Fast Activation Kinetics Research Articles

High-speed solution switching using piezo-based micropositioning stages.

Motion-induced vibration is a critical limitation in high-speed micropositioning stages used to achieve solution switching. Controlled rapid solution switching is used to study the fast activation and deactivation kinetics of ligand-gated ion-channel populations isolated in excised membrane patches--such studies are needed to understand fundamental mechanisms that mediate synaptic excitation and inhibition in the central nervous system. However, as the solution-switching speed is increased, vibration induced in the piezo-based positioning stages can result in undesired, repeated, ligand application to the excised patch. The article describes a method to use knowledge of the piezo-stage's vibrational dynamics to compensate for and reduce these unwanted vibrations. The method was experimentally verified using an open-electrode technique, and fast solution switching (100 micros range) was achieved.

Pentobarbital has curare-like effects on adult-type nicotinic acetylcholine receptor channel currents.

Pentobarbital (PB) is widely used as a short-term sedative and anticonvulsive drug with a side-effect of relaxing muscle tone. We investigated block of nicotinic acetylcholine receptor (nAChR) channel currents by PB using the patch-clamp technique in combination with an ultrafast system for solution exchange. As a preparation, recombinant rat adult-type nAChR channels transiently expressed in HEK293 cells were used. Appli-cation of 1 mM acetylcholine to small cells or outside-out patches showed a transient current with fast activation and desensitization kinetics. Adding PB to the acetylcholine-containing solution resulted in a decrease of the time constant of current decay and of the peak current amplitude starting at concentrations >0.01 mM PB. Preincubation of nAChR channels with PB led to a decrease of the peak current amplitude without alteration of activation and desensitization kinetics caused by competitive block of nAChR channels. In conclusion, similar to the effect of d-Tubocurarine, block of nAChR channel currents by PB can be explained by a combination of open-channel and competitive block. The interaction between adult-type nicotinic acetylcholine receptors, acetylcholine, and pentobarbital was biophysically investigated by using the patch-clamp technique in combination with tools for ultrafast solution exchange. PB elicited open-channel block and competitive block of nicotinic acetylcholine receptor channel currents, whereas the latter seems to be effective in clinically relevant concentrations.

Regional distribution of ionic currents and membrane voltage responses of type II hair cells in the vestibular neuroepithelium.

Basolateral ionic currents and membrane voltage responses were studied in pigeon vestibular type II hair cells using a thin slice through either the semicircular canal (SCC) crista or utricular macular epithelium. Whole cell tight-seal patch-clamp recording techniques were used. Current-clamp and voltage-clamp studies were carried out on the same cell. One hundred ten cells were studied in the peripheral (Zone I) and central (Zone III) zones of the SCC crista, and 162 cells were studied in the striolar (S Zone) and extrastriolar (ES Zone) zones of the utricular macula. One of the major findings of this paper is that hair cells with fast activation kinetics of their outward currents are found primarily in one region of the SCC crista and utricular macula, whereas hair cells with slow activation kinetics are found in a different region. In Zone I of the crista, 95% of the cells have fast activation kinetics ("fast" cells) and in Zone III of the crista, 86% of the cells have slow activation kinetics ("slow" cells). In the utricular macula slice, 100% of the cells from the S Zone are slow cells, whereas 86% of the cells from the ES Zones are fast cells. Oscillation frequency (f) and quality factor (Q) of the damped oscillations of the membrane potential during extrinsic current injections were studied in hair cells in the different regions. The slow cells in Zone III and in the S Zone have a statistically significantly lower f, as a function of the amplitude of injected current, when compared with the fast cells in Zone I and the ES Zone. Although Q varied over a small range and was <2.6 for all cells tested, there was a statistically significant difference between Q for the membrane oscillations of the slow cells and fast cells in response to a range of current injections.

Fast BK-type channel mediates the Ca(2+)-activated K(+) current in crayfish muscle.

