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PubMed1983

A voltage-clamp analysis of inward (anomalous) rectification in mouse spinal sensory ganglion neurones.

Mouse embryo dorsal root ganglion neurones were grown in tissue culture and voltage-clamped with two micro-electrodes. Hyperpolarizing voltage commands from holding potentials of -50 to -60 mV evoked slow inward current relaxations which were followed by inward tail currents on repolarization to the holding potential. These relaxations are due to the presence of a time- and voltage-dependent conductance provisionally termed Gh. Gh activates over the membrane potential range -60 to -120 mV. The presence of Gh causes time-dependent rectification in the current-voltage relationship measured between -60 and -120 mV. Gh does not inactivate within this range and thus generates a steady inward current at hyperpolarized membrane potentials. The current carried by Gh increases when the extracellular K+ concentration is raised, and is greatly reduced in Na+-free solutions. Current-voltage plots show considerably less inward rectification in Na+-free solution; conversely inward rectification is markedly enhanced when the extracellular K+ concentration is raised. The reversal potential of Ih is close to -30 mV in media of physiological composition. Tail-current measurement suggests that Ih is a mixed Na+-K+ current. Low concentrations of Cs+ reversibly block Ih and produce outward rectification in the steady-state current-voltage relationship recorded between membrane potentials of -60 and -120 mV. Cs+ also reversibly abolishes the sag and depolarizing overshoot that distort hyperpolarizing electrotonic potentials recorded in current-clamp experiments. Impermeant anion substitutes reversibly block Ih; this block is different from that produced by Cs+ or Na+-free solutions: Cs+ produces outward rectification in the steady-state current-voltage relationship recorded over the Ih activation range; in Na+-free solutions inward rectification, of reduced amplitude, can still be recorded since Ih is a mixed Na+-K+ current; in anion-substituted solutions the current-voltage relationship becomes approximately linear. It is concluded that in dorsal root ganglion neurones anomalous rectification is generated by the time-and voltage-dependent current Ih. The possible function of Ih in sensory neurones is discussed.

Action Potentials↗
PubMed1983

Calcium transients studied under voltage-clamp control in frog twitch muscle fibres.

1. Intracellular calcium transients were recorded from frog twitch muscle fibres in response to voltage-clamped depolarizing pulses, using arsenazo III as an intracellular calcium monitor. The object was to investigate the time- and voltage-dependent characteristics of the coupling process between membrane depolarization and calcium release from the sarcoplasmic reticulum (s.r.)2. To examine the extent to which the T-tubule membrane potential was controlled during clamp pulses, the dye NK 2367 was used as an optical probe of tubular potential. This indicated that the tubular time constant is about 0.6 msec.3. Strength-duration curves were obtained for depolarizing pulses required to give both threshold mechanical contraction and calcium signal. Curves measured in these two ways were closely similar.4. Changes in holding potential altered the strength-duration curve for calcium release so that at more positive holding potentials a shorter pulse was needed to obtain a response for any given pulse amplitude.5. A latency of a few milliseconds was observed between the onset of depolarization and the initial rise of the calcium signal. This became shorter with stronger depolarizations, but approached a minimum at potentials above about +25 mV.6. Subthreshold depolarizations applied before a test pulse increased the size and decreased the latency of the calcium signal. Conditioning hyperpolarizations had opposite effects.7. The rate of build-up of potentiation or depression of response size seen with subthreshold de- and hyperpolarizing conditioning pulses was examined using conditioning pulses of different durations. For both pulses this process showed a time constant of about 3 msec (at 10 degrees C).8. The rate of decay of potentiation or depression was similarly measured, using a gap of variable duration between conditioning and test pulses. For both de- and hyperpolarizing pulses this showed a time constant of about 5 msec (10 degrees C).9. The relationship between conditioning pulse potential, and the size of calcium signal elicited by a following test pulse was non-linear.10. Subthreshold pulses immediately following a brief test pulse affected the size of the calcium signal in a similar way to preceding conditioning pulses.11. The relationship between potential and size of the calcium signal was examined using pulses of 3 and 20 msec duration. With the long pulse the relation was roughly sigmoid, but with the short pulse continued to rise even at strongly positive potentials.12. The results are discussed in terms of a model in which the exponential build-up of a hypothetical coupler in the excitation-contraction (e.-c.) coupling process is presumed to lead to calcium release when a threshold level is exceeded.

Animals↗
PubMed1983

Voltage-clamp study of the conductance activated at fertilization in the starfish egg.

