computed functions expressing voltage and current transients with constant phase angle capacitance, a parallel leakage conductance, and a series resistance, at nine values of alpha from 0.5 to 1.0. A series in powers of t(alpha) provided a good approximation for short times; one in powers of t(-alpha), for long times; for intermediate times, a rational approximation matching both series for a finite number of terms was used. These computations may help in determining experimental series resistances and parallel leakage conductances from membrane voltage or current clamp data.

Animals↗

PubMed1976

Voltage clamp with double sucrose gap technique. External series resistance compensation.

In this paper we deal with the double sucrose-gap voltage clamp technique. To perform a reliable clamp or to analyze the intracellular potential distribution, any external series resistance in the artificial node must be taken into account for it induces an instability in the external potential as soon as a current develops. A circuit was designed to compensate for this error, it has been found effective on an analog model and on experimental uni- or multicellular preparations. The attenuation in series resistance frequently causes ringing in the step response. This behavior was studied theoretically and also simulated with analog models where a selective bridged-T network was found to represent the electrical characteristics of the preparation when associated with the chamber and control electronics. A residual series resistance was found and is considered to be a part of the preparation. Characteristics necessary to obtain best results are proposed, for a preparation to be studied in experiments utilizing the double sucrose gap technique with external series resistance compensation.

Cell Physiological Phenomena↗

PubMed1981

Currents recorded through small areas of squid axon membrane with an internal virtual ground voltage clamp.

A new voltage-clamp apparatus for the squid axon has been implemented to enable recording of currents through small areas of axon membrane. The performance of this clamp was tested by recording total sodium currents from perfused axons (I total) and sodium currents from small membrane patches (I patch), which were recorded from inside the axon with an L-shaped pipette. The I patch records, although four orders of magnitude smaller than I total, were stable and showed normal kinetics and voltage dependence, and appeared to reflect the activation of a small population of normal sodium channels. The size of the current recorded from the patch was mainly a function of the tip diameter of the L-shaped pipette and of the shunt resistance between inside the pipette and the axoplasm.

Animals↗

PubMed1983

Phospholipid bilayers made from monolayers on patch-clamp pipettes.

Phospholipid bilayers were made from phospholipid monolayers at the air/water interface on patch-clamp pipettes. Lipid bilayers were characterized using the K+ carrier nonactin and the channel formers gramicidin and alamethicin. Bilayers were also formed from monolayers spontaneously assembled in a suspension of native vesicles from cardiac sarcolemma and lobster axonal membranes and an excess of lipids. In these types of bilayers we observed several different channels including one contained in the axonal membrane that shows delayed rectifier behavior. This technique permits the study of reconstituted channels on a time scale and noise comparable to cellular patch-clamp standards.

Alamethicin↗

PubMed1984

Extracellular [K+] fluctuations in voltage-clamped canine cardiac Purkinje fibers.

Membrane currents and extracellular [K+] were measured in canine Purkinje strands during voltage-clamp steps to plateau or diastolic potentials. Extracellular [K+] increased during step depolarizations and decreased during step hyperpolarizations. On hyperpolarization, the largest fraction of the K+ depletion occurred during the initial 500 ms of the voltage-clamp step and was correlated with a potassium depletion current, the id. A slower component of the depletion also occurred on hyperpolarization and had a time constant consistent with cylindrical diffusion of potassium within the Purkinje strands. On depolarization, there is an accumulation of K+ that is correlated with the plateau current ix. On termination of depolarizing test pulses, the K+ accumulation decays with a time course similar to the ix tail current. Surprisingly, no accumulation of K+ occurred during the arrhythmogenic transient inward current, TI, suggesting that the selectivity of this current should be reevaluated.

Animals↗

PubMed1984

Rapid sodium channel conductance changes during voltage clamp steps in squid giant axons.

The sodium conductance of the membrane of the giant axon of squid was isolated by the use of potassium-free solutions and voltage-clamped with pulses containing three levels of depolarization. The conductance appears to undergo rapid changes during certain repolarizing clamp steps whose voltage reach at least partially overlaps the gating range. The percentage change in conductance increases with time of depolarization from approximately 0 to approximately 25-30% at 7 ms for a potential step from +70 to -30 mV. Conductance steps were also observed for voltage steps from various depolarized levels to -70 mV. All observed shifts were in the direction of a decreased conductance. The conductance steps appear to be a weak function of the concentration of external calcium, which also acts as a voltage-dependent channel blocker for inwardly directed sodium currents. A number of possible mechanisms are suggested. One of these is discussed in some detail and postulates a voltage- and time-dependent molecular process that does not itself yield open or closed channel conformations, but that affects the magnitude of the rate constants that do connect open and closed state conformations.

