Potassium channels of myenteric neurons in guinea-pig small intestine

Patch-clamp recording was used to study rectifying K+ currents in myenteric neurons in short-term culture. In conditions that suppressed Ca2+ -activated K+ current, three kinds of voltage-activated K+ currents were identified by their voltage range of activation, inactivation, kinetics and pharmacology. These were A-type current, delayed outwardly rectifying current (I(K),dr) and inwardly rectifying current (I(K),ir). I(K),ir consisted of an instantaneous component followed by a time-dependent current that rapidly increased at potentials negative to -80 mV. Time-constant of activation was voltage-dependent with an e-fold decrease for a 31-mV hyperpolarization amounting to a decrease from 800 to 145 ms between -80 and -100 mV. I(K),ir did not inactivate. I(K),ir was abolished in K+ -free solution. Increases in external K+ increased I(K),ir conductance in direct relation to the square root of external K+ concentration. Activation kinetics were accelerated and the activation range shifted to more positive K+ equilibrium potentials. I(K),ir was suppressed by external Cs+ and Ba2+ in a concentration-dependent manner. Ca2+ and Mg+ were less effective than Ba2+. I(K),ir was unaffected by tetraethylammonium ions. I(K),dr was activated at membrane potentials positive to - 30 mV with an e-fold decrease in time-constant of activation from 145 to 16 ms between -20 and 30 mV. It was half-activated at 5 mV and fully activated at 50 mV. Inactivation was indiscernible during 2.5 s test pulses. I(K),dr was suppressed in a concentration-, but not voltage-dependent manner by either tetraethylammonium or 4-aminopyridine and was insensitive to Cs+. The results suggest that I(K),ir may be important in maintaining the high resting membrane potentials found in afterhyperpolarization-type enteric neurons. They also suggest importance of I(K),ir channels in augmentation of the large hyperpolarizing after-potentials in afterhyperpolarization-type neurons and the hyperpolarization associated with inhibitory postsynaptic potentials. I(K),dr in afterhyperpolarization-type enteric neurons has overall kinetics and voltage behaviour like delayed rectifier currents in other excitable cells where the currents can also be distinguished from A-type and Ca2+ -activated K+ current.     

Kinetics and distribution of voltage-gated Ca,Na and K channels on the somata of rat cerebellar Purkinje cells

Voltage gated ion channels on the somatic membrane of rat cerebellar Purkinje cells were studied in dissociated cell culture with the combination of cell-attached and whole-cell variation of patch clamp technique. The method enables us to record local somatic membrane current under an improved space clamp condition. Transient (fast-inactivating) and steady (slow inactivating) Ca channel currents, Na current, transient (fast-inactivating) and steady (slow-inactivating) K currents, were observed. Transient and steady Ca channel currents were activated at test potentials more positive than –40 mV and –20 mV, respectively (in 50 mM external Ba). The transient current inactivated with a half-decay time of 10–30 ms during maintained depolarizing pulses, while the steady current showed relatively little inactivation. Na current was activated at more positive potentials than –60 mV, and inactivated with a half-decay time of less than 5 ms. Transient and steady K outward currents were recorded at more positive potential than –20 mV and –40 mV, respectively. The transient current inactivated with a half-decay time of 2–8 ms. Ca, Na and K channels showed different patterns of distribution on the somatic membrane. Steady Ca channels tended to cluster compared with Na or K channels.     

Patch-clamp study of the tetrodotoxin-resistant sodium current in group C sensory neurones

