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PubMed1984

Outward currents in voltage-clamped rat sympathetic neurones.

Outward membrane currents were studied in neurones of the isolated rat superior cervical ganglion by using a two-micro-electrode or single-micro-electrode voltage-clamp technique. Under current clamp, depolarization elicited electrotonic potentials that displayed marked outward rectification. From negative resting potentials (-70 mV) a short latency, short duration outward rectification was observed. From more positive potentials (-40 mV) a longer latency persistent outward rectification could be demonstrated. Under voltage clamp, four distinct outward currents were observed: a delayed rectifier (IK); a transient outward current (IA); a Ca2+-activated current (IC) and the M-current (IM). The maximum amplitude of IK, IA and IC was 1-2 orders of magnitude greater than IM. Depolarizing from -40 mV to potentials more positive than -20 mV co-activated IK and IC, producing a characteristic N-shaped current voltage curve with a minimum at about +80 mV. Superfusion with Mn2+-containing solutions reduced outward current at all voltages and abolished the N-characteristic; the remaining current (IK) slowly inactivated (tau greater than 1 s). Raising [K+]o from 6 to 36 mmol/l reversed outward tail currents observed in normal solution. Addition of tetraethylammonium ions (1-3 mmol/l) strongly reduced the amplitude of IK and IC. IA was characterized by very rapid activation at potentials more positive than -60 mV and by fast and complete inactivation at potentials in the activation range. The amplitude of IA was dependent on [K+]o and was reduced by external 4-aminopyridine (1-3 mmol/l). The activation appeared to depend on the nature and concentration of divalent cations present in the superfusate. It is concluded that the soma membrane of rat sympathetic neurones, like many other vertebrate and invertebrate neurones, contains multiple populations of K+ channels. The possible functions of these in the control of ganglion cell excitability are discussed.

4-Aminopyridine↗
PubMed1985

Voltage clamp of bull-frog cardiac pace-maker cells: a quantitative analysis of potassium currents.

Spontaneously active single cells have been obtained from the sinus venosus region of the bull-frog, Rana catesbeiana, using an enzymic dispersion procedure involving serial applications of trypsin, collagenase and elastase in nominally 0 Ca2+ Ringer solution. These cells have normal action potentials and fire spontaneously at a rate very similar to the intact sinus venosus. A single suction micro-electrode technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981; Hume & Giles, 1983) has been used to record the spontaneous diastolic depolarizations or pace-maker activity as well as the regenerative action potentials in these cells. This electrophysiological activity is completely insensitive to tetrodotoxin (TTX; 3 X 10(-6) M) and is very similar to that recorded from an in vitro sinus venosus preparation. The present experiments were aimed at identifying the transmembrane potassium currents, and analysing their role(s) in the development of the pace-maker potential and the repolarization of the action potential. Depolarizing voltage-clamp steps from the normal maximum diastolic potential (-75 mV) elicit a time- and voltage-dependent activation of an outward current. The reversal potential of this current in normal Ringer solution [( K+]0 2.5 mM) is near -95 mV; and it shifts by 51 mV per tenfold increase in [K+]0, which strongly suggests that this current is carried by K+. We therefore labelled it IK. The reversal potential of IK did not shift in the positive direction following very long (20 s) depolarizing clamp steps to +20 mV, indicating that 'extracellular' accumulation of [K+]0 does not produce any significant artifacts. The fully activated instantaneous current-voltage (I-V) relationship for IK is approximately linear over the range of potentials -130 to -30 mV. Thus, the ion transfer mechanism of IK may be described as a simple ohmic conductance in this range of potentials. Positive relative to -30 mV, however, the I-V exhibits significant inward rectification. A Hodgkin-Huxley analysis of the kinetics of IK, including a demonstration that the envelope of tails quantitatively matches the time course of the onset of IK during a prolonged depolarizing clamp step has been completed. The steady-state activation variable (n infinity) of IK spans the voltage range approximately -40 to +10 mV. It is well-fitted by a Boltzmann distribution function with half-activation at -20 mV. The time course of decay of IK is a single exponential. However, the activation or onset of IK shows clear sigmoidicity in the range of potentials from the activation threshold (-40 mV) to 0 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1987

A patch-clamp study of mammalian platelets and their voltage-gated potassium current.