The role of the Ca(2+)-activated K(+) current (I(K(Ca))) in crayfish opener muscle fibers is functionally important because it regulates the graded electrical activity that is characteristic of these fibers. Using the cell-attached and inside-out configurations of the patch-clamp technique, we found three different classes of channels with properties that matched those expected of the three different ionic channels mediating the depolarization-activated macroscopic currents previously described (Ca(2+), K(+), and Ca(2+)-dependent K(+) currents). We investigated the properties of the ionic channels mediating the extremely fast activating and persistent I(K(Ca)). These voltage- and Ca(2+)-activated channels had a mean single-channel conductance of approximately 70 pS and showed a very fast activation. Both the single-channel open probability and the speed of activation increased with depolarization. Both parameters also increased in inside-out patches, i.e., in high Ca(2+) concentration. Intracellular loading with the Ca(2+) chelator bis(2-aminophenoxy) ethane-N, N,N',N'-tetraacetic acid gradually reduced and eventually prevented channel openings. The channels opened at very brief delays after the pulse depolarization onset (<5 ms), and the time-dependent open probability was constant during sustained depolarization (< or =560 ms), matching both the extremely fast activation kinetics and the persistent nature of the macroscopic I(K(Ca)). However, the intrinsic properties of these single channels do not account for the partial apparent inactivation of the macroscopic I(K(Ca)), which probably reflects temporal Ca(2+) variations in the whole muscle fiber. We conclude that the channels mediating I(K(Ca)) in crayfish muscle are voltage- and Ca(2+)-gated BK channels with relatively small conductance. The intrinsic properties of these channels allow them to act as precise Ca(2+) sensors that supply the exact feedback current needed to control the graded electrical activity and therefore the contraction of opener muscle fibers.

Molecular and functional characterization of s-KCNQ1 potassium channel from rectal gland of Squalus acanthias.

Functional and pharmacological data point to the involvement of KCNQ1/IsK potassium channels in the basolateral potassium conductance of secretory epithelia. In this study, we report the cloning and electrophysiological characterization of the KCNQ1 protein from the salt secretory rectal gland of the spiny dogfish (Squalus acanthias). The S. acanthias KCNQ1 (s-KCNQ1) cDNA was cloned by polymerase chain reaction (PCR) intensive techniques and showed overall sequence similarities with the KCNQ1 potassium channel subunits of Man, mouse and Xenopus laevis of 64, 70 and 77%, respectively, at the translated amino acid level. Analysis of s-KCNQ1 expression on a Northern blot containing RNA from heart, rectal gland, kidney, brain, intestine, testis, liver and gills revealed distinct expression of 7.4-kb s-KCNQ1 transcripts only in rectal gland and heart. Voltage-clamp analysis of s-KCNQ1 expressed in Xenopus oocytes showed pronounced electrophysiological similarities to human and murine KCNQ1 isoforms, with a comparable sensitivity to inhibition by the chromanol 293B. Coexpression of s-KCNQ1 with human-IsK (h-IsK) induced currents with faster activation kinetics and stronger rectification than observed after coexpression of human KCNQ1 with h-IsK, with the voltage threshold of activation shifted to more negative potentials. The low activation threshold at approximately -60 mV in combination with the high expression in rectal gland cells make s-KCNQ1 a potential candidate responsible for the basolateral potassium conductance.

Delayed rectifier potassium current in undiseased human ventricular myocytes

The purpose of the study was to investigate the properties of the delayed rectifier potassium current (IK) in myocytes isolated from undiseased human left ventricles. The whole-cell configuration of the patch-clamp technique was applied in 28 left ventricular myocytes from 13 hearts at 35 degrees C. An E-4031 sensitive tail current identified the rapid component of IK (IKr) in the myocytes, but there was no evidence for an E-4031 insensitive slow component of IK (IKs). When nifedipine (5 microM) was used to block the inward calcium current (ICa), IKr activation was fast (tau = 31.0 +/- 7.4 ms, at +30 mV, n = 5) and deactivation kinetics were biexponential and relatively slow (tau 1 = 600.0 +/- 53.9 ms and tau 2 = 6792.2 +/- 875.7 ms, at -40 mV, n = 7). Application of CdCl2 (250 microM) to block ICa altered the voltage dependence of the IKr considerably, slowing its activation (tau = 657.1 +/- 109.1 ms, at +30 mV, n = 5) and accelerating its deactivation (tau = 104.0 +/- 18.5 ms, at -40 mV, n = 8). In undiseased human ventricle at 35 degrees C IKr exists having fast activation and slow deactivation kinetics; however, there was no evidence found for an expressed IKs. IKr probably plays an important role in the frequency dependent modulation of repolarization in undiseased human ventricle, and is a target for many Class III antiarrhythmic drugs.