Conditions were devised to isolate in cranial sensory neurones transfer of Na ions: K and Ca were omitted from the extracellular medium, and simultaneously cells were intracellularly loaded with 120 mM caesium and 20 mM TEA at [Ca]i = 10(-8) M. A tetrodotoxin (TTX)-resistant current was shown to be elicited by step depolarization from -25 MV upwards. This current successively activates and inactivates at increasing rates on further depolarization and at 0 mV (where peak amplitude is reached) its time course is of 20-50 ms. Absence of TTX-sensitivity (up to 15 microM), slow time course and an activation curve shifted by 15 mV towards the depolarized potentials differentiate this current from the more classical fast Na current which can be elicited on the same cells. Inactivation was provoked by a prepulse of varying amplitude and duration: with a prepulse command to -20 mV, inactivation was of 50% within a delay of 300 ms and almost 100% in about 1 min. After complete inactivation by command to 0 mV for 300 ms, recovery by holding the potential at -80 mV was of 50% in 205 ms, and of 100% after 1-4 s. It is concluded that a charge transfer of Na accounts for most of the hump which prolongs the action potential of these sensory neurones, and thus it can be proposed that spike duration as modulated by neurotransmitters may also involve Na in addition to Ca.     

Blocking and modifying actions of octanol on Na channels in frog myelinated nerve

The actions of externally applied n-octanol on Na channels in myelinated frog nerve fibres were studied under voltage clamp conditions. Upon octanol application peak Na inward currents declined in two phases: 90% of the reduction occurred in less than 2 min but a steady-state was reached only after 15 min. During washout the currents came to a stable level within 10 min. The reduction of Na inward currents by octanol was dependent on the amplitude and duration of prepotentials. At the resting potential (VH = 0 mV) 0.4 mM octanol reduced peak Na inward currents at V = 60 mV by 50%. After a prepulse of -60 mV and 50 ms duration Na currents decreased only by 20%. At a hyperpolarizing holding potential of VH = -28 mV 0.7 mM octanol reduced peak inward Na currents to one half. Octanol depressed Na currents at all potentials by approximately the same factor. The Na reversal potential VNa remained unchanged. 0.7 mM external octanol shifted the Na activation curve m infinity (V) by 5 mV to more positive and the inactivation curve h infinity (V) by 14 mV to more negative potentials. The midpoint slopes of both curves were reduced. The time constants of Na activation and inactivation at small depolarizations were decreased. The conductance gamma of a single Na channel and the number No of conducting Na channels per node were determined from nonstationary Na current fluctuations. 0.7 mM octanol increased gamma by a factor of 1.6 and reduced No by a factor of 0.34. It is concluded that octanol blocks some Na channels and modifies the remaining unblocked channels.     

Removal of Ca current inactivation in dialysed guinea-pig atrial cardioballs by Ca chelators

Ca currents flowing during voltage clamp depolarizations were studied in cultured guinea-pig atrial cardioballs by means of single low resistance patch clamp pipettes. The pipettes were filled with solutions containing Cs+ as major cation in order to block K+ currents and high concentrations of various Ca chelating agents (EGTA, nitrilotriacetic acid, citrate, dipicolinic acid) to prevent rises of the intracellular Ca-activity by Ca-entry. Ca currents of myocytes loaded with 20 mM of either EGTA [(ethylenedioxy)-diethylenedinitrilo)tetra-acetic acid] or NTA (nitrilotriacetic acid) display a biphasic time course of inactivation at membrane potentials between -25 and +45 mV. The fast phase is reduced with increasingly positive membrane potentials. In cells loaded with either citrate or DPA (dipicolinic acid, pyridine-2,6-dicarboxylic acid) inactivation is negligible or absent for small depolarizations. In the range of membrane potentials where maximum current flows (0-+10 mV) a monophasic slow time course of inactivation is observed. At more positive membrane potentials inactivation is slowed. The amount of inactivation under this condition is related to the current density of the cell. Conditions, which for a given membrane potential reduce the amplitude of ICa such as extracellular application of blocking ions (Co2+, Cd2+), a conditioning depolarization, or 'rundown' of Ca-channels lead to a slowing or a complete removal of inactivation in cells dialysed with citrate or DPA respectively. Cells loaded with these Ca chelators did not show any symptom of voltage dependent inactivation of ICa. Under the conditions described action potentials were recorded in the current clamp mode. Upon dialysis with EGTA the typical 'triangular shaped' atrial action potential develops a plateau of 500 to 800 ms in duration. With citrate-containing pipette solutions the action potential duration usually is several seconds. The results for the first time demonstrate that inactivation of cardiac ICa can be considerably slowed or even removed. They provide further strong support for the hypothesis that inactivation of this current depends on Ca entry rather than membrane potential. The fast phase of inactivation observed with EGTA (NTA) possibly reflects the slow kinetics of the binding reaction of this type of Ca chelators.     