Ionic currents underlying the fertilization potential of the egg of the starfish Mediaster aequalis were studied using a two-micro-electrode voltage clamp. Mature eggs were fertilized in vitro under voltage-clamp conditions. The fertilization current, here termed IF, was induced by adding sperm to sea water bathing the egg. At a holding potential of -70 mV, IF was inward. It reached a peak within 2-4 min and then decayed over the next approximately 20 min with a rate which depended on the holding membrane potential. Instantaneous current-voltage relations measured at different times during IF were approximately linear and reversed at a potential of +6.0 +/- 5.8 mV (mean +/- S.D., n = 11). Membrane chord conductance was highest at the peak of inward current and the declining phase of IF was due to a decrease in conductance towards the pre-fertilization level. When the membrane potential was rapidly stepped to levels more positive than about -45 mV, the conductance underlying IF decreased in a manner which depended on both membrane potential and time. The fertilization-specific conductance showed a sigmoidal activation curve between -50 and +10 mV with a half-activation level of -25 mV. Analysis of the steady-state voltage dependence indicated that at the peak of the fertilization potential (+10 to +15 mV) only 4-5% of the total available channels would be open. Current relaxations followed first-order kinetics and the relaxation time constant depended upon the membrane potential during the voltage pulse. The relation between the time constant and voltage was bell-shaped, decreasing at potentials more negative than -40 and more positive than 0 mV. Both the steady-state conductance-voltage relation and the kinetics of the current relaxations were consistent with a simple two-state gating model in which the probability of a channel being open is determined by a single gating particle with an effective valency of -1.7 moving through the entire membrane field. The shifts in reversal potential with changes in external Na (at 10 mM-external K) were analysed using the constant field expression, which gave a relative permeability of Na to K of approximately 0.6.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1983

Calcium tail currents in voltage-clamped intact nerve cell bodies of Aplysia californica.

Calcium tail currents were measured in axotomized Aplysia neurones L2-L6 using a two-electrode voltage clamp and micro-electrodes of specially low resistance. Measurements were made at -40 mV following depolarizing pulses of 7 or 10 ms duration in the presence of 45 microM-tetrodotoxin and 200 mM-tetraethylammonium. Symmetrical currents were eliminated by addition of digitally stored current traces produced in response to equivalent hyperpolarizations. The remaining current, identified as a tail current, was blocked by replacement of extracellular calcium with cobalt or manganese. Computer fits showed that the tail current closely approximated the sum of two exponentially decaying components. The first had a time constant, tau 1, of 0.38 +/- 0.05 ms, which may have been frequency-limited by the speed of the clamp; the second had a time constant, tau 2, of 2.0 +/- 0.8 ms. A more slowly decaying third tail current component (tau 3 = 30 ms), which developed more slowly, may be related to the non-specific current rather than the calcium current. The tau 1 and tau 2 components of the tail current lost amplitude with increasing pulse duration along an approximately bi-exponential time course that resembled the time course of relaxation of the calcium current during a prolonged depolarization. The slow third component of the tail current showed no such inactivation. The amplitudes of the first and second components of the calcium tail current both increased as sigmoidal functions of the test pulse voltage, reaching half maximum at +20 mV and plateauing above +60 mV. The voltage dependencies of the two components were similar. The rate of decay of the tau 1 component increased with increasing temperature and with increasing negative potential, whereas tau 2 showed little dependence on these parameters. The rates of decay of neither the tau 1 nor the tau 2 component were affected by large changes in the amplitude of the test depolarization or in the amplitude of the tail current or by injection of calcium ions or EGTA. Thus, the kinetics of the tail current as resolved under our conditions appear to be virtually independent of calcium-mediated inactivation. Activation time constants (tau m) predicted from tau 1 are 3 to 5 times longer than the values of tau m determined from the half-time to peak of activation. These kinetics are slower than those reported for Limnaea by factors of 2.5 to 3.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1984

Single cardiac Purkinje cells: general electrophysiology and voltage-clamp analysis of the pace-maker current.