1. Mammalian platelets were freshly isolated from human, rabbit, or rat blood. The whole-cell and cell-attached voltage-clamp variations of the patch-clamp technique were employed to study the passive electrical properties and ion channels of unstimulated platelets. 2. The input capacitance of a platelet measured by the phase-sensitive detection method was about 128 fF, the input resistance of a platelet was about 59 G omega and the resting membrane potential was about -50 mV which was directly measured by a whole-cell recording in the current-clamp mode. 3. The predominant ion channel was a voltage-gated K+ channel resembling the delayed rectifier K+ channel of nerve, muscle and T-lymphocyte. There was no indication of any inward current in the platelet membrane. The activation of the K+ current could be fitted by n4 kinetics, and was half-maximal at about -35 mV. 4. The time constant of K+ current inactivation was virtually independent of voltage and varied from cell to cell. Recovery from inactivation was slow and dependent on the size and duration of the preceding conditional voltage step. Steady-state inactivation was half-maximal at about -50 mV and was complete at positive potentials. 5. The predominant single-K+-channel conductance was 9 pS and the estimated number of K+ channels per platelet was about 325, corresponding to a density of 25/micron2 apparent membrane area.

Action Potentials↗
PubMed1993

Calcium clamp in isolated neurones of the snail Helix pomatia.

1. Intracellular free calcium concentration ([Ca2+]i) in isolated non-identified Helix pomatia neurones has been clamped at different physiologically significant levels by a feedback system between the fluorescent signal of fura-2 probe loaded into the cell and ionophoretic injection of Ca2+ ions through a CaCl2-loaded microelectrode. The membrane potential of the neurone has also been clamped using a conventional two-microelectrode method. 2. Special measurements have shown that the transport indices of injecting microelectrodes filled with 50 mM CaCl2 are quite variable (0.11 +/- 0.06, mean +/- S.D.). However, for each electrode the transport indices remained stable during several injection trials into a solution drop having the size of a neurone. The spread of calcium ions from the tip of the microelectrode across the cytosol of the neurone terminated within 2-4 s. The spatial difference in [Ca2+]i at this time did not exceed 10%. 3. Clamping of [Ca2+]i at a new increased level was accompanied by a transient of the Ca(2+)-injecting current. To increase [Ca2+]i by 0.1 microM, the amount of calcium ions injected during this stage had to be 36 +/- 20 microM Ca2+ per cell volume. Obviously, this transient represents the filling of a fast cytosolic buffer which has to be saturated to reach a new increased level of [Ca2+]i. It was followed by a steady component of Ca(2+)-injecting current, which was quite low (corresponding to injection of 0.39 +/- 0.20 microM s-1 for a 0.1 microM change of [Ca2+]i). This may represent the functioning of Ca(2+)-eliminating systems and corresponds to a similar amount of Ca2+ extruded from the cytoplasm. 4. Changes in the injection current also developed when Ca2+ influx through the membrane was triggered by the activation of voltage-gated calcium channels. The amount of Ca2+ entering the cell during the first seconds of depolarization to--15 mV was equal to 0.59 +/- 0.31 microM s-1 per cell volume. 5. No activation of Ca(2+)-dependent potassium current was observed during the changes in [Ca2+]i to levels exceeding the basal one by several times. Obviously, to activate this current, a much stronger increase in [Ca2+]i is needed in the immediate vicinity of the corresponding channels.

Animals↗
PubMed1991

The anion paradox in sodium taste reception: resolution by voltage-clamp studies.