Ionic currents underlying fast action potentials in the obliquely striated muscle cells of the octopus arm

The octopus arm provides a unique model for neuromuscular systems of flexible appendages. We previously reported the electrical compactness of the arm muscle cells and their rich excitable properties ranging from fast oscillations to overshooting action potentials. Here we characterize the voltage-activated ionic currents in the muscle cell membrane. We found three depolarization-activated ionic currents: 1) a high-voltage-activated L-type Ca(2+) current, which began activating at approximately -35 mV, was eliminated when Ca(2+) was substituted by Mg(2+), was blocked by nifedipine, and showed Ca(2+)-dependent inactivation. This current had very rapid activation kinetics (peaked within milliseconds) and slow inactivation kinetics (tau in the order of 50 ms). 2) A delayed rectifier K(+) current that was totally blocked by 10 mM TEA and partially blocked by 10 mM 4-aminopyridine (4AP). This current exhibited relatively slow activation kinetics (tau in the order of 15 ms) and inactivated only partially with a time constant of ~150 ms. And 3) a transient A-type K(+) current that was totally blocked by 10 mM 4AP and was partially blocked by 10 mM TEA. This current exhibited very fast activation kinetics (peaked within milliseconds) and inactivated with a time constant in the order of 60 ms. Inactivation of the A-type current was almost complete at -40 mV. No voltage-dependent Na(+) current was found in these cells. The octopus arm muscle cells generate fast (~3 ms) overshooting spikes in physiological conditions that are carried by a slowly inactivating L-type Ca(2+) current.     

Characterization of voltage-dependent calcium currents in mouse motoneurons.

1. Calcium channel currents were measured with the whole-cell patch clamp technique in cultured, identified mouse motoneurons. Three components of current were operationally defined on the basis of voltage dependence, kinetics, and pharmacology. 2. Test potentials to -50 mV or greater (10 mM external Ca2+) elicited a low-voltage activated T-type current that was transient (decaying to baseline in less than 200 ms) and had a relatively slow time to peak (20-50 ms). A 1-s prepulse to -45 mV produced approximately half-maximal inactivation of this T current. 3. Two high-voltage activated (HVA) components of current (1 transient and 1 sustained) were activated by test potentials to -20 mV or greater (10 mM external Ca2+). A 1-s prepulse to -35 mV produced approximately half-maximal inactivation of the transient component without affecting the sustained component. 4. When Ba2+ was substituted for Ca2+ as the charge carrier, activation of the HVA components was shifted in the hyperpolarizing direction, and the relative amplitude of the transient HVA component was reduced. 5. Amiloride (1-2 mM) caused a reversible, partial block of the T current without affecting the HVA components. 6. The dihydropyridine agonist isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3- pyridine-carboxylate [(+)-SDZ 202-791, 100 nM-1 microM)] shifted the activation of the sustained component of HVA current to more negative potentials and increased its maximal amplitude. Additionally, (+)-SDZ 202-791 caused the appearance of a slowed component of tail current.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic properties of T-type Ca2+ currents in isolated rat hippocampal CA1 pyramidal neurons.

1. T-type Ca2+ channels producing a transient inward current were studied in pyramidal neurons acutely isolated from the ventral portion of rat hippocampal CA1 region. Membrane currents were recorded by the suction-pipette technique, which allows for internal perfusion under a single-electrode voltage clamp. 2. In all cells superfused with external solution containing 10 mM Ca2+, the T-type Ca2+ current was evoked by step depolarization to potentials more positive than -60 mV from a holding potential of -100 mV and reached a peak in the current-voltage relationship around -30 mV at 20-22 degrees C. 3. Activation and inactivation processes of T-type Ca2+ current were highly potential dependent, and the latter was fitted by a single exponential function. 4. Steady-state inactivation of T-type Ca2+ current could be fitted by a Boltzmann's equation with a slope factor of 6.0 and a half-inactivated voltage of -79 mV. 5. Recovery from inactivation of T-type Ca2+ current was not a single exponent. The major component of recovery (60-90% of total) was voltage sensitive with a time constant of 215 ms at -100 mV. 6. Amplitude of the T-type Ca2+ current depended on the external Ca2+ concentration. The ratio of peak amplitude in the individual current-voltage relationships of Ca2+, Ba2+, and Sr2+ currents passing through T-type Ca2+ channel was 1.0:0.85:1.32. The current kinetics were much the same. 7. All kinetic properties, including activation and inactivation, as well as the amplitude of T-type Ca2+ current, were temperature sensitive with Q10 (temperature coefficient) values of 1.7-2.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