Single Purkinje cells from dog, sheep and cow hearts were isolated by injecting a Ca-free collagenase containing Tyrode solution in the space between the connective tissue sheath and the Purkinje cells. A small proportion of these cells survived the isolation procedure and these cells were used for further investigation. The cells showed electrophysiological properties similar to intact Purkinje fibres as indicated by the following results. Maximum diastolic potentials between -70 and -85 mV and specific membrane resistances of 21-32 k omega cm2 indicated that the single cells were not leaky or hyperpermeable . The action potential showed a rapid upstroke, with a maximum rate of rise, Vmax' between 150 and 750 V/s, and two phases of fast repolarization separated by a plateau phase with a duration of about 200 ms. Each action potential was followed by a spontaneous depolarization with an amplitude between 1 and 10 mV. The upstroke of the action potential could be blocked by tetrodotoxin (TTX) in a dose-dependent manner. The rate of depolarization of the action potential was sensitive to changes in membrane potential; the resulting S-shaped curve showed a half-maximum potential of -65 mV and a steepness of 0.46 mV-1. The duration of the action potential was sensitive to external K concentrations, catecholamines and TTX in a way similar to intact Purkinje fibres. Both application of catecholamines and lowering the external K concentration induced spontaneous activity. The cells were used to study the ionic nature of the pace-maker current under voltage-clamp conditions using the two-micro-electrode technique. This pace-maker current was blocked in a voltage-dependent manner by 1 mM-Cs, and was not affected by 1 mM-Ba. The steady-state activation curve was shifted in the depolarizing direction by application of adrenaline. In contrast to voltage-clamp data obtained on the pace-maker current of intact Purkinje fibres, the pace-maker current in a single cell did not reverse near the presumed equilibrium potential for K ions; no reversal could be seen in the voltage range negative to -50 mV. These observations together with preliminary results on the Na and K dependence of the pace-maker current are strong arguments in favour of the hypothesis that the pace-maker current in cardiac Purkinje fibres is an inward current carried by Na and K ions and activates upon hyperpolarization.

Action Potentials↗
PubMed1984

Voltage-clamp analysis of a calcium-mediated potassium conductance in cockroach (Periplaneta americana) central neurones.

The electrical properties of motoneurone cell bodies in the metathoracic ganglion of the cockroach, Periplaneta americana, have been studied by the voltage-clamp technique. Most experiments were carried out on a single identified cell (cell 28), the cell body of which, as for most other insect motoneurones, is electrically inexcitable. For comparison, some experiments were also carried out on dorsal unpaired median (d.u.m.) cells, the cell bodies of which are excitable. The two cell types differed only in that the d.u.m. cells developed a transient net inward current when depolarized towards zero membrane potential. This current was reduced but not abolished by Ca-free saline. Both cell types had an N-shaped current-voltage relation, as typically seen for molluscan and other neurones, but the location of the falling phase of the relation showed an unusually strong time dependence. The N-shape was abolished by prolonged exposure to Ca-free saline, suggesting it to be due to a K conductance that was activated by the entry of Ca ions through voltage-dependent channels. An outward current was also elicited by ionophoretic injection of Ca ions. The reversal potential of this current varied with the saline K concentration, in the manner expected if the current was carried by K ions. The Ca-mediated K current was blocked by La ions and by the organic Ca antagonist D-600. A series of double-pulse experiments on voltage-clamped cell bodies of cell 28 suggested that very short periods of Ca entry were sufficient to activate the K conductance fully. These experiments also suggested that the K conductance activation was due to intracellular Ca accumulation rather than to its being directly linked to the inward Ca current. The activation of the K conductance by intracellular Ca ions was made more effective by cell membrane depolarization. The Ca-mediated conductance did not inactivate significantly under conditions in which substantial inactivation has been observed in other neurones. The physiological significance of the electrical properties of cell 28 is discussed.

Action Potentials↗
PubMed1984

A patch-clamp study of potassium channels and whole-cell currents in acinar cells of the mouse lacrimal gland.

Individual acinar cells were isolated enzymatically from the mouse exorbital lacrimal gland. Their electrical characteristics were studied by the patch-clamp methods of single-channel and whole-cell recording as described by Hamill, Marty, Neher, Sakmann & Sigworth (1981). Recording from cell-attached and excised inside-out patches of acinar membrane with quasi-physiological ion gradients demonstrated large outward current events that correspond to single-channel openings. The amplitude, frequency and duration of channel events increased as the membrane patch was depolarized and were reduced by hyperpolarization of the patch membrane. The reversal potential for these channel events is more negative than -40 mV. In excised inside-out patches exposed to quasi-physiological ion gradients single-channel events were abolished when K+ was replaced by Rb+. Since there was no Cl- gradient the channel is clearly highly selective for K+. In excised inside-out patches, when the free Ca2+ concentration bathing the physiological inside of the membrane was raised from less than 10(-9) M to 10(-8) M the frequency and duration of opening of the K+ channel was increased. The channel was almost continuously open when the membrane was exposed to 10(-7) M-free Ca2+. 'Whole cell' recording of lacrimal acinar cells containing 140 mM-KCl and 1 mM-EGTA (with no added Ca2+) provided cell resting membrane potentials of -30 to -40 mV. Depolarizing voltage jumps from the resting membrane potential evoked large outward currents. Hyperpolarizing voltage jumps only evoked small inward currents. Whole-cell recording where RbCl replaced KCl in the pipette provided resting membrane potentials of -20 to -30 mV, reduced the amplitude of outward currents evoked by cell-depolarizing voltage steps by 60% and slowed the time course of the currents. Isolated cells containing 140 mM-KCl and 1 mM-EGTA were voltage clamped at their resting membrane potentials. Acetylcholine (ACh) was applied locally and immediately evoked a strong outward current which rapidly declined to a steady-state level. Sustained agonist responses were obtained by exposing the isolated cell to a solution containing 10(-6) M-ACh. In both K+- and Rb+-filled cells, where the intracellular Ca2+ concentration was buffered by the inclusion of 1 mM-EGTA, 10(-6) M-ACh evoked sustained outward currents that corresponded to cell hyperpolarizations of 5-15 and 10-20 mV, respectively. Increasing intracellular Ca2+ buffering by including 10 mM-EGTA abolished secretagogue-induced outward current in both K+- and Rb+-filled cells. It is concluded that the lacrimal acinar cell membrane contains voltage- and Ca2+-activated K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1985

