Single Exponential Time Course Research Articles

Thiosulfate elimination and permeability in a sulfide-adapted marine invertebrate.

Oxidation of hydrogen sulfide to thiosulfate is one of the best-characterized mechanisms by which animals adapted to sulfide minimize its toxicity, but the mechanism of thiosulfate elimination in these animals has remained unclear. In this study, we examined the accumulation and elimination of thiosulfate in the sulfide-adapted marine worm Urechis caupo. The coelomic fluid of U. caupo exposed to 50-100 micromol L-1 sulfide in hypoxic seawater (Po2 ca. 10 kPa) accumulated (mean+/-SD) 132+/-41 micromol L-1 thiosulfate after 2 h, reaching 227+/-113 micromol L-1 after an additional 4 h in aerated, sulfide-free seawater. In whole-animal thiosulfate clearance studies, the rate of thiosulfate elimination from the coelomic fluid followed a single exponential time course with a half-life of 6 h. The thiosulfate permeability coefficient of isolated preparations mounted in diffusion chambers was 7.6x10-5+/-7. 7x10-5 cm s-1 for the hindgut and 5.5x10-7+/-2.7x10-7 cm s-1 for the body wall. These rates were independent of the direction of net efflux (mucosal-to-serosal or serosal-to-mucosal). Using a simple mathematical model of U. caupo that incorporates the thiosulfate permeability coefficients, the thiosulfate half-life was calculated to be 23 h without hindgut ventilation but less than 1 h with normal hindgut ventilation. Based on this information, we propose that passive thiosulfate diffusion across the hindgut is adequate to explain the observed rates of thiosulfate elimination.

Fast voltage gating of Ca2+ release in frog skeletal muscle revealed by supercharging pulses.

1. In single frog skeletal muscle fibres, we utilized supercharging voltage clamp command pulses to boost the rate of depolarization in the transverse tubular system (T-system) such that 95 % of steady-state potential is achieved in < 2 ms (as indicated by fluorescent potentiometric dye signals detected from a global illumination region). Signals detected near the edge of muscle fibres indicate that peripheral regions of the T-system are not significantly overcompensated under these conditions. 2. We explored the impact of accelerating T-system depolarization on voltage-dependent events of excitation-contraction (E-C) coupling by measuring charge movement currents (CMCs) and Ca2+ fluorescence transients in response to both supercharging and conventional step pulses. 3. When compared with CMCs elicited by step pulses, supercharging CMCs are larger, and their kinetics more closely resemble those of gating current records reported for ionic channels. Furthermore, they decay bi-exponentially (tau fast range, 1.3-1.8 ms; tau slow range, 7.3-11.9 ms), whereas step CMCs fall with a single exponential time course (tau range, 12.5-26.7 ms). 4. Similarly, supercharging produces a distinct acceleration in Ca2+ release transients, which show little evidence of the voltage-dependent onset latencies previously encountered using step pulses. 5. The use of this novel methodology in skeletal muscle unveils a previously undetected component of charge movement, the rapid, voltage-dependent recruitment of which may provide the basis for understanding the fast gating of physiological E-C coupling.

Voltage-induced slow activation and deactivation of mechanosensitive channels in Xenopus oocytes.

1. The relationship between stretch and voltage activation of mechanosensitive (MS) channels from Xenopus oocytes was studied in excised patches of membrane using the patch clamp technique. 2. As is characteristic of MS channels to oocytes, stretching the membrane by applying negative pressure to the patch pipette at -50 mV activated the MS channels rapidly. The channels then deactivated rapidly when the stretch was removed. The stretch-activated MS channels entered a main conductance level (45 pS) and one or more subconductance levels in the range of about 75-90% of the main conductance level. 3. In the absence of stretch, a depolarizing step from -50 to +50 mV activated apparent MS channels after long delays of typically 1-20 s (range, 100 ms to 6 min). Upon repolarization, the channels deactivated slowly with a single exponential (mean time constant of 4 s) or double exponential (mean time constants of 0.8 and 3 s) time course. 4. Delayed activation with depolarization and slow deactivation upon repolarization were also observed for apparent MS channels in on-cell patches. 5. The voltage-activated channels were cation selective and had the same selectivity and conductance levels as the stretch activated MS channels. Applying stretch during voltage-induced channel activity did not activate any additional channels, and the same maximal number of channels were typically activated by either stretch or by voltage. These observations suggest that voltage activates the same MS channels that are activated by stretch. 6. The opening of MS channels following steps to +50 mV occurred in an apparently co-operative manner in 70% of the excised patches containing multiple MS channels. 7. In the absence of stretch, the opening frequency and open probability of MS channels increased with depolarization in the examined voltage range of -60 to -20 mV. 8. Applying a brief stretch during the delay to activation at +50 mV activated the MS channels rapidly, which then remained active when the stretch was removed. In contrast, applying a brief stretch during the slow deactivation induced by stepping from +50 to -50 mV abruptly terminated the voltage induced channel activity upon release of the stretch and inhibited subsequent depolarization-induced activity. 9. Depolarizing steps from -50 to +50 mV inhibited any spontaneous channel activity that was present before the depolarizing step. If the potential was stepped back to -50 mV before the channels activated at +50 mV, a delayed activation could occur at -50 mV, followed by normal deactivation, indicating that the depolarizing step initiated activation processes that were initially masked by inhibition. 10. These observations suggest that voltage and stretch can induce different functional gating configurations of MS channels with associated structures, and that these different gating configurations can interconvert.