Sodium salts are potent taste stimuli, but their effectiveness is markedly dependent on the anion, with chloride yielding the greatest response. The cellular mechanisms that mediate this phenomenon are not known. This "anion paradox" has been resolved by considering the field potential that is generated by restricted electrodiffusion of the anion through paracellular shunts between taste-bud cells. Neural responses to sodium chloride, sodium acetate, and sodium gluconate were studied while the field potential was voltage-clamped. Clamping at electronegative values eliminated the anion effect, whereas clamping at electropositive potentials exaggerated it. Thus, field potentials across the lingual epithelium modulate taste reception, indicating that the functional unit of taste reception includes the taste cell and its paracellular microenvironment.

Animals↗
PubMed1984

Voltage clamp effects on bacterial chemotaxis.

To examine whether or not sensory signaling in bacteria is by way of fluctuations in membrane potential, we studied the effect of clamping the potential on bacterial chemotaxis. The potential was clamped by valinomycin, a K+ -specific ionophore, in the presence of K+. Despite the clamped potential, sensory signaling did occur: both Escherichia coli and Bacillus subtilis cells were still excitable and adaptable under these conditions. It is concluded that signaling in the excitation and adaptation steps of chemotaxis is not by way of fluctuations in the membrane potential.

Bacillus subtilis↗
PubMed2004

Photo-cross-linking of a purified preinitiation complex reveals central roles for the RNA polymerase II mobile clamp and TFIIE in initiation mechanisms.

The topological organization of a TATA binding protein-TFIIB-TFIIF-RNA polymerase II (RNAP II)-TFIIE-promoter complex was analyzed using site-specific protein-DNA photo-cross-linking of gel-purified complexes. The cross-linking results for the subunits of RNAP II were used to determine the path of promoter DNA against the structure of the enzyme. The results indicate that promoter DNA wraps around the mobile clamp of RNAP II. Cross-linking of TFIIF and TFIIE both upstream of the TATA element and downstream of the transcription start site suggests that both factors associate with the RNAP II mobile clamp. TFIIE alpha closely approaches promoter DNA at nucleotide -10, a position immediately upstream of the transcription bubble in the open complex. Increased stimulation of transcription initiation by TFIIE alpha is obtained when the DNA template is artificially premelted in the -11/-1 region, suggesting that TFIIE alpha facilitates open complex formation, possibly through its interaction with the upstream end of the partially opened transcription bubble. These results support the central roles of the mobile clamp of RNAP II and TFIIE in transcription initiation.

Animals↗
PubMed1980

The load clamp analysis of mammalian heart contraction in the presence of noradrenaline.

In experiments on isolated guinea pig papillary muscles superfused by Tyrode's solution the effects of abrupt alterations in load on length transients were investigated in controls and the presence of noradrenaline (2 mg/L) at 25 +/- 2 degrees C. In controls short load clamps (50 ms, 0.6 g) had no effect on the isotonic contraction if they were imposed in the fast shortening phase but accelerated relaxation when applied at the later phases of the contractile response. The timing of this stretch intervention determined the effect of the load clamps on length transients. The load clamp analysis revealed two components after the addition of noradrenaline even if splitting was absent on the contraction curve. The amplitude of the first component increased by 81 +/- 22% (n = 9) when the stimulation frequency was raised from 0.1 to 1.0 Hz; its time to peak tension did not change and was equal to 169 +/- 6ms(n = 12). The second component did not change between 0.1 and 0.5 Hz but significantly decreased to 19 +/- 10% (n = 4) at stimulation frequency 1.0 Hz; its time to peak tension correlated with action potential duration (r = 0.98) and decreased from 518 +/- 12 ms at 0.1 Hz to 314 +/- 20 ms at 1.0 Hz (n = 12). It was concluded that the first component is induced by calcium release from sarcoplasmic reticulum and the second one is determined by the action potential duration.

Action Potentials↗
PubMed1986

Skull radiography in infants: potential hazards of the use of head clamps.