A fast transient outward current in the rat sympathetic neurone studied under voltage-clamp conditions.

Post-ganglionic neurones of the isolated rat superior cervical ganglion were voltage clamped at 37 degrees C using separate intracellular voltage and current micro-electrodes. Control experiments in current clamp suggested that the neurone is electrotonically compact, the soma and the proximal dendritic membranes being under good spatial voltage uniformity. Depolarizing voltage steps from membrane potentials near -50 mV evoked: (i) a voltage-dependent inward Na+ current, (ii) an inward Ca2+ current, (iii) a voltage-dependent outward K+ current, (iv) a Ca2+-activated K+ outward current. Depolarizations from holding potentials more negative than -60 mV elicited, besides the currents mentioned above, a fast transient outward current IA which peaked in 1-2.5 ms and then decayed to zero following an exponential time course. The IA current was shown to be primarily, if not exclusively, carried by K+. It was unaffected by removal of external Ca2+ or addition of Cd2+ and was weakly blocked by tetraethylammonium ions and partially by 4-aminopyridine. The IA current showed a linear instantaneous current-voltage relationship. Its activation ranged from -60 to 0 mV with a mid-point at -30 mV. The A conductance could be described in terms of a simple Boltzmann distribution for a single gating particle with a valency of +3. Both the development and removal of inactivation followed a single exponential time course with a voltage-dependent time constant which was large near the resting potential (42 ms at -70 mV) and small (11 ms) near -100 and -40 mV. Steady-state inactivation h infinity ranged from -100 to -50 mV, with a mid-point at -78 mV, suggesting that approximately 50% of the IA channels are available at the physiological resting potential. Action potentials elicited from various holding potentials showed maximal repolarization rates dependent on the holding potential itself. This voltage dependence was found to be in reasonably good agreement with that of h infinity curve. These data are consistent with the view that in the rat sympathetic neurone, under physiological conditions, it is the IA current rather than the delayed outward current that is responsible for the fast action potential repolarization.

4-Aminopyridine↗
PubMed1985

A voltage-clamp analysis of membrane currents in solitary bipolar cells dissociated from Carassius auratus.

Membrane properties of solitary bipolar cells, mechanically dissociated from the enzyme-treated goldfish retina, were studied under current- and voltage-clamp conditions with 'giga-seal' suction pipettes (pipette solution 138 mM-K). The resting potential of solitary bipolar cells was about -30 mV. They responded to depolarizing current pulses with sustained depolarization, and to hyperpolarizing current pulses with an initial hyperpolarizing transient followed by a sag to a less hyperpolarized level. The current-voltage relationship determined under voltage-clamp conditions showed strong outward and inward rectification. The membrane currents consisted of four components; Ca current (ICa), voltage- and Ca-dependent K currents (IK(V) and IK(Ca), respectively), and an inward current activated by membrane hyperpolarization (Ih). ICa was activated by membrane depolarization beyond -40 mV, was maximum at +10 mV and became smaller with further depolarization. No polarity reversal was seen. ICa was enhanced by equimolar replacement of Ca with Ba, and was blocked by 4 mM-Co. IK(Ca) was observed by membrane depolarization beyond -10 mV, was maximum at about +40 mV, and became smaller with further depolarization. This current was suppressed by 4 mM-Co, 1.6 mM-Ba, 35 mM-TEA or 30 microM-quinine. IK(V) was activated by membrane depolarization beyond -60 mV, and had slower kinetics that ICa or IK(Ca). The reversal potential of the tail current was close to the K equilibrium potential (EK), suggesting that this current is carried purely by K ions. IK(V) was inactivated slowly and nearly completely by sustained depolarization. IK(V) was blocked by 35 mM-TEA. Ih was activated by membrane hyperpolarization (less than -60 mV). The current showed a time-dependent increase. It was also dependent on the membrane potential, but not on the driving force of K ions. This current seems to be carried by a mixture of Na and K ions, since (1) in low Na solution, Ih became small in amplitude, and (2) the reversal potential of the tail current was between the Na equilibrium potential (ENa) and EK X Ih was blocked by 10 mM-Cs, but was resistant to 0.2 mM-Ba. The resting potential and voltage responses of solitary bipolar cells are discussed in reference to the characteristics of each membrane conductance isolated in the present study.