Transbilayer movement of fully ionized taurine-conjugated bile salts depends upon bile salt concentration, hydrophobicity, and membrane cholesterol content.

Taurine-conjugated bile salts mediate rapid transmembrane flux of divalent cations, irrespective of whether bile salts and divalent cations are initially on the same or opposite side of the membrane. We therefore hypothesized that ionized bile salts can equilibrate between membrane hemileaflets. We quantitated bile salt binding to large unilamellar egg yolk phosphatidylcholine (EYPC) +/- cholesterol (Ch) vesicles under conditions in which one or both hemileaflets were initially exposed to bile salts. At unbound taurodeoxycholate (TDC) concentrations >0.2 mM, the dependence of binding on TDC concentration after 30 min was indistinguishable for vesicles prepared by either method and did not change from 30 minutes to 24 h. At unbound TDC concentrations <0.1 mM, the ratio of bound/free TDC to EYPC vesicles doubled over a single exponential time course. Equilibration times were greater for the more hydrophilic bile salts taurocholate and tauroursodeoxycholate, for EYPC/Ch vesicles, and at lower temperatures. For glycine-conjugated bile salts, time-dependent changes in binding did not occur, consistent with more rapid equilibration of the small fraction of the protonated form. We conclude that fully ionized conjugated bile salts translocate between lipid bilayer hemileaflets, in contrast to previous observations that equilibration of fully ionized unconjugated bile salts occurs at a negligible rate in small unilamellar vesicles. The rate of "flip-flop" increases with increases in intramembrane bile salt concentration and hydrophobicity but decreases with cholesterol content and lower temperature. We speculate that physiologically, even in the absence of a specific membrane transporter, bile salts can gain access to intracellular compartments and mediate increases in divalent cation flux that may underlie cytotoxicity.

Gap junction channel gating at bass retinal electrical synapses.

To further characterize the properties of retinal horizontal cell electrical synapses, we have studied the gating characteristics of gap junctions between cone-driven horizontal cells from the hybrid striped bass retina using double whole-cell voltage-clamp techniques. In a total of 105 cell pairs, the macroscopic conductance ranged from 0.4-100 nS with most cell pairs exhibiting junctional conductances between 10 and 30 nS. The junctional current-voltage relationship showed that peak or instantaneous currents (Iinst) were linear within the Vj range of +/- 100 mV and that steady-state junctional currents (Iss) exhibited rectification with increasing voltage beginning around +/- 30-40 mV Vj. The normalized junctional current-voltage relationship was well fit by a two-state Boltzmann distribution, with an effective gating charge of 1.9 charges/channel, a half-maximal voltage of approximately +/- 55 mV, and a normalized residual conductance of 0.28. The decay of junctional current followed a single exponential time course with the time constant decreasing with increasing Vj. Recovery of junctional current from voltage-dependent inactivation takes about 1 s following a pulse of 80 mV, and is about five times slower than the inactivation time course at the same Vj. Single-channel analysis showed that the unitary conductance of junctional channels is 50-70 pS. The overall open probability decreased in a voltage-dependent manner. Both the mean channel open time and the frequency of channel opening decreased, while the channel closure time increased. The ratio of closed time/total recording time significantly increased as Vj increased. Increased Vj reduced the number of events at all levels and shifted the unitary conductance to a lower level. Kinetic analysis of channel open duration showed that the distribution of channel open times was best fit by two exponentials and increased Vj significantly reduced the slower time constant. These results indicate that bass retina horizontal cells exhibit voltage-dependent inactivation of macroscopic junctional current. The inactivation occurs at the single-channel level mainly by increasing the rate of closure of voltage-sensitive channels.