Properties of human glial cells associated with epileptic seizure foci.

We studied physiological properties of glial cells from acute slices of biopsies from patients operated for intractable mesio-temporal lobe epilepsy using whole-cell patch-clamp recordings. Cells were filled with Lucifer Yellow (LY) during recordings to allow morphological reconstruction and immunohistochemical cell identification. Seizure-associated astrocytes had complex, arborized, highly branched processes giving them a stellate appearance, and cells stained intensely for the intermediate filament GFAP as previously reported for 'reactive' astrocytes. GFAP-positive astrocytes from epilepsy biopsies consistently expressed voltage-activated, TTX-sensitive Na+ channels that showed fast activation and inactivation kinetics. Unlike comparison astrocytes, derived from tissues that were not associated with seizure foci, these astrocytes expressed Na+ channels at densities sufficient to generate slow action potentials (spikes) in current clamp studies. In these cells, the ratio of Na+ to K+ conductance was consistently 3-4-fold higher than in comparison human or control rat astrocytes. Four of 17 astrocytes from epilepsy patients versus 14/14 from control rat hippocampus and four of five in comparison human tissue showed a lack of inwardly rectifying K+ currents, which in normal astrocytes are implicated in the control of extracellular K+ levels. These results suggest that astrocytes surrounding seizure foci differ in morphological and physiological properties, and that glial K+ buffering could be impaired at the seizure focus, thus contributing to the pathophysiology of seizures.

Extremely slow inactivation of the ion channels formed by transfected α2 of L-type Ca2+ channelsof L-type Ca2+ channels

Barium currents through ion channels formed by α1-subunit of L-type Ca2+ channel (Iα1) were recorded from cultured chinese hamster ovary (CHO) cells. The cells were stably transfected with either a cardiac or a smooth muscle (SM) variant of α1-subunit. TheIα1 in both cases exhibited similar fast voltage-dependent activation kinetics and slow apparent inactivation kinetics. With 10 mM Ba2+ in the bath solution,Iα1 was activated at potentials more positive than −40 mV, peaked between 0 and +10 mV, and reversed at about +50 mV. In addition to slow apparent inactivation of inward current, both subunits provided an extremely slow voltage-dependent inactivation at potentials more positive than −100 mV, with half-maximum inactivation at −43.4 mV for cardiac and −41.4 mV for SM α1-subunits. The onset of inactivation as well as recovery from this process were within a time range of minutes. The voltage dependence of steady-state inactivation could be fitted by the sum of two Boltzmann's equations with slope factors of about 12 mV and 5 mV. A less sloped component has its midpoints at −75.6 and −63.7 mV, and a steeper component has its midpoints at −42.8 and −37.7 mV for cardiac and SM α1-subunits, respectively. Relative contribution of the steeper component was higher in both subunits (0.86 and 0.66 for cardiac and SM subunits, respectively). For comparison, the inactivation curves for 5-sec-long conditioning prepulses could be fitted by single Boltzmann's distribution with a 20 mV more positive midpoint and a slope factor of about 13 mV. In contrast to the steady-state inactivation curves, they showed considerable overlap with the steady-state activation curve. Our results reflect functional consequences of known sequence differences between α1-subunits of the cardiac and SM L-type Ca2+ channels and could be used in structural modeling of Ca2+ channel gating. In addition, they show that depolarization-induced window current has a transient nature and decays with the development of extremely slow inactivation. This is the first demonstration that slow inactivation of the L-type Ca2+ channel is an intrinsic property of its α1-subunits.

Roles of outward potassium currents in the action potentials of guinea pig ureteral myocytes.

Outward currents of freshly dissociated ureteral myocytes consist mainly of Ca(2+)-activated K+ current (IKCa) and a transient outward current (ITO). No delayed rectifier current was apparent. IKCa is small and nondecaying and fluctuates actively and irregularly. Blocking IKCa decreased resting membrane conductance and prolonged action potential plateaus, showing its roles in maintaining the resting potential and in repolarizing action potentials. It is also responsible for the membrane potential fluctuations on action potential plateaus. Neither 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride nor caffeine reduced the fluctuations in the outward current or in the action potentials, indicating that internal Ca2+ storage contributes little to the fluctuations. ITO has fast activation and inactivation kinetics with inactivation time constants of approximately 15 and 150 ms, respectively. Its highly negative voltage-availability relationship (V0.5 = -70.5 mV) suggests a low availability (< 5%) at normal resting potentials. It has only trivial effects on action potentials.