Action Potentials↗
PubMed1985

Calcium entry and transmitter release at voltage-clamped nerve terminals of squid.

Presynaptic and post-synaptic cells of the squid giant synapse were voltage-clamped simultaneously to study the relationship between presynaptic Ca current and transmitter-induced post-synaptic current (p.s.c.). Local Ca application was used to restrict Ca current and transmitter release to a limited region of the presynaptic terminal and thus minimize errors due to spatial heterogeneity of presynaptic membrane potential. Presynaptic terminals were depolarized by brief (3-6 ms) voltage-clamp pulses of varying amplitude to collect graded series of presynaptic Ca current and p.s.c. records. During presynaptic depolarization at 14 degrees C, Ca current activation preceded initial onset of p.s.c. (on-p.s.c.) by an interval of approximately 1 ms. The main component of on-p.s.c. followed Ca current activation by about 2 ms. The delay between a brief Ca tail current and peak response of the p.s.c. produced after pulse termination (off-p.s.c.) was also approximately 2 ms. Curves relating both Ca current and p.s.c. magnitudes to presynaptic potential were bell shaped with peaks near -10 mV, but the p.s.c. curve showed stronger voltage dependence on both sides of the peak. With very small and very large presynaptic command pulses, Ca current could be observed without measureable p.s.c. Synaptic transfer curves, plotting p.s.c. as a function of presynaptic Ca current, resembled third-power functions. On the average, p.s.c.s fit a curve representing the 2.9 power of Ca current (range 2.4-3.5 in eighteen experiments). Transfer curves consisted of two limbs: one from presynaptic pulses below -10 mV and the other from more positive pulses. These two limbs were similar and generally resembled power functions of identical exponent. It is thus likely that the third-power function accurately reflects synaptic current transfer, rather than interference from some other voltage-dependent process. Power functions fitting small-pulse and large-pulse limbs of some transfer curves had different scale coefficients, even though exponent values were the same. Consideration of synaptic transmission kinetics suggests that the voltage dependence of Ca channel opening rates can probably explain the difference in transfer curve limbs. Our experiments provide no evidence for an intrinsic voltage dependence of the transmitter release process.

Action Potentials↗
PubMed1985

Patch-clamp recordings of the light-sensitive dark noise in retinal rods from the lizard and frog.

In cell-attached recordings from rods in the intact lizard retina, light decreased a standing inward membrane current with a reversal potential approximately 60 mV more positive than the resting potential. The peak amplitude of saturating responses depended upon the area of recorded membrane and varied from cell to cell over approximately 100-fold range. Small patches of membrane gave variable responses to identical moderately intense flashes. Whole-cell voltage-clamp recordings were obtained on isolated frog rods with intact ellipsoids. Peak whole-cell photocurrent was related to flash intensity by a Michaelis equation with saturating response amplitudes ranging up to 30 pA in 0.1 mM-Ca2+ Ringer solution. In darkness the steady-state current-voltage relation, determined with whole-cell voltage clamp, showed outward rectification. Photocurrent had nearly constant amplitude between -80 and -10 mV, a mean reversal potential of +8 mV and recovered from flashes more slowly at positive holding potentials. Although it was not possible to resolve light-sensitive single-channel current events, power spectral analysis revealed both low- and high-frequency components of the light-sensitive noise in both cell-attached and whole-cell recordings. The low-frequency component was described by the product of two Lorentzians using time constants derived from the kinetics of the dim flash response. The high-frequency component of the light-sensitive noise was described by a single Lorentzian with a half-power frequency of 62 Hz in lizard and 212 Hz in frog. The half-power frequency was not appreciably affected by steady illumination. The Lorentzian nature of the noise suggests that the light-sensitive channel is a pore rather than a shuttle-type carrier. In cell-attached recordings the high-frequency component declined monotonically with increasing light intensity, suggesting that less than one-half of the channels are open in darkness. Furthermore, the ratio of the variance of the high-frequency noise to the mean photocurrent was independent of light intensity. Changing external Ca2+ from 0.1 to 0.5 mM reduced the ratio from 19.7 to 9.0 fA without a significant effect on the cut-off frequency of the noise. The results support the conclusion that the light-sensitive pore is opened by an internal transmitter that acts as an agonist and that both open and closed states of the pore may be blocked by external Ca2+. The conductance of the light-sensitive pore in the absence of external Ca2+ is estimated to be 1.25-2 pS.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1986

Calcium and delayed potassium currents in mouse pancreatic beta-cells under voltage-clamp conditions.