Layer I neurons of the rat neocortex. II. Voltage-dependent outward currents

1. Whole cell patch-clamp techniques, combined with direct visualization of neurons, were used to study voltage-dependent potassium currents in layer 1 neurons and layer II/III pyramidal cells. 2. In the presence of tetrodotoxin, step depolarizations evoked an outward current. This current had a complex waveform and appeared to be a composite of early and late components. The early peak of the composite K+ outward current was larger in layer I neurons. 3. In both layer I and pyramidal cells, the composite outward K+ current could be separated into two components based on kinetic and pharmacological properties. The early component was termed I(A) because it was a transient outward current activating rapidly and then decaying. I(A) was more sensitive to blocking by 4-aminopyridine (4-AP) than tetraethylammonium (TEA). The second component, termed the delayed rectifier or I(DR), activated relatively slowly and did not decay significantly during a 200-ms test pulse. I(DR) was insensitive to blocking by 4-AP at concentrations up to 4 mM and blocked by > 60% by 40-60 mM TEA. 4. I(A) kinetics were examined in the presence of 40-60 mM TEA. Under these conditions, I(A) began to activate between -40 and -30 mV. Half-maximal activation occurred around 0 mV. In both layer I and pyramidal cells, the half-inactivation potential (Vh-inact) was around or more positive than -50 mV. At -60 mV, > 70% of I(A) conductance was available. I(A) decayed along a single exponential time course with a time constant of approximately 15 ms. This decay showed little voltage dependence. 5. In both layer I and pyramidal cells, I(DR) was studied in the presence of 4 mM 4-AP to block I(A) and in saline containing 0.2 mM Ca2+ and 3.6 mM Mg2+ to reduce contributions from Ca2+-dependent K+ currents. Under these conditions, I(DR) began to activate at -35 to -25 mV with Vh-act of 3.6 +/- 4.5 mV (mean +/- SD). The 10-90% rise time of I(DR) was 15 ms at 30 mV. At 2.2 ms after the onset of the command potential to +30 mV, I(DR) could reach a significant amplitude (approximately 1.5 nA in layer I neurons and 2.2 nA in pyramidal cells depending on the cell size). When long test pulses (> or = 1,000 ms) were used, a decay time constant approximately 800 ms at +40 mV was observed. In both layer I and pyramidal cells, steady state inactivation of I(DR) was minimal. 6. These results indicate that I(A) and I(DR) are the two major hyperpolarizing currents in layer I and pyramidal cells. The kinetics and pharmacological properties of I(A) and I(DR) were not significantly different in fast-spiking layer I neurons and regular-spiking layer II/III pyramidal cells. The relatively positive activation threshold (more than or equal to -40 mV) of both I(A) and I(DR) suggest that they do not play a role in neuronal behavior below action potential (AP) threshold and that their properties are more suitable to repolarize AP. The greater density of I(A) in layer I neurons appears responsible for fast spike generation.

The depolarizing mechanoreceptor potential and Ca/Mg receptor-current of the marine ciliate Euplotes vannus

The anterior depolarizing mechanoreceptor potential and the correlated receptor currents have been studied in the marine ciliate Euplotes vannus. Mechanical stimuli that mimicked cell-cell collisions depolarized the resting potential of about — 25 mV to maximally — 5 mV, with a speed of 1.2 mV/ms, a delay to the stimulus of about 15 ms, and a repolarization within 30 to 300 ms. The power-stroke direction of the cirri-beat reversed from backward to forward during this response. The receptor current rose to an average amplitude of 1.4 nA with a speed of 0.1–0.3 nA/ms and decayed with a single exponential time course with a time constant between 7 and 9 ms. Similar current-reversal potentials, after substitution of extracellular Ca2+ by Mg2+ and vice versa, indicate that the mechanically activated conductance is identical for Ca2+ or Mg2+. The current can be carried by Ba2+ as well, but not by K+ or Na+. Decirriation experiments have shown that the mechanosensitivity is located within the soma membrane.

A fast transient outward current in cultured cells from human pulmonary artery smooth muscle.

A voltage-dependent transient outward current (I(to)) was observed in cells cultured from human pulmonary artery smooth muscle when K+, but not Cs+, was the dominant cation in the pipette solution. In 30% of cells investigated using the Cs+ pipette solution, a tetrodotoxin-sensitive inward current (I(in)) dependent on extracellular Na+ was evoked from depolarizations positive to -30 mV.Iin showed voltage-dependent inactivation with a membrane potential at 50% of the evoked current (V50%) of -75.53 +/- 0.81 mV and slope factor potential (Vs) of -10.73 +/- 0.01 mV. In the presence of 1 microM tetrodotoxin, I(to) was rapidly evoked by depolarizations positive to -40 mV and decayed with a single exponential time course (tau = 9.9 +/- 1.1 ms, pulse potential = +50 mV). I(to) also showed voltage-dependent inactivation with a V50% of -70.9 +/- 2.63 mV and Vs of -7.7 +/- 0.03 mV. I(to) was inhibited concentration dependently by the K+ channel blocker, 4-aminopyridine (4-AP), with a concentration of 4-AP at which I(to) was reduced to 50% of control of 36.5 +/- 2.8 microM. These cells possess voltage-dependent currents characteristic of the K(+)-selective fast transient outward and Na(+)-selective inward currents of smooth muscle cells.