A fast transient potassium current in thalamic relay neurons: kinetics of activation and inactivation.

1. Whole-cell voltage-clamp techniques were used to record K+ currents in relay neurons (RNs) that had been acutely isolated from rat thalamic ventrobasal complex and maintained at 23 degrees C in vitro. Tetrodoxin (TTX; 0.5 microM) was used to block Na+ currents, and reduced extracellular levels of Ca2+ (1 mM) were used to minimize contributions from Ca2+ current (ICa). 2. In RNs, depolarizing commands activate K+ currents characterized by a substantial rapidly inactivating (time constant approximately 20 ms) component, the features of which correspond to those of the transient K+ current (IA) in other preparations, and by a smaller, more slowly activating K+ current, "IK". IA was reversibly blocked by 4-aminopyridine (4-AP, 5 mM), and the reversal potential varied with [K+]o as predicted by the Nernst equation. 3. IA was relatively insensitive to blockade by tetraethylammonium [TEA; 50%-inhibitory concentration (IC50) much much greater than 20 mM]; however, two components of IK were blocked with IC50S of 30 microM and 3 mM. Because 20 mM TEA blocked 90% of the sustained current while reducing IA by less than 10%, this concentration was routinely used in experiments in which IA was isolated and characterized. To further minimize contamination by other conductances, 4-AP was added to TEA-containing solutions and the 4-AP-sensitive current was obtained by subtraction. 4. Voltage-dependent steady-state inactivation of peak IA was described by a Boltzman function with a slope factor (k) of -6.5 and half-inactivation (V1/2) occurring at -75 mV. Activation of IA was characterized by a Boltzman curve with V1/2 = -35 mV and k = 10.8. 5. IA activation and inactivation kinetics were best fitted by the Hodgkin-Huxley m4h formalism. The rate of activation was voltage dependent, with tau m decreasing from 2.3 ms at -40 mV to 0.5 ms at +50 mV. Inactivation was relatively voltage independent and nonexponential. The rate of inactivation was described by two exponential decay processes with time constants (tau h1 and tau h2) of 20 and 60 ms. Both components were steady-state inactivated with similar voltage dependence. 6. Temperature increases within the range of 23-35 degrees C caused IA activation and inactivation rates to become faster, with temperature coefficient (Q10) values averaging 2.8. IA amplitude also increased as a function of temperature, albeit with a somewhat lower Q10 of 1.6. 7. Several voltage-dependent properties of IA closely resemble those of the transient inward Ca2+ current, IT. (ABSTRACT TRUNCATED AT 400 WORDS)     

Single voltage-dependent potassium channels in cultured rat hippocampal neurons

Single-channel recordings using the gigohm seal patch-clamp technique were carried out on the somatic membranes of dissociated embryonic rat hippocampal neurons grown in cell culture. The recording medium contained tetrodotoxin to block the voltage-dependent Na+ conductance and Cd2+ to block Ca2+ and Ca2+-activated conductances. In the cell-attached configuration, depolarizing voltage steps activated outward directed single-channel currents with conductance 15-20 pS. The channel openings exhibited a moderate degree of flickering. The mean burst lifetimes ranged from 5 to 13 ms with a tendency to increase slightly at more depolarized potentials (T = 21-25 degrees C). Reversal potential measurements using excised membrane patches indicated that the channels behaved as expected of a K+-selective membrane pore. Channel opening occurred in Ca2+-free EGTA-containing solutions but was never observed in the presence of tetraethylammonium (TEA; 20 mM). The frequency of channel opening increased as the membrane was depolarized by up to 50 mV from resting potential; the fraction of time spent in the open state during the first 300 ms following a step depolarization increased e-fold for a 8-25 mV change in potential. First-latency histograms and simulations of the macroscopic current based on channel data obtained during repeated depolarizing voltage steps indicated that the probability of the channel being in the open state increases gradually with time after a step depolarization. During repeated depolarizing steps the channels appeared to randomly enter and exit a long-lived inactive state. It is concluded that these channels may underly the slowly activating, very slowly inactivating, TEA-sensitive voltage-dependent K+ current (IK) in cultured hippocampal neurons.     