Amino terminal-dependent gating of the potassium channel rat eag is compensated by a mutation in the S4 segment.

1. Rat eag potassium channels (r-eag) were expressed in Xenopus oocytes. They gave rise to delayed rectifying K+ currents with a strong Cole-Moore effect. 2. Deletions in the N-terminal structure of r-eag either shifted the activation threshold to more negative potentials and slowed the activation kinetics (delta 2-190, delta 2-12 and delta 7-12) or resulted in a shift to more positive potentials and faster activation kinetics (delta 150-162). 3. The impact of the deletion delta 7-12 was investigated in more detail: it almost abolished the Cole-Moore effect and markedly slowed down channel deactivation. 4. Unlike wild-type channels, the deletion mutants delta 7-12 exhibited a rapid inactivation which, in combination with the slow deactivation, resulted in current characteristics which were similar to those of the related potassium channel HERG. 5. Both the slowing of deactivation and the inactivation induced by the deletion delta 7-12 were compensated by a single histidine-to-arginine change in the S4 segment, while this mutation (H343R) only had minor effects on the gating kinetics of the full-length r-eag channel. 6. These results demonstrate a functional role of the N-terminus in the voltage-dependent gating of potassium channels which is presumably mediated by an interaction of the N-terminal protein structure with the S4 motif during the gating process.

Presynaptic Recordings fromDrosophila: Correlatin of Macroscopic and Single-Channel K+Currents

We have performed direct electrophysiological recordings from Drosophila peptidergic synaptic boutons in situ, taking advantage of a mutation, ecdysone, which causes an increase in size of these terminals. Using patch-clamp techniques, we have analyzed voltage-dependent potassium currents at the macroscopic and single-channel level. The synaptic membrane contained at least two distinct voltage-activated potassium currents with different kinetics and voltage sensitivity: an IA-like current with fast activation and inactivation kinetics and voltage-dependent steady-state inactivation; a complex delayed current that includes a slowly inactivating component, resembling the IK described in other preparations; and a noninactivating component. The IA-like current in these peptidergic boutons is not encoded by the gene Shaker, because it is not affected by null mutations at this locus. Rather, synaptic IA has properties similar to those of the Shal-encoded IA. Single-channel recordings revealed the presence in synaptic membranes of three different potassium channel types (A2, KD, KL), with biophysical properties that could account for the macroscopic currents and resemble those of the Shal, Shab, and Shaw channels described in heterologous expression systems and Drosophila neuronal somata. A2 channels (6-9 pS) have brief open times, and like the macroscopic IA they exhibited voltage-dependent steady-state inactivation and a rapidly inactivating ensemble average current profile. KD channels (13-16 pS) had longer open times, activate and inactivate with much slower kinetics, and may account for the slowly inactivating component of the macroscopic current. KL (44-54 pS) channels produced a noninactivating ensemble average and may contribute to the delayed macroscopic current observed.

Low-threshold, persistent sodium current in rat large dorsal root ganglion neurons in culture.

Dorsal root ganglion neurons from adult rats (> or = 200 g) were maintained in culture for between 1 and 3 days. Membrane currents generated by large neurons (50-75 microns apparent diameter) were recorded with the whole cell patch-clamp technique. Large neurons generated transient Na+ currents and at least two types of inward current that persisted throughout 200-ms voltage-clamp steps to +20 mV. One persistent current activated close to -35 mV (high threshold), whereas in about half of the cells another persistent current began to activate negative to -70 mV (low threshold). The high-threshold persistent current was identified as a Ca2+ current, as previously described in these neurons. The low-threshold current was reversibly suppressed either by replacing external Na+ with tetramethylammonium ions or by reducing external Na+ concentration ([Na+]) and simultaneously raising external [Ca2+]. It was blocked by tetrodotoxin (TTX) with an apparent equilibrium dissociation constant in the single nanomolar range. We conclude that the low-threshold current is a TTX-sensitive, persistent Na+ current. The persistent TTX-sensitive current contributed to steady-state membrane current from at least -70 mV to 0 mV, a wider potential range than predicted by activation-inactivation gating overlap for transient Na+ current. Because of its low threshold and fast activation kinetics, the persistent Na+ current is expected to play an important role in determining membrane excitability.