Potassium currents across the plasma membrane of protoplasts derived from rye roots: a patch-clamp study

Epidermal-cell protoplasts from rye (Secale cereale L.) roots were voltage-clamped in both the whole-cell and outside-out membrane-patch modes. Time-dependent inwardly-rectified (IR) and outwardly-rectified (OR) K+-currents were recorded, as well as a ubiquitous, timeindependent (instantaneous) K+-current. The IR current activated at voltages more negative than —100mV with two exponentially rising components. The time-constant of the shorter component was voltage-independent, whereas the time-constant of the longer component was voltage-dependent, increasing as the activating voltage became more negative. The IR current showed no inactivation. The IR current deactivated with a single exponential timecourse. The steady-state IR current could be fitted to a Boltzmann function with —135 mV as the voltage at which the current was half-maximal and a minimal gating charge of 1.93. These parameters were insensitive to changes in EK. One component of the IR current was K + , but other ions were also permeable. The IR current was inhibited by extracellular Ca2+ , Ba2+ , Cs+, and TEA+, but was insensitive to quinine. Single channels with unitary conductances of 56 pS and 110 pS (in c.100 mM K+) were recorded at negative voltages. Two OR currents were observed. One had sigmoidal activation kinetics and activated at low positive voltages. The other activated more rapidly, with apparently exponential kinetics, at voltages 50–100 mV more positive than the first. Neither current showed inactivation and deactivation of OR currents followed a double exponential time-course. Unitary-conductances of the channels mediating these OR currents were 24 pS and 57 pS (in c.100 mM K+), respectively. Only the first type of OR current was studied in detail. This current activated with a sigmoidal time-course, which could be described using a Hodgkin-Huxley function with the activation variable raised to the second power. Its voltage-dependence was modulated in response to changes in EK and analysis of single-channel recordings indicated that the channel was K+-selective. The current was inhibited by Ba2+ and TEA+, but not Ca2+, Cs+ or quinine. The instantaneous current was selective for monovalent cations and K+ , Na+ and Cs+ were all permeant. It was inhibited by extracellular quinine and the instantaneous inward K+-current was reduced by extracellular Ca2+, Ba2+ and TEA+, as well as by competing permeant monovalent cations. The kinetics and pharmacology of these currents are compared with K+-currents across the plasma membrane of protoplasts from other root-derived cells and with K+ channels in the plasma membrane of rye roots studied following incorporation into artificial, planar lipid bilayers.

Paired-pulse facilitation in the dentate gyrus: a patch-clamp study in rat hippocampus in vitro.

1. Whole-cell patch-clamp recordings were used to study paired-pulse facilitation (PPF) of the lateral perforant path input to the dentate gyrus in thin hippocampal slices. 2. Orthodromic stimulation of the lateral perforant pathway evoked a excitatory postsynaptic current (EPSC) with a latency of 3.3 +/- 0.1 ms (mean +/- SE) that fluctuated in amplitude. The EPSC had a rise time (10-90%) of 2.79 +/- 0.06 ms (n = 35) and decayed with a single exponential time course with a time-constant of 9.14 +/- 0.24 ms (n = 35). No correlation was found between the amplitude of the EPSC and the rise time or decay time-constant. The non-N-methyl-D-aspartate (NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione completely blocked the EPSC whereas the NMDA antagonist D-aminophosphonovaleric acid (APV) had modest effects. 3. When a test (T-)EPSC was preceded at an interval of 100 ms by a conditioning (C-)EPSC, a significant increase in the amplitude of the T-EPSC was seen in 38 out of 44 trials analyzed from a total of 27 granule cells. The average amount of PPF was 35.7 +/- 2.1%. There was no apparent correlation between the amount of PPF and the stimulation intensity or mean amplitude of the C-EPSC. The time course of the facilitated T-EPSC was not significantly different from that of the C-EPSC. 4. No correlation was found between the amplitude of the C-EPSC and that of the T-EPSC. Estimates of quantal content (mcv) were determined by calculating the ratio of the squared averaged EPSC amplitude (from 48 responses) to the variance of these responses (M2/sigma 2) whereas quantal amplitudes (qcv) were estimated by calculating the ratio of the response variance to average EPSC amplitude (sigma 2/M). PPF was found to be associated with an average increase in mcv of 64.8 +/- 7.2% (n = 38) whereas qcv was decreased by 12.1 +/- 3.8%. 5. The time course of PPF was studied by varying the interval between the C- and T-pulse from 10 to 400 ms while keeping the stimulation intensity constant. Maximal facilitation of the T-EPSC was obtained with interpulse intervals < or = 25 ms where the average facilitation amounted to approximately 70% (n = 6). The decline of facilitation was nearly exponential and was no longer evident with intervals > 350 ms.(ABSTRACT TRUNCATED AT 400 WORDS)

Pump and K+ inward rectifiers in the plasmalemma of wheat root protoplasts.