Animals↗

PubMed1992

Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels.

The electrical properties of gap junctions in cell pairs are usually studied by means of the dual voltage clamp method. The voltage across the junctional channels, however, cannot be controlled adequately due to an artificial resistance and a natural resistance, both connected in series with the gap junction. The access resistances to the cell interior of the recording pipettes make up the artificial resistance. The natural resistance consists of the cytoplasmic access resistances to the tightly packed gap junction channels in both cells. A mathematical model was constructed to calculate the actual voltage across each gap junction channel. The stochastic open-close kinetics of the individual channels were incorporated into this model. It is concluded that even in the ideal case of complete compensation of pipette series resistance, the number of channels comprised in the gap junction may be largely underestimated. Furthermore, normalized steady-state junctional conductance may be largely overestimated, so that transjunctional voltage dependence is easily masked. The model is used to discuss conclusions drawn from dual voltage clamp experiments and offers alternative explanations for various experimental observations.

Animals↗

PubMed1992

Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.

The present report describes a novel technique for voltage-clamping amphibian oocytes in which part of the membrane is isolated by a vaseline gap and the cytoplasmic fluid is exchanged by cutting or permeabilizing the remaining membrane. The main features of this open-oocyte, vaseline-gap voltage clamp are: (a) low current noise (1 nA at 3 kHz), (b) control of the ionic composition of both the internal and external media, (c) fast time resolution (20-100 microseconds time constant of decay of the capacity transient) and (d) stable recordings for several hours. These features allow reliable measurements of tail or gating currents and the new method is especially suitable when either of these currents must be measured to test the effects of mutations introduced into the cDNAs of cloned ion channels.

Animals↗

PubMed1994

Lipid-glass adhesion in giga-sealed patch-clamped membranes.

Adhesion between patch-clamped lipid membranes and glass micropipettes is measured by high contrast video imaging of the mechanical response to the application of suction pressure across the patch. The free patch of membrane reversibly alters both its contact angle and radius of curvature on pressure changes. The assumption that an adhesive force between the membrane and the pipette can sustain normal tension up to a maximum Ta at the edge of the free patch accounts for the observed mechanical responses. When the normal component of the pressure-induced membrane tension exceeds Ta membrane at the contact point between the free patch and the lipid-glass interface is pulled away from the pipette wall, resulting in a decreased radius of curvature for the patch and an increased contact angle. Measurements of the membrane radius of curvature as a function of the suction pressure and pipette radius determine line adhesion tensions Ta which range from 0.5 to 4.0 dyn/cm. Similar behavior of patch-clamped cell membranes implies similar adhesion mechanics.

Adhesiveness↗

PubMed2003

The diverse spectrum of sliding clamp interacting proteins.

DNA polymerase sliding clamps are a family of ring-shaped proteins that play essential roles in DNA metabolism. The proteins from the three domains of life, Bacteria, Archaea and Eukarya, as well as those from bacteriophages and viruses, were shown to interact with a large number of cellular factors and to influence their activity. In the last several years a large number of such proteins have been identified and studied. Here the various proteins that have been shown to interact with the sliding clamps of Bacteria, Archaea and Eukarya are summarized.

Archaea↗

PubMed1987

Cell-attached patch clamp measurement of macroscopic rapid inward sodium current in cultured heart cell reaggregates.

A megaohm seal, cell-attached patch clamp technique utilizing plastic suction pipettes for measuring macroscopic rapid inward sodium current (INa) was applied to cell membrane patches 30 to 100 micron2 in area at the periphery of reaggregates of myocardial cells from newborn rats cultured for 3 to 7 days. The reaggregates were composed of about 20 to several thousand of electrically well-coupled cells, the latter forming spheroidal reaggregates with a diameter of maximally 300 microns. This system was shown to guarantee satisfactory voltage-clamp control during ionic current measurements. The time and voltage dependence of the INa recorded during periods of up to 90 min was similar to that observed in single cardiac cell preparations from adult rats. INa inactivation was described by two time constants (tau h1, and tau h2). For maximal INa tau h1 and tau h2 had values of 1.5 +/- 0.25 ms and 8.7 +/- 3.9 ms (n = 4), respectively. The time course for recovery of INa from inactivation at the reaggregate resting potential exhibited also two time constants (tau re1 = 9.8 +/- 3 ms, tau re2 = 193 +/- 50 ms, n = 5). Estimated current density was about 10 pA/micron2. The concentration of tetrodotoxin needed to reduce maximal INa by 75% was 85 times lower in the reaggregates than it was in freshly isolated heart muscle cells from adult rats. The present system offers a combination of features that should make it well-suited for the study of both short- and long-term effects on the sodium channels in neonatal heart cells: Easy handling of the object, great electric stability, high time and amplitude resolution during ionic current measurements, and viability for at least one week.