Fast activation and inactivation of inositol trisphosphate-evoked Ca2+ release in rat cerebellar Purkinje neurones.

1. Calcium release from stores via inositol trisphosphate (InsP3) activation of intracellular Ca2+ receptor-channels is thought to have a role in regulating the excitability of cerebellar Purkinje neurones. The kinetic characteristics of InsP3 receptor activation in Purkinje neurones are reported here. 2. InsP3 was applied by flash photolysis of caged InsP3 during whole-cell patch clamp. Ca2+ flux into the cytosol was measured with a low-affinity fluorescent Ca2+ indicator and by activation of Ca(2+)-dependent membrane conductance. 3. InsP3 produced Ca2+ release from stores with an initial well-defined delay (mean, 85 ms at 10 microM InsP3), which decreased to less than 20 ms at high InsP3 concentrations. 4. The rate of rise of free [Ca2+], which provides a measure of Ca2+ efflux and InsP3 receptor activation, increased with increasing InsP3 concentration in each cell and had a high absolute value of up to 1400 microM s-1 at 40 microM InsP3. The period of fast efflux was brief, inactivating in 25 ms at low and in 9 ms at high InsP3 concentration. 5. Peak free [Ca2+] was high (mean, 23 microM with a pulse of 40 microM InsP3) and increased with InsP3 concentration up to 80 microM InsP3 tested here. 6. Experiments with a flash-released, stable 5-thio-InsP3 confirm that the low InsP3 sensitivity of Purkinje neurones does not result from metabolism of InsP3. 7. The low functional affinity and fast activation by InsP3 suggest a difference in InsP3 receptor properties from non-neuronal cells tested in the same way. The large Ca2+ efflux and high peak [Ca2] probably result from high InsP3 receptor-channel density. 8. Elevated cytosolic [Ca2+] produced by Ca2+ influx through plasmalemmal Ca2+ channels strongly suppressed InsP3-evoked Ca2+ release from stores. Rapid termination of InsP3-evoked efflux results mainly from inhibition by high [Ca2+]. 9. The fast InsP3 activation kinetics and rapid, strong inactivation by Ca2+ influx suggest that interactions between InsP3-mediated and membrane Ca2+ signalling could occur on a time scale compatible with neuronal excitation.

Auditory cortex neurons: primary culture and ion channel activity in rat.

We have developed a primary dissociated cell culture of the fetal (E17) and post-natal (P0-P10) rat auditory cortex. Pyramidal and non-pyramidal cells had a mean cross-sectional diameter of 12.73 +/- 1.80 microns (mean +/- S.D., n = 25) and 17.58 +/- 1.67 microns (mean +/- S.D., n = 10), respectively, measured at 6 days in culture. These cells were viable for as long as 18-21 days. They expressed voltage-gated sodium and potassium channel currents as early as one day in culture, and at various phases in cell culture. Sodium current, activated at membrane potentials more positive than -60 mV, displayed fast activation and inactivation kinetics. Fifty percent inactivation of sodium channels occurred at a pre-pulse potential of -63 mV. Delayed rectifier potassium channels were activated at potentials positive to -40 mV. Large hyperpolarizing constant current pulses elicited anode break action potentials, and large depolarizing constant current pulses exhibited rectification indicative of the delayed rectifying potassium channel activity.

Synaptic integration in a model of cerebellar granule cells.