Pancreatic islets of NMRI mice were dissociated into single cells which were kept in tissue culture for 1-3 days. The whole-cell configuration of the patch-clamp technique was used to study inward and delayed outward currents of beta-cells under voltage-clamp conditions at 20-22 degrees C. Outward currents were suppressed by substituting the impermeant cation N-methyl-D-glucamine for intracellular K+. The remaining inward current had a V-shaped current-voltage relation reaching a peak value of 39 +/- 4 pA (mean +/- S.E. of mean) around -15 mV. It was identified as a Ca2+ current, because the peak amplitude was increased 1.6 times by increasing external [Ca2+] ([Ca2+]o) from 2.6 mM to 10 mM and it was blocked by Co2+ (5 mM) or nifedipine (5 microM) but not by TTX (20 microM). The activation time constant of the inward current at -10 mV was 1.28 +/- 0.08 ms. The relation between the degree of activation (estimated from the size of the tail currents) and membrane potential V followed the sigmoidal function f = 1/(1 + exp [(Vh-V)/k]) with half-maximal activation potential, Vh = 4 +/- 1 mV and slope factor, k = 14 +/- 1 mV (for [Ca2+]o 10 mM). The inward current inactivated only weakly during depolarizing pulses of 0.1-1 s duration. The delayed outward current (in experiments with 155 mM-internal [K+] ([K+]i)) had a linear voltage dependence at potentials above -20 mV; its amplitude at -10 mV was 210 +/- 30 pA. Tail currents related to the activation of the outward current had K+-dependent reversal potentials. The current was blocked by extracellularly applied tetraethylammonium (20 mM) and 4-aminopyridine (2 mM). It was not affected by glibenclamide (3 microM), tolbutamide (0.2 mM) and alterations of intracellular [Ca2+] (1 nM-1 microM). The activation time constant of the outward current at -10 mV was 21 +/- 3 ms. The voltage dependence of activation could be described by the sigmoidal function (see above) with Vh = 19 +/- 1 mV and k = 5.6 +/- 0.4 mV. The outward current inactivated during long (15 s) depolarizing pre-pulses (time constant at -10 mV: 2.6 +/- 0.6 s). 50% inactivation occurred at Vh = -36 +/- 2 mV, k was -4.1 +/- 0.2 mV. Inward and outward currents during depolarizing voltage pulses in beta-cells are similar to Ca2+ and delayed K+ currents in other cell types. These currents seem sufficient to generate the action potentials of the beta-cell.

Action Potentials↗
PubMed1989

Voltage-gated and agonist-mediated rises in intracellular Ca2+ in rat clonal pituitary cells (GH3) held under voltage clamp.

1. Intracellular free calcium (Ca2+i) was estimated in single GH3 cells by dual wavelength emission spectrofluorimetry using the Ca2+ indicator dye Indo-1, while cells were held under voltage clamp using patch clamp techniques. 2. Depolarization of cells evoked a transient rise in Ca2+i that increased with increasing duration of depolarization to a peak at about 10 s. 3. Calcium transients showed a bell-shaped dependence on the amplitude of the depolarizing pulse. They were abolished in the absence of extracellular calcium and by application of 10 microM-nifedipine. 4. Thyrotrophin-releasing hormone (TRH) evoked a transient rise in Ca2+i that was followed by a more sustained period of elevated Ca2+i in some cells. The transient phase of the response but not the sustained phase was seen in the absence of extracellular calcium. 5. Ca2+i transients evoked by depolarization were not affected by pre-release of internal Ca2+ stores with TRH. 6. The results demonstrate that voltage-gated Ca2+ entry and Ca2+ store release can each elevate cytoplasmic free calcium in GH3 cells and may both be important for stimulus-secretion coupling. Non-voltage-gated Ca2+ entry is not a major source of Ca2+ under these conditions.

Action Potentials↗
PubMed1991

Two types of potassium currents seen in isolated Necturus enterocytes with the single-electrode voltage-clamp technique.