Patch-clamp study of the calcium-dependent chloride current in AtT-20 pituitary cells

1. Voltage-clamp recordings were made from cultured AtT-20 pituitary cells using the whole-cell patch-clamp technique. Cells were perfused internally with Cs+ to block K+ currents and bathed externally with either 1 microM tetrodotoxin or with tetraethylammonium (TEA) as a Na+ substitute to block voltage-activated Na+ currents. 2. Depolarizing voltage steps from a holding potential of -80 mV to potentials positive to -30 mV evoked two currents: a fast inward current that activated between -30 and +70 mV and a slowly activating current (designated "slow step current") that was inward between -30 and near 0 mV (the Cl- equilibrium potential) and outward positive to about 0 mV. Repolarization to -80 mV revealed a slowly decaying, inward tail current, whose magnitude with respect to step potential closely matched the current-voltage relationship of the voltage-activated Ca2+ current. 3. Activation of the fast inward current, slow step current, and tail current, was prevented by extracellular application of Cd2+ or removal of extracellular Ca2+. Replacement of extracellular Ca2+ with Ba2+ potentiated the fast inward current but blocked the slow step and tail currents. Intracellular perfusion with greater than 1 mM of the Ca2+ chelators ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) or [1,2-bis(2)aminophenoxy]ethane N,N,N',N'-tetraacetic acid (BAPTA) prevented activation of the slow step and tail currents, but not the fast inward current. 4. The reversal potential of the slow inward current was sensitive to changes in the Cl- equilibrium potential but not to substitution of TEA for Na+. The slow step current, but not the fast inward current, was partially blocked by the Cl- channel blocker, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. 5. These data indicate that both the slow inward tail current and the slowly activating, reversible step current were a Ca2+-dependent Cl- current, similar to that described in other neuronal and nonneuronal cell types. The fast inward current was a voltage-activated Ca2+ current, described previously in these and other cells. 6. In the absence of intracellular EGTA, the tail current decayed with complex kinetics, its time course apparently dependent on the magnitude of the voltage-activated Ca2+ current. In the presence of 200 microM intracellular EGTA, the tail current decayed significantly faster and often decayed exponentially.     

Calcium current inactivation in insulin-secreting cells is mediated by calcium influx and membrane depolarization

Inactivation of voltage-dependent calcium currents was studied in single, dissociated insulin-secreting HIT cells voltage-clamped by the whole-cell patch-clamp method at room temperature. Na and K currents were suppressed by tetrodotoxin, tetraethylammonium, ATP, 4-aminopyridine and Cs. Ca currents activated in less than 10 ms by depolarizations beyond –50 mV from a holding potential of –100 mV and were identified, as in previous studies, by their sensitivity to divalent cation blockade and permeability to Ba as a charge carrier. Sustained depolarization revealed two kinetically distinct phases of inactivation: a rapid phase inactivated approximately 50% of the current in less than 100 ms while the remaining current was inactivated over the next 10–20 s. Rapid inactivation appeared to be due to Ca²⁺ influx since it was slowed markedly when Ba²⁺ was used as the current carrier, while the degree of inactivation mercased and decreased with increasing depolarization in direct parallel with the U-shaped current-voltage relationship for inward Ca current. Slow inactivation appeared to be voltage-dependent since current could be inactivated (by 20%) by 10 s long depolarizations to potentials below the threshold for activating Ca current, slow time constants of inactivation were voltage-dependent and slow inactivation persisted when Ca was replaced with Ba. Ca currents with low activation thresholds (in the –50 to –30 mV range) appeared to be preferentially inactivated by the rapid Ca-dependent mechanism. Recovery of slowly inactivated Ca current was very slow and currents inactivated by larger depolarizations required longer recovery time than those elicited by smaller depolarizations. Rapid and slow inactivation mechanisms may be important in understanding the fast spiking and slow plateau depolarizations seen in pancreatic B-cells exposed to stimulatory levels of glucose.     