An electrogenic pump, a slowly activating K+ inward rectifier and an intermittent, "spiky," K+ inward rectifier, have been identified in the plasmalemma of whole protoplasts from root cortical cells of wheat (Triticum) by the use of patch clamping techniques. Even with high external concentrations of K+ of 100 mM, the pump can maintain the membrane potential difference (PD) down to -180mV, more negative than the electrochemical equilibrium potentials of the various ions in the system. The slowly activating K+ inward rectifier, apparent in about 23% of protoplasts, allows inward current flow when the membrane PD becomes more negative than the electrochemical equilibrium potential for K+ by about 50 mV. The current usually consists of two exponentially rising components, the time constant of one about 10 times greater than the other. The longer time constant is voltage dependent, while the smaller time constant shows little voltage dependence. The rectifier deactivates, on return of the PD to less negative levels, with a single exponential time course, whose time constant is strongly voltage dependent. The spiky K+ inward rectifier, present in about 68% of protoplasts, allows intermittent current, of considerable magnitude, through the plasmalemma at PDs usually more negative than about -140mV. Patch clamp experiments on detached outside-out patches show that a possibly multi-state K+ channel, with maximum conductance greater than 400 pS, may constitute this rectifier. The paper also considers the role of the pump and the K+ inward rectifiers in physiological processes in the cell.

Intracellular calcium transients recorded with Fura-2 in spontaneously active myocytes isolated from the atrioventricular node of the rabbit heart

We have used the fluorescent Ca indicator Fura-2 to assess the changes in intracellular calcium (Cai) in single spontaneously active myocytes isolated from the rabbit atrioventricular node (AVN). Simultaneous recordings of membrane potential and the Fura-2 ratio signal (which reflects Cai) showed that a transient rise of Cai occurred with each spontaneous action potential (AP). The AP upstroke preceded the rise in Cai and repolarization of the AP occurred faster than the decline of Cai. The level of Cai remained raised and progressively declined towards a baseline diastolic level during the subsequent pacemaker depolarization. The Fura-2 (Cai) transient in spontaneously active AVN cells had a time-to-peak of 49.2 +/- 5.4 ms (mean +/- s.e.m.; n = 7) and declined with a single exponential time course (time constant = 139.8 +/- 23.9 ms; n = 7). Application of 10 microM ryanodine completely and irreversibly abolished the Cai transient, identifying the sarcoplasmic reticulum (SR) as the major source of releasable Ca. Both removal of external Ca and block of L-type Ca channels (with 2 microM nifedipine) also abolished Cai transients, suggesting that Ca entry via L-type Ca-channels is involved in triggering the SR Ca release underlying the Cai transient. Removal of external Na (in the presence of 20 microM nifedipine to block L-type Ca channels) caused a reversible increase in Cai, showing that Na/Ca exchange is present in AVN cells and that it is involved in Cai regulation. Spontaneous Cai transients were abolished by 1 microM acetylcholine, and this was associated with a hyperpolarization of membrane potential and cessation of action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of high-threshold transmission between heart interneurons of the medicinal leech by FMRF-NH2

1. We examined high-threshold synaptic transmission between oscillatory pairs of leech heart interneurons. Inhibitory postsynaptic currents (IPSCs) could be reliably evoked by depolarizing the presynaptic neuron in voltage clamp from a holding potential of -35 mV. At this presynaptic potential, the Ca2+ currents underlying graded transmission are completely inactivated, and we conclude that a high-threshold Ca2+ current is extant in heart interneurons. Further evidence for this was that inhibitory postsynaptic currents were blocked when Co2+ replaced Ca2+ in the saline and thus high-threshold transmission was dependent on the presence of external Ca2+. 2. When IPSCs were evoked by a 200-ms duration voltage step from a holding potential of -35 mV in the presynaptic neuron, the time course of turn-on of the IPSC consisted of a fast (time-to-peak = 17.5 +/- 1.93 (SE) ms [n = 7]) and a slow (time-to-peak = 250 +/- 28.5 ms [n = 8]) component. FMRF-NH2 reduced the amplitude of the fast component but did not affect the slow component. When the presynaptic voltage step was ended the IPSC turned off with a single exponential time course. FMRF-NH2 slowed the time course of turn-off of the IPSC. 3. When IPSCs were evoked by a 1500-ms duration voltage step from a holding potential of -35 mV in the presynaptic neuron, these IPSCs peaked around 300 ms. Following the peak, the IPSC decayed with a single exponential time course. FMRF-NH2 accelerated the time course of this decay. At potentials of 0 mV and +5 mV, FMRF-NH2 produced a significant decrease in the peak current and at potentials of -5 mV and 0 mV, produced a significant decrease in the current integral. 4. High-threshold IPSCs could also be evoked by a spike in the presynaptic neuron. Bath application of 1 microM FMRF-NH2 decreased the amplitude of the spike-evoked IPSC and slowed the time course of its falling phase. 5. We examined the effect of FMRF-NH2 on the quantal synaptic transmission. Bath-application of FMRF-NH2 increased binomial p, the probability of release, and decreased binomial n, the number of units available for release. FMRF-NH2 had no effect on q, the unit size, when calculated from the distributions of PSPs, and increased the coefficient of variation (CV). 6. The lack of a change in q and the increase in CV suggested that FMRF-NH2 acted at a presynaptic location.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane rectification in single smooth muscle cells from the rat tail artery