1. The ionic permeability of Necturus maculosus small intestine epithelial cells was investigated using intracellular microelectrodes to measure membrane potential in intact tissue or by the single-electrode voltage-clamp technique in isolated cells. 2. The basolateral membrane of enterocytes appears to be K+ selective as demonstrated by the dependence of membrane potential and fractional serosal resistance measured in the intact epithelium on serosal K+ concentration. 3. Isolated cells had membrane potentials similar to those measured in the intact tissue. Voltage-clamp experiments in a physiological Ringer solution showed the presence of both large outward and inward currents. 4. Removal of Cl- from the bathing medium, linear subtraction or the use of a Cl- channel blocker revealed outwardly rectifying currents. The quasi-linear component was also revealed following K+ channel inhibition; it reversed near ECl, suggesting that the charge carrier was Cl-. 5. Outwardly rectifying currents could be kinetically resolved into two components. A fast component (tau for activation < 4 ms) accounted for 60-80% of the total current at positive potentials. A slowly activating component appeared at voltages positive to 50 mV with tau for activation of > 25 ms. 6. The slow outward current showed strong voltage dependence of both activation and relaxation, which were faster at more depolarized potentials. 7. Both fast and slow outward currents seem to be carried by K+ as they were blocked by Ba2+ and tetraethylammonium (TEA). Tail current analysis of the slow component indicated a reversal potential very similar to EK. 8. Fast outward currents were half-activated at about -40 mV whereas slow outward currents were only apparent at more positive potentials. It is proposed that the fast outward K+ current plays a role, together with Cl- currents, in determining the resting membrane potential of Necturus enterocytes.

Animals↗
PubMed1992

A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture.

1. Acetylcholine-induced currents recorded from bovine adrenal medullary chromaffin cells maintained in culture were studied during pressure or ionophoretic applications of ACh, using the 'whole-cell' and 'outside-out' configurations of the patch clamp technique. In standard salines, ACh evoked whole-cell currents of -38 pA to -1 nA at -60 mV, which had a reversal potential (EACh) of -7.1 +/- 0.6 mV. The ACh current-voltage relationship was characteristically linear at negative holding potentials and biphasic at positive holding potentials, displaying a region of almost zero slope conductance between 0 and +40 mV followed by a region of positive slope conductance at more positive potentials. 2. Relative permeation to cations was examined. Substitution of external Na+ by sucrose resulted in a -42 mV shift of EACh for a 10-fold reduction in [Na+]o. Using isotonic substitutions, the permeability ratios (relative to Na+) for monovalent cations were determined to be 1.32 +/- 0.02 for Cs+ (n = 11), 1.03 +/- 0.02 for Li+ (n = 8) and 0.18 +/- 0.02 for Tris+ (n = 7). Elevated external Ca2+ salines were found to shift EACh to more positive potentials, especially in the presence of low external Na+. 3. The nicotinic agonists nicotine, tetramethylammonium and lobeline evoked inward currents in bovine chromaffin cells. In contrast, decamethonium and the muscarinic agonist, methacholine, had no effect. 4. The nicotinic antagonists mecamylamine, trimetaphan, (+)-tubocurarine and hexamethonium caused dose-dependent reductions in the amplitude of ACh-evoked inward currents. The estimated IC50's were 0.25, 0.33, 0.63 and 2.2 microM respectively, for cells voltage clamped at -60 mV. High concentrations (> 2 microM) of the muscarinic antagonist, atropine, also produced a dose-dependent reduction in the amplitude of ACh-induced currents. 5. Inhibition by trimetaphan was voltage independent. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The voltage sensitivity was most marked for hexamethonium. Neither hexamethonium nor mecamylamine were found to depress ACh-evoked outward currents at concentrations which severely depressed inward currents. In addition to its antagonist actions, (+)-tubocurarine activated unitary currents in these cells and on isolated membrane patches. 6. The results indicate that nicotinic ion channels of bovine chromaffin cells have a similar ionic selectivity to monovalent cations, but that Ca2+ ions permeate the channels to a greater degree than at the motor endplate. The ACh current-voltage relationship resembles that described for other types of 'neuronal' nicotinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1993

Electrical and integrative properties of rabbit sympathetic neurones re-evaluated by patch clamping non-dissociated cells.

1. Voltage recordings were performed on non-dissociated sympathetic neurones from rabbit coeliac ganglia using the whole-cell configuration of the patch clamp technique. 2. Cells were classified depending on their firing pattern as silent cells (63%) producing either phasic (24%) or tonic (76%) spike discharge in response to depolarizing currents, and pacemaker cells (37%). 3. All the cells produced large overshooting spikes and prolonged postspike after-hyperpolarization. The peak-to-peak spike amplitude was 113.8 +/- 1 mV. Spikes were shortened and the after-hyperpolarization was suppressed when calcium channel blockers (Cd2+ and La3+) were added. 4. Silent cells have a resting potential of -58.8 +/- 1.5 mV. At potentials ranging from -50 to -90 mV, the input impedance was 490 +/- 27 M omega at 22-24 degrees C and 426 +/- 47 M omega at 35-36 degrees C. The time constant at voltages corresponding to the high input impedance region was 126 +/- 7 ms at 22-24 degrees C and 86 +/- 7 ms at 35-36 degrees C. 5. The firing frequency of the pacemaker cells was 3.2 +/- 0.5 Hz at 35-36 degrees C in the presence of nicotinic blockers. Evidence is given that the firing did not result from cell injury but was induced by an intrinsic pacemaker mechanism. Input impedance of pacemaker neurones was 580 +/- 47 M omega at 22-24 degrees C and 473 +/- 56 M omega at 35-36 degrees C. 6. Most of the pacemaker cells (63%) were motoneurones, since they were antidromically fired by stimulating post-ganglionic nerves. In addition, they received synaptic inputs from both preganglionic fibres (splanchnic nerves) and the periphery (postganglionic nerves). Long-lasting depolarizations were induced in either silent or pacemaker cells by single shocks applied to pre- and postganglionic nerves. 7. Slowly rising voltage ramps revealed the presence of an N-shaped current-voltage relationship in voltage clamped pacemaker cells. The negative slope was located in a subthreshold voltage range, between -83.4 +/- 1.4 and -59.0 +/- 1.8 mV. It was induced by the activation of a low threshold persistent inward current. Although it was tiny (22 +/- 3 pA at its peak level) this current brought the null-current voltage up to -41.0 +/- 1.4 mV, which resulted in continuous firing. 8. Due to the instability introduced by the N-shaped I-V relationship, pacemaker cells can display bistable behaviour characterized by hyperpolarizing responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1994