1. We have developed a compartmental model of a turtle cerebellar granule cell consisting of 13 compartments that represent the soma and 4 dendrites. We used this model to investigate the synaptic integration of mossy fiber inputs in granule cells. 2. The somatic compartment contained six active ionic conductances: a sodium conductance with fast activation and inactivation kinetics, gNa; a high-voltage-activated calcium conductance, gCa(HVA); a delayed potassium conductance, gK(DR); a transient potassium conductance, gK(A); a slowly relaxing mixed Na+/K+ conductance activating at hyperpolarized membrane potentials, gH, and a calcium- and voltage-dependent potassium conductance, gK(Ca). The kinetics of these conductances was derived from electrophysiological studies in a variety of preparations, including turtle and rat granule cells. 3. In the soma, dynamics of intracellular free Ca2+ was modeled by incorporation of a Na+/Ca2+ exchanger, radial diffusion, and binding sites for Ca2+. 4. The model of the turtle granule cell exhibited depolarization-induced action potential firing with properties closely resembling those seen with intracellular recordings in turtle granule cells in vitro. 5. In the most distal compartments of the dendrites, mossy fiber activity induced synaptic currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-type of glutamate receptors. The strength of synaptic inputs chosen was such that the synaptic potential induced by synchronous activation of two mossy fiber synapses reached threshold for induction of a single action potential. 6. The slow time course of the NMDA synaptic current together with the slow relaxation kinetics of gH significantly affected the temporal summation of excitatory synaptic potentials. A priming action potential evoked by mossy fiber stimulation increased the maximal time interval between two synaptic potentials capable to reach again threshold for a subsequent action potential. This time interval then decreased in parallel with the decay of the NMDA synaptic current, reached a minimum after 200 ms, and slowly recovered with reactivation of gH. 7. Repetitive, steady activation of synaptic conductances by a single mossy fiber at different frequencies induced action potential firing with a sharp threshold at 12 Hz. Activity of a single or of several mossy fibers induced firing of the granule cell at a frequency close to that induced when the average synaptic current was directly injected into the cell. The mossy fiber activity-granule cell firing frequency curve was close to linear with a slope of about one-half for input frequencies < or = 400 Hz.(ABSTRACT TRUNCATED AT 400 WORDS)

Fast release of 45Ca2+ induced by inositol 1,4,5-trisphosphate and Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle: evidence for two types of Ca2+ release channels

The kinetics of Ca2+ release induced by the second messenger D-myoinositol 1,4,5 trisphosphate (IP3), by the hydrolysis-resistant analogue D-myoinositol 1,4,5 trisphosphorothioate (IPS3), and by micromolar Ca2+ were resolved on a millisecond time scale in the junctional sarcoplasmic reticulum (SR) of rabbit skeletal muscle. The total Ca2+ mobilized by IP3 and IPS3 varied with concentration and with time of exposure. Approximately 5% of the 45Ca2+ passively loaded into the SR was released by 2 microM IPS3 in 150 ms, 10% was released by 10 microM IPS3 in 100 ms, and 20% was released by 50 microM IPS3 in 20 ms. Released 45Ca2+ reached a limiting value of approximately 30% of the original load at a concentration of 10 microM IP3 or 25-50 microM IPS3. Ca(2+)-induced Ca2+ release (CICR) was studied by elevating the extravesicular Ca2+ while maintaining a constant 5-mM intravesicular 45Ca2+. An increase in extravesicular Ca2+ from 7 nM to 10 microM resulted in a release of 55 +/- 7% of the passively loaded 45Ca2+ in 150 ms. CICR was blocked by 5 mM Mg2+ or by 10 microM ruthenium red, but was not blocked by heparin at concentrations as high as 2.5 mg/ml. In contrast, the release produced by IPS3 was not affected by Mg2+ or ruthenium red but was totally inhibited by heparin at concentrations of 2.5 mg/ml or lower. The release produced by 10 microM Ca2+ plus 25 microM IPS3 was similar to that produced by 10 microM Ca2+ alone and suggested that IP3-sensitive channels were present in SR vesicles also containing ruthenium red-sensitive Ca2+ release channels. The junctional SR of rabbit skeletal muscle may thus have two types of intracellular Ca2+ releasing channels displaying fast activation kinetics, namely, IP3-sensitive and Ca(2+)-sensitive channels.

Whole-cell clamp of dissociated photoreceptors from the eye of Lima scabra.

Voltage-dependent membrane currents were investigated in enzymatically dissociated photoreceptors of Lima scabra using the whole-cell clamp technique. Depolarizing steps to voltages more positive than -10 mV elicit a transient inward current followed by a delayed, sustained outward current. The outward current is insensitive to replacement of a large fraction of extracellular Cl- with the impermeant anion glucuronate. Superfusion with tetraethylammonium and 4-aminopyridine reversibly abolishes the outward current, and internal perfusion with cesium also suppresses it, indicating that it is mediated by potassium channels. Isolation of the inward current reveals a fast activation kinetics, the peak amplitude occurring as early as 4-5 ms after stimulus onset, and a relatively rapid, though incomplete inactivation. Within the range of voltages examined, spanning up to +90 mV, reversal was not observed. The inward current is not sensitive to tetrodotoxin at concentrations up to 10 microM, and survives replacement of extracellular Na with tetramethylammonium. On the other hand, it is completely eliminated by calcium removal from the perfusing solution, and it is partially blocked by submillimolar concentrations of cadmium, suggesting that it is entirely due to voltage-dependent calcium channels. Analysis of the kinetics and voltage dependence of the isolated calcium current indicates the presence of two components, possibly reflecting the existence of separate populations of channels. Barium and strontium can pass through these channels, though less easily than calcium. Both the activation and the inactivation become significantly more sluggish when these ions serve as the charge carrier. A large fraction of the outward current is activated by preceding calcium influx. Suppression of this calcium-dependent potassium current shows a small residual component resembling the delayed rectifier. In addition, a transient outward current sensitive to 4-aminopyridine (Ia) could also be identified. The relevance of such conductance mechanisms in the generation of the light response in Lima photoreceptors is discussed.