Membrane rectification to depolarization was studied by voltage recording with patch electrodes in freshly isolated cells from the rat tail artery. Injection of depolarizing currents elicited electrotonic potentials that developed with a single-exponential time course (time constant of 94.8 ms). When the cell was depolarized beyond -30 mV, delayed rectification was observed. A second type of rectification, characterized by oscillations, was observed when the cell was depolarized positive to +30 mV. The threshold of this rectification and the oscillations were sensitive to changes in intracellular Ca2+. Delayed rectification was more sensitive to 4-aminopyridine but more resistant to tetraethylammonium and charybdotoxin than the Ca(2+)-sensitive rectification. A 4-aminopyridine-sensitive outward current (IK,dr) with a threshold of around -30 mV and a second Ca(2+)-sensitive outward current (IK,Ca) activated at around +30 mV were observed from whole-cell voltage clamp recordings. IK,Ca was blocked by tetraethylammonium and charybdotoxin. An 11-pS and a 122-pS channel, having characteristics similar to IK,dr and IK,Ca respectively, were identified from single-channel recordings. These observations showed how membrane depolarization of vascular smooth-muscle cells was regulated by these two populations of K+ channels under various conditions.

Glutamate-activated currents in outside-out patches from spiny versus aspiny hilar neurons of rat hippocampal slices

The desensitization rate of non-NMDA glutamate receptors was investigated in outside-out membrane patches obtained from morphologically identified spiny "mossy cells" (SMCs) and aspiny hilar interneurons (AHIs) in young rat hippocampal slices. The fast application of a 1 mM step of L-glutamate for 50-100 msec in the presence of TTX and dizolcipine (MK-801) onto patches excised from these neurons produced large glutamate-activated currents (GACs) that decayed with a single or double exponential time course despite the continued presence of agonist. These desensitization rates of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-sensitive receptors differed markedly between patches obtained from the two cell types. The fast time constant of desensitization in AHIs (n = 34) averaged 3.3 +/- 0.93 msec (mean +/- SD), while that of SMCs (n = 57) averaged 6.8 +/- 2.0 msec. Current-voltage relationships of the GACs did not differ between SMCs and AHIs, with comparable reversal potentials and no evidence of inward rectification. We also failed to observe significant Ca2+ permeability in either cell type. However, brief (< 1 msec) pulses of 1 mM glutamate produced rapidly decaying GACs with distinct kinetics in the two neuronal classes. Furthermore, analysis of the single glutamate-activated channel currents in outside-out patches from hilar neurons revealed a larger predominant single-channel current in AHIs versus SMCs. Lastly, we observed a greater sensitivity to cyclothiazide in SMCs versus AHIs, with half-maximal removal of desensitization being 90 mM and 200 mM, respectively. Taken together, these differences in GACs between SMCs and AHIs might indicate a functional correlate to the substantial heterogeneity in the molecular structure of glutamate receptor subunits or might be related to posttranslational modifications of these subunits, perhaps provided by the unique microenvironment in the spines covering SMCs.

Hyperpolarization-activated currents in neurons of the rat basolateral amygdala.

1. A single microelectrode was used to obtain current-clamp or voltage-clamp recordings from two neuronal cell types (pyramidal and late-firing neurons) in the basolateral nucleus of the amygdala (BLA) in slices of the rat ventral forebrain. Conductances activated by hyperpolarizing voltage steps from a holding potential of -70 mV were identified and their sensitivity to muscarinic modulation was determined using bath superfusion of carbachol. 2. Unclamped pyramidal neurons exhibited anomalous rectification, seen as a slowly developing depolarizing sag in the electronic potential in response to a hyperpolarizing current pulse. 3. Stepping voltage-clamped pyramidal neurons to command potentials of between -70 and -100 mV activated a slowly developing inward current (ISlow) that followed a single exponential time course. Larger hyperpolarizing voltage steps evoked a rapidly developing inward current (IFast) that preceded the development of ISlow. 4. The ISlow component reversed at a level positive to the -70 mV holding potential. Its rate of activation accelerated as the hyperpolarizing voltage step was made more negative. The threshold for activation of the conductance underlying ISlow was approximately -60 mV, with half-activation occurring at -90 mV. 5. Extracellular Cs+ (2 mM) blocked ISlow and eliminated anomalous rectification in unclamped pyramidal neurons. The inhibition of ISlow by Cs+ was also associated with membrane hyperpolarization and reduction of the medium afterhyperpolarization. ISlow was unaffected by extracellular Ba2+ (100 microM). The properties of this current appeared similar to that of the mixed cationic H-current previously identified in other neurons. 6. In comparison with pyramidal cells, unclamped late-firing neurons displayed a lesser but more rapidly developing anomalous rectification in response to large hyperpolarizations from rest. In voltage clamp, hyperpolarizing steps to command potentials more negative than -100 mV elicited IFast. Late-firing neurons expressed little or no ISlow. 7. The properties of IFast were identical in both pyramidal and late-firing neurons. This current reversed at a potential negative to -70 mV. Its rate of current activation increased with the magnitude of the hyperpolarizing voltage step. This rate was approximately sevenfold faster than ISlow activation recorded at the same membrane potential. IFast was blocked by 2 mM extracellular Cs+ and reduced by 100 microM extracellular Ba2+. The threshold for activation of the underlying conductance was approximately -85 mV, with half-activation occurring at -112 mV. The properties of IFast were similar to those of the inward rectifier current previously identified in other central neurons. 8. Carbachol (40 microM) largely blocked IFast without affecting its rate of activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Two kinetically distinct components of hyperpolarization-activated current in rat superior colliculus-projecting neurons.