Gradation of Ca(2+)-induced Ca2+ release by voltage-clamp pulse duration in potentiated guinea-pig ventricular myocytes.

1. This study tests the hypothesis that whole-cell cardiac SR Ca2+ release is graded by recruitment of independent 'release units'. Structurally, an individual release unit may comprise ca four sarcolemmal L-type Ca2+ channels, adjacent ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels and the junctional gap between them. After depolarization, the first opening of a single L-type Ca2+ channel of the unit provides sufficient Ca2+ influx to increase local [Ca2+] beyond the threshold activating Ca(2+)-induced Ca2+ release (CICR), which amplifies local [Ca2+] until all release channels of the unit are active. This all-or-none activation does not spread to other release units. Gradation of whole-cell Ca2+ release is predicted to correlate with the cumulative probability density distribution of first latency of L-type Ca2+ channels or the activation time course of the calcium current, ICa. 2. Guinea-pig ventricular myocytes were potentiated by paired voltage-clamp pulses (1 Hz, 2 mM [Ca2+]o, 40 microM K5-indo-1, 36 degrees C). When the cellular Ca2+ load was at a steady high level, cytosolic calcium concentration ([Ca2+]c) transients were measured in response to test pulses of varied pulse duration (PD, 1-180 ms) and amplitude (-20, 0, 20 and 50 mV). The maximal rate of rise (RRmax) of the [Ca2+]c transient was used as an indicator for SR Ca2+ release. 3. Fast [Ca2+]c transients due to 4 ms pulses to 0 or 50 mV were blocked by 1 mM cadmium suggesting that these Ca2+ release signals are triggered by Ca2+ influx through L-type Ca2+ channels and not by Ca2+ influx through Na(+)-Ca2+ exchange. 4. RRmax increased with longer PD along the sigmoidal curve [1-exp(-PD/tau)]kappa(exponent k: 2 < k < 3). The time constant, tau, resembled the activation time constant of whole-cell ICa (Cs(+)-dialysed cells). A PD longer than a limiting duration did not modify RRmax. That is, inactivation of ICa was not reflected in the duration dependence. 5. Single L-type Ca2+ channels (cell-attached patches, 36 degrees C, -20 mV, 3.6 mM CaCl2 and 1 microM Bay K 8644 in patch pipette) opened with a waiting time the cumulative probability distribution of which resembled the duration dependence of RRmax, suggesting that the first opening of L-type Ca2+ channels determines whether the corresponding release unit contributes to the [Ca2+]c transient activated during a short voltage-clamp pulse. 6. The time constant, tau, of the duration dependence was shorter at positive than at negative potentials.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Strain fields on cell stressing devices employing clamped circular elastic diaphragms as substrates.

Solutions are presented for the surface strain fields on inflated elastomeric circular diaphragms used for in vitro cell stressing experiments. It is shown, by using the method developed by Way (1934) to solve the nonlinear von Karman plate equations, that the surface strains due to bending are not negligible and that large negative radial strains arise near the clamped edge for center deflection-to-thickness ratios (w/h) less than 10. The method of Hart-Smith and Crisp (1967) was used for w/h greater than 10 to solve the nonlinear equations for symmetrical deformation of axially symmetrical rubber-like membranes. In the membrane solutions the circumferential strains drop parabolically to zero at the clamped edge of the diaphragm, while the radial strains increase slightly with the radius. The solutions for w/h greater than 10 are compared to optical measurements of in-plane displacements used to calculate the circumferential strains on the diaphragm, yielding excellent agreement with the theory.

Biomechanical Phenomena↗