Animals↗

PubMed2002

The mechanism of transcriptional activation by the topologically DNA-linked sliding clamp of bacteriophage T4.

Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the sigma-family protein gp55 provides promoter recognition, gp33 is the co-activator, and gp45 is the activator of transcription; gp33 also represses transcription in the absence of gp45. Transcriptional activation by gp45, the toroidal sliding clamp of the T4 DNA polymerase holoenzyme, requires assembly at primer-template junctions by its clamp loader. The mechanism of transcriptional activation has been analyzed by examining rates of formation of open promoter complexes. The basal gp55-RNA polymerase holoenzyme is only weakly held in its initially formed closed promoter complex, which subsequently opens very slowly. Activation ( approximately 320-fold in this work) increases affinity in the closed complex and accelerates promoter opening. Promoter opening by gp55 is also thermo-irreversible: the T4 late promoter does not open at 0 degrees C, but once opened at 30 degrees C remains open upon shift to the lower temperature. At a hybrid promoter for sigma(70) and gp55-holoenzymes, only gp55 confers thermo-irreversibility of promoter opening. Interaction of gp45 with a C-terminal epitope of gp33 is essential for the co-activator function of gp33.

Bacteriophage T4↗

PubMed1997

Hemodynamic effects of isovolemic hemodilution during descending thoracic aortic cross clamping and lower torso reperfusion.

BACKGROUND: Isovolemic hemodilution has been suggested for blood conservation and to improve hemodynamic tolerance to abdominal aortic cross clamping. However, the hemodynamic effects of hemodilution during descending thoracic aortic cross clamping (DAC) have not been established. We evaluated them in anesthetized swine. METHODS: Hemodilution (n = 7) was produced by the isovolemic exchange of blood for 6% hetastarch to a target hematocrit of 20%. Hematocrit in control pigs (n = 7) remained at 30%. DAC was performed at the T9 level for 45 minutes. During a 60-minute reperfusion period, control pigs were infused with lactated Ringer's solution; shed blood was returned to hemodilution pigs, followed by lactated Ringer's. If hypotension occurred despite left atrial pressure of 10 mm Hg or greater, boluses of phenylephrine were given to keep mean arterial pressure above 60 mm Hg. RESULTS: Hemodilution caused a marked reduction in hematocrit and in global oxygen delivery (DO2). DAC produced a significant increase in proximal arterial pressure, cardiac index, and DO2 and oxygen consumption (VO2) was markedly reduced in both groups. A significant increase in systemic vascular resistance during DAC occurred only in control pigs. After reperfusion, vascular resistance was significantly lower than baseline in hemodilution pigs, requiring a sixfold greater dose of phenylephrine to avoid hypotension. A lower global DO2 and supply-limited VO2 were also observed in hemodilution pigs. CONCLUSIONS: Isovolemic hemodilution maintains hemodynamic stability during DAC. During lower torso reperfusion, however, hemodilution caused hemodynamic instability, decreased global DO2, and limited VO2, which may offset its potential benefits.

Animals↗

PubMed2002

Incisionless in-office castration using a veterinary castration device (Burdizzo clamp).

Nonoperative ablation of the testicular artery has been described in animals using the Burdizzo clamp, but not in humans. We report a case of incisionless castration performed using local anesthesia, in a transsexual who wished to avoid the pain, expense, and scar associated with the open procedure. The Burdizzo clamp crushed the spermatic cord bilaterally, and the testis was allowed to atrophy. The soft-tissue damage was minimal, and the serum testosterone fell to castrate levels. This method has proved useful for castration in a transgender patient, but could also be used for castration in patients with advanced prostate cancer.

Adult↗