In six infants, aged 2 days to 3 months, the use of head clamps for immobilization during radiography resulted in depression of the cranial vault. In all of the infants the depression disappeared after removal or repositioning of the clamp. However, because of the possible unfavorable consequences, head clamps with spongy, larger surface areas should be used in infants.

Craniocerebral Trauma↗
PubMed1992

Postcatheterization femoral artery injuries: repair with color flow US guidance and C-clamp assistance.

Color flow ultrasound-guided compression repair of postcatheterization femoral artery injuries was attempted with the assistance of a C-clamp device in 10 patients. The C-clamp device was designed to hold the transducer in the optimal compression position and eliminate operator fatigue. In nine patients, the lesion was eliminated in a mean compression time of 59 minutes. The C-clamp maintained the transducer in an effective compression position in six procedures; in the other four, intervention to prevent slippage was required.

Aged↗
PubMed1991

Ca2+ current and Ca2+ transients under action potential clamp in guinea pig ventricular myocytes.

Precise characterization of the magnitude and kinetics of transsarcolemmal Ca2+ influx during an action potential (AP) is essential for a complete understanding of excitation-contraction coupling in heart. Using a voltage-clamp protocol that simulated a physiological AP (AP clamp), we characterized the properties of the Ca2+ current (ICa) in guinea pig ventricular myocytes. The AP-generated ICa showed a complex time course that was different from ICa generated by a square pulse. ICa activated rapidly during the upstroke of the AP and then partially inactivated during the plateau. The fast component of ICa reached a peak value of -7.6 +/- 1.0 pA/pF at 2.40 +/- 0.30 ms after depolarization, followed by a slow component with a peak value of -2.9 +/- 0.4 pA/pF during the plateau. ICa generated by an AP was composed of both L- and T-type Ca2+ channels. T-type Ca2+ current contributed to the fast component of ICa and L-type Ca2+ current contributed to both fast and slow components of ICa. Activation of beta-adrenoceptors enhanced ICa with a maximal effect lasting throughout the entire plateau of the AP. Measurements of cytosolic Ca2+ transients using fura-2 indicated that the ICa was responsible for triggering Ca2+ release from the sarcoplasmic reticulum. The AP clamp provides a new approach for investigation of the relationship between ICa and Ca2+ transients under more physiological conditions.

Action Potentials↗
PubMed1993

Receptor-activated Ca influx in human airway smooth muscle: use of Ca imaging and perforated patch-clamp techniques.

Previous studies have demonstrated a dihydropyridine-insensitive, receptor-activated calcium influx pathway in cultured human airway smooth muscle (ASM) cells. To further define the biophysical characteristics of this pathway, the relationship between membrane potential and cytosolic free calcium ([Ca2+]i) was studied with the combined methods of the patch-clamp technique and single cell calcium imaging. The nystatin perforated-patch method was used to maintain normal intracellular calcium buffering and receptor-activated signal transduction processes in voltage-clamped cells. Single voltage-clamped human ASM cells responded to exposure to histamine (200 microM) with an initial transient rise in [Ca2+]i followed by a secondary sustained elevation that was dependent on extracellular calcium. Before agonist activation, step changes in holding potential produced only slight changes in [Ca2+]i, whereas, after activation, cells developed a sustained rise in [Ca2+]i that showed a large variation as a function of membrane potential. Depolarization from -80 to 0 mV caused a fall in the steady-state [Ca2+]i to basal levels or slightly below. Repolarization to -80 mV caused the redevelopment of the sustained phase of the calcium response. When calcium was removed from the extracellular fluid by the addition of a stoichiometric excess of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the voltage dependence of the sustained phase was abolished. In a series of experiments, agonist addition evoked a 54-fold increase in the voltage dependence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchi↗
PubMed1994

Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated-patch configurations.

A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.

Animals↗
PubMed1984

Voltage clamping induces resistance and current-voltage plot changes in frog gastric mucosa.