1. Whole-cell and perforated patch recording techniques were used to examine the activation, deactivation and inactivation of the time-dependent hyperpolarization-activated inward currents (Ih) in isolated superior colliculus-projecting (SCP) neurons from rat primary visual cortex. 2. Examination of inward current waveforms revealed the presence of two kinetically distinct components of Ih: one that activates with a time constant of the order of hundreds of milliseconds, and one that activates with a time constant of the order of seconds. We have termed these Ih,f and Ih,s, to denote the fast and slow components, respectively, of current activation. The time constants of activation of both Ih,f and Ih,s decrease with increasing membrane hyperpolarization. 3. Following the onset of hyperpolarizing voltage steps, a delay is evident prior to time-dependent inward current activation. This delay is voltage dependent and decreases with increasing membrane hyperpolarization. 4. The sigmoidal inward current waveforms are well fitted by the sum of two exponentials in which the faster term, corresponding to the activation of Ih,f, is raised to the power 1.34 +/- 0.26 (mean +/- S.D.). The non-integral exponent suggests that Ih,f activation involves at least two energetically non-equivalent gating transitions prior to channel opening. 5. Over a limited voltage range, tail currents could also be resolved into two distinct components. The faster component, which corresponds to the deactivation of Ih,f, decayed over a single exponential time course with a mean (+/- S.D.) time constant of 355 +/- 161 ms at -70 mV. Ih,s decay also followed a single exponential time course with a mean (+/- S.D.) time constant of 2428 +/- 1285 ms at -70 mV. Both deactivation time constants decreased with increasing depolarization. 6. The separation of inward current activation and deactivation into two distinct components and the lack of correlation between the relative amplitudes of these components suggest that Ih,f and Ih,s reflect the presence of two functionally distinct channel populations. 7. No decrements in time-dependent hyperpolarization-activated inward currents were observed during hyperpolarizations lasting up to 18 s, suggesting that neither Ih,f nor Ih,s inactivates from the open state. In addition, 10 s depolarizations to 0 mV prior to activation did not alter the waveforms of the inward currents activated directly from -40 mV, suggesting that Ih,f and Ih,s also do not inactivate from closed states. 8. The hyperpolarization-activated currents in rat SCP neurons are ideally suited to contribute to the control of the resting membrane potential and input resistance.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic study and numerical reconstruction of A-type current in granule cells of rat cerebellar slices.

1. Whole-cell voltage-clamp techniques were used to study voltage-activated transient potassium currents in a large sample (n = 143) of granule cells (GrC) from rat cerebellar slices. Tetrodotoxin (TTX; 0.1 microM) was used to block sodium currents, while calcium current was too small to be seen under ordinary conditions. 2. Depolarizing pulses from -50 mV evoked a slow, sustained outward current, developing with a time constant of 10 ms, inactivating over a time scale of seconds and which could be suppressed by 20 mM tetraethylammonium (TEA). By preventing the Ca2+ inflow, this slow outward current could be further separated into a Ca(2+)-dependent and a Ca(2+)-independent component. 3. After conditioning hyperpolarizations to potentials negative to -60 mV, depolarizations elicited transient outward current, peaking in 1-2 ms and inactivating rapidly (approximately 10 ms at 20 degrees C), showing the overall kinetic characteristics of the A-current (IA). The current activated following third-order kinetics and showed a maximal conductance of 12 nS per cell, corresponding to a normalized conductance of 3.8 nS/pF. 4. IA was insensitive to TEA and to the Ca(2+)-channel blockers. 4-Aminopyridine (4-AP) reduced the A-current amplitude by approximately 20%, and the delayed outward currents by > 80%. 5. Voltage-dependent steady-state inactivation of peak IA was described by a Boltzmann function with a slope factor of 8.4 mV and half-inactivation occurring at -78.8 mV. Activation of IA was characterized by a Boltzmann curve with the midpoint at -46.7 mV and with a slope factor of 19.8 mV. 6. IA activation and inactivation was best fitted by the Hodgkin-Huxley m3h formalism. The rate of activation, tau a, was voltage-dependent, and had values ranging from 0.55 ms at -40 mV to 0.2 ms at +50 mV. Double-pulse experiment showed that development and removal of inactivation followed a single-exponential time course; the inactivation time constant, tau ha, was markedly voltage-dependent and ranged from approximately 10 ms at -40 and -100 mV and 70 ms at -70 mV. 7. A set of continuous equations has been developed describing the voltage-dependence of both the steady-state and time constant of activation and inactivation processes, allowing a satisfactory numerical reconstruction of the A-current over the physiologically significant membrane voltage range.(ABSTRACT TRUNCATED AT 400 WORDS)