Calcium currents in normal and dystrophic human skeletal muscle cells in culture

Human muscle cells obtained from biopsy specimens were grown in a primary culture system and electrophysiologically studied. Whole cell patch-clamp recordings revealed the presence of two types of calcium currents: (i) a low-threshold (−60 mV) one (I Ca,T) with fast activation and inactivation kinetics (time-to-peak: 39 ms at −30 mV); and (ii) a high-threshold (−10 mV) one (I Ca,L) with slower kinetics (time-to-peak: 550 ms at 20 mV). These two types of calcium currents could be also distinguished by their pharmacological characteristics since I Ca,L was sensitive to the antagonist and agonist dihydropyridine derivatives contrary to I Ca,T which was completely resistant to these compounds. These functional calcium channels existed both in normal and Duchenne dystrophic (DMD) human skeletal muscle cells in culture. We discuss a possible role of these two types of calcium channels in the myoplasmic calcium accumulation observed in the Duchenne muscular dystrophy.

Voltage‐dependent potassium currents in developing neurones from quail mesencephalic neural crest.

Neurones in explants cultured from quail mesencephalic neural crest were studied at different stages of their development using the voltage-clamp technique. A voltage-dependent outward current activated by membrane depolarization was identified as a potassium current by the sensitivity of its reversal potential to extracellular potassium. The voltage-dependent potassium current is made up of two components which differ in their sensitivity to 4-aminopyridine (4-AP) and tetraethylammonium (TEA). The component most sensitive to 4-AP has fast activation kinetics and inactivates quickly at sustained depolarized voltages. By analogy with a current described in other preparations, this current was called IA. The component most sensitive to TEA has slower activation kinetics and inactivates more slowly at sustained depolarized voltages. This current was called IK. IA and IK were already present in neurones cultured for 24 h. The ratio between the peak of IK and that of IA increased significantly between 24 h and 4 days in culture. This means that the two components of the voltage-dependent potassium current follow a different time course during development.

Na and Ca channels in a transformed line of anterior pituitary cells.

The ionic conductances of GH3 cells, a transformed line from rat anterior pituitary, have been studied using the whole-cell variant of the patch-clamp technique (Hamill et al., 1981). Pipettes of very low resistance were used, which improved time resolution and made it possible to control the ion content of the cell interior, which equilibrated very rapidly with the pipette contents. Time resolution was further improved by using series resistance compensation and "ballistic charging" of the cell capacitance. We have identified and partially characterized at least three conductances, one carrying only outward current, and the other two normally inward. The outward current is absent when the pipette is filled with Cs+ instead of K+, and has the characteristics of a voltage-dependent potassium conductance. One of the two inward conductances (studied with Cs+ inside) has fast activation, inactivation and deactivation kinetics, is blocked by tetrodotoxin (TTX), and has a reversal potential at the sodium equilibrium potential. The other inward current activates more slowly and deactivates with a quick phase and a very slow phase after a short pulse. Either Ca++ or Ba++ serves as current carrier. During a prolonged pulse, current inactivates fairly completely if there is at least 5 mM Ca++ outside, and the amplitude of the current tails following the pulse diminishes with the time course of inactivation. When Ba++ entirely replaces Ca++ in the external medium, there is no inactivation, but deactivation kinetics of Ca channels vary as pulse duration increases: the slow phase disappears, the fast phase grows in amplitude. Inactivation (Ca++ outside) is unaltered by 50 mM EGTA in the pipette: inactivation cannot be the result of internal accumulation of Ca++.