Changing the potential across the isolated frog gastric mucosa by voltage clamping changes the measured resistance of the tissue in two ways. An immediate change in resistance results from changing the measuring position on the nonlinear current-voltage (I-V) plot. Subsequent to this, the resistance changes slowly with a half-time of about 3 min, a change that is not predicted by a previous model for voltage transients and that implies slow changes in membrane resistance following changes in intracellular ion content. The I-V plot over the range examined shows three breakpoints; changing clamp voltage alters the position of two of these breakpoints as well as the slope of the connecting resistances. The central breakpoint agrees with the potential at zero current and varies with it as the clamp potential is changed, as predicted from a diode model for breakpoint generation.

Animals↗
PubMed1992

Patch-clamp recording in myenteric neurons of guinea pig small intestine.

The results of our research established the feasibility of applying patch-clamp methods in the study of the cellular neurophysiology of myenteric neurons enzymatically dissociated from adult guinea pig small intestine. Recording in current-clamp mode revealed two populations of neurons. One population discharged repetitively during depolarizing current pulses and displayed anodal-break excitation reminiscent of S/type 1 myenteric neurons. In the second population, spike discharge was limited to one or two spikes at the onset of depolarizing pulses and was similar to the behavior of AH/type 2 neurons. Recording in voltage-clamp mode revealed a complex of overlapping inward and outward whole cell currents. Fast and slow components of inward current were interpreted as sodium and calcium currents, respectively. Outward currents were blocked by cesium and consisted of components with properties of delayed rectifier current and A-type potassium current.

Animals↗
PubMed1990

Force measurements from voltage-clamped guinea pig ventricular myocytes.

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

Administration, Topical↗
PubMed1996

Restoring forces assessed with left atrial pressure clamps.

A negative pressure (P) in the fully relaxed left ventricle (LV) indicates the presence of restoring forces generated during contraction. To assess restoring forces in the intact LV under physiological filling conditions, a servomotor system was used in anesthetized open-chest dogs (n = 8) to produce nonfilling diastoles by left atrial pressure (LAP) clamping during systole such that LAP was less than left ventricular pressure (LVP) during the subsequent diastole. Steady-state LV end-diastolic pressure (EDP) was varied by volume infusion from 4.0 +/- 1.5 (+/-SD) to 12.8 +/- 2.1 mmHg. The corresponding fully relaxed LVPs increased from -2.1 +/- 1.9 to 1.1 +/- 3.2 mmHg, P < 0.001. LAP clamping increased the rate of LVP fall by 34 +/- 28% (P < 0.001) during 10 ms after the LVP dropped below the level of the LVP-LAP crossover of the preceding normal beat. During clamped beats, two-dimensional echo revealed substantial downward displacement of the mitral valve (MV) leaflets despite the reversed LA-LV gradient and absence of filling. Thus 1) restoring forces are present at low physiological EDP but absent at high physiological EDP; 2) filling retards the rate of fall of LVP; 3) even in the absence of filling, the process of LV relaxation facilitates MV opening.

Animals↗
PubMed1983

Maximum shortening velocity of smooth muscle: zero load-clamp vs. afterloaded method.

Previous reports from this laboratory of force-velocity relationships of canine tracheal smooth muscle (TSM) have presented maximum shortening velocities (Vmax) mathematically derived from the linearized transformation of the Hill equation (A. V. Hill, Proc. Roy. Soc. London, Ser. B., 126:136-195, 1938). Recent technical advances enable us to measure Vmax directly using an electromagnetic lever system that can instantaneously clamp to a zero load, thus we compared values of Vmax derived mathematically and those directly measured on the same TSM strips. Derived Vmax values from afterloaded isotonic shortening curves for loads greater than preload were 0.328 +/- 0.021 optimal length (lO)/s and were not significantly different from zero load-clamp measurements of 0.301 +/- 0.022 lO/s from the same (n = 15) muscles. These data indicate that Vmax values mathematically derived for TSM from conventional isotonic afterloaded force-velocity curves are valid estimates of zero load velocity, because they were not significantly different from values obtained by direct measurement using the zero load-clamp technique.

Animals↗