Postsynaptic activation at the squid giant synapse by photolytic release of L-glutamate from a 'caged' L-glutamate.

1. Pharmacological evidence suggests L-glutamate is a strong candidate as a transmitter at the giant synapse of the squid. Postsynaptic activation at the giant synapse cannot be effected by conventional application of putative neurotransmitters by iontophoresis or perfusion, apparently because the complex structure of the synapse prevents a sufficiently rapid change in concentration at the postsynaptic membrane. Flash photolytic release of L-glutamate from a pharmacologically inert 'caged' L-glutamate pre-equilibrated in the stellate ganglion of Alloteuthis or Loligo was used to determine whether L-glutamate can produce postsynaptic activation when released rapidly in the synaptic clefts. 2. The preparation, reaction mechanism and properties of the caged L-glutamate, N-1-(2-nitrophenyl)ethoxycarbonyl-L-glutamate, are described. The product quantum yield on photolysis was 0.65 (+/- 0.05). On flash photolysis glutamate release followed a single exponential time-course in the pH range 5.5-7.8. The rate constant was proportional to [H+] and was 93 s-1 at pH 5.5 and 16 degrees C in artificial sea water (ionic strength, I = 0.68 M). 3. At pH 7.8 flash photolysis of caged glutamate pre-equilibrated in the synapse caused only a slow depolarization. A second photolytic release of L-glutamate or transsynaptic activation produced no further depolarization, suggesting desensitization and inactivation of postsynaptic mechanisms by the initial pulse of L-glutamate. 4. Synaptic transmission in the giant synapse was normal at pH 5.5. Flash photolysis at pH 5.5 caused rapid production of L-glutamate within the synaptic cleft and a fast postsynaptic depolarization which generated postsynaptic action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Interconversion between distinct gating pathways of the high threshold calcium channel in rat ventricular myocytes.

1. High-voltage-activated Ca2+ current (ICa) waveforms in adult rat ventricular myocytes comprise two components, referred to here as ICa(fc) and ICa(sc) to denote the fast and slow components, respectively, of ICa decay. At all test potentials, the two time constants of ICa decay, tau fc and tau sc, differ by approximately an order of magnitude. Neither tau fc nor tau sc varies appreciably with test potential, however, suggesting that current inactivation is not markedly voltage dependent. 2. Current activation at all test potentials follows a sigmoidal time course and is best described by a power function with n = 4. Deactivation of the currents, examined following variable length depolarizations to various test potentials, however, follows a single exponential time course. In addition, the kinetics of activation and deactivation of ICa(fc) and ICa(sc) are indistinguishable. 3. Although both begin to activate at approximately -30 mV, the voltage dependences of ICa(fc) and ICa(sc) are distinct: ICa(fc) peaks at -10 mV and ICa(sc) peaks at +10 mV. 4. The relative amplitudes of ICa(fc) and ICa(sc) vary with the holding potential from which the currents are evoked and with the frequency of current activation: hyperpolarized holding potentials and low stimulation frequencies reveal preferential activation of ICa(fc), whereas depolarized holding potentials and high stimulation frequencies potentiate ICa(sc). In addition, the observed voltage- and frequency-dependent changes in ICa(fc) and ICa(sc) amplitudes are reciprocal. 5. The apparent voltage dependences of steady-state inactivation of ICa(fc) and ICa(sc) are also distinct. ICa(fc) is reduced to approximately 50% of its maximal amplitude at -45 mV, whereas ICa(sc) is approximately 50% inactivated at -30 mV. 6. Recovery of ICa(peak) from steady-state inactivation follows a complex time course. Following inactivation at -10 mV, ICa(peak) recovers at -90 mV to its maximal value over a biexponential time course; ICa(peak) then decreases over the next several seconds to a steady-state level. 7. The time course of recovery from steady-state inactivation of ICa(fc) at -90 mV is best described by the sum of two exponentials; the two time constants of recovery differ by approximately a factor of 25. ICa(sc), in contrast, recovers rapidly and over a single exponential time course to its maximal value. When the recovery time at -90 mV is increased, however, ICa(sc) amplitude decreases slowly and over a single exponential time course to a steady-state level.(ABSTRACT TRUNCATED AT 400 WORDS)