Biophysical characterization of whole-cell currents in O2-sensitive neurons from the rat glossopharyngeal nerve

In this study we use nystatin perforated-patch and conventional whole-cell recording to characterize the biophysical properties of neuronal nitric oxide synthase (nNOS)-expressing paraganglion neurons from the rat glossopharyngeal nerve (GPN), that are thought to provide NO-mediated efferent inhibition of carotid body chemoreceptors. These GPN neurons occur in two populations, a proximal one near the bifurcation of the GPN and the carotid sinus nerve, and a more distal one located further along the GPN. Both populations were visualized in whole mounts by vital staining with the styryl pyridinium dye, 4-Di-2-ASP (D289). Following isolation in vitro, proximal and distal neurons had similar input resistances (mean: 1.5 and 1.6 GOmega, respectively), input capacitances (mean: 25.0 and 27.4 pF, respectively), and resting potentials (mean: -53.9 and -53.3 mV, respectively). All neurons had similar voltage-dependent currents composed of: tetrodotoxin (TTX)-sensitive Na+ currents (IC50 approximately 0.2 microM), prolonged and transient Ca2+ currents, and delayed rectifier-type K+ currents. Threshold activation for the Na+ currents was approximately -30 mV and they were inactivated within 10 ms. Inward Ca2+ currents consisted of nifedipine-sensitive L-type, omega-agatoxin IVA-sensitive P/Q-type, omega-conotoxin GVIA-sensitive N-type, SNX-482-sensitive R-type, and Ni2+-sensitive, but SNX-482-insensitive, T-type channels. The voltage-dependent outward K+ currents were sensitive to tetraethylammonium (TEA; 10 mM) and 4-aminopyridine (4-AP; 2 mM). Exposure to a chemosensory stimulus, hypoxia (PO2 range: 80-5 Torr), caused a dose-dependent decrease in K+ current which persisted in the presence of TEA and 4-AP, consistent with the involvement of background K+ channels. Under current clamp, GPN neurons generated TTX-sensitive action potentials, and in spontaneously active neurons, hypoxia caused membrane depolarization and an increase in firing frequency. These properties endow GPN neurons with an exquisite ability to regulate carotid body chemoreceptor function during hypoxia, via voltage-gated Ca2+-entry, activation of nNOS, and release of NO.     

The effect of yohimbine on sodium and gating currents in frog Ranvier node membrane

The indolalkylamine alkaloid yohimbine induced two phenomenologically-different types of sodium current (INa) inhibition in the voltage-clamped frog node of Ranvier, a tonic and a phasic ('use-dependent') block. The latter developed during a repetitive membrane stimulation with short (5 ms) depolarizing pulses at frequencies at 1 to 10 Hz. Unlike repetitive pulsing, a single-long lasting (1 s) depolarizing step did not produce a phasic block. Turning on a hyperpolarizing prepulse (50 ms to E = -123 mV) immediately before each test pulse produced a gradual unblocking of Na channels, while a depolarizing prepulse (to -86 mV) enhanced the phasic block. Yohimbine blocked the outward INa much more strongly than the inward ones. Reduction of external Na+ ions concentration from 112 to 55 mM caused a shift in the voltage-department of yohimbine block to more negative voltages, which coincided with the shift of INa reversal potential. Sodium current inhibition produced by yohimbine was accompanied by partial depression of the intramembrane charge movements ('ON-response'). Modification of Na channels by batrachotoxin made the Na channels resistant to both tonic and phasic blocking action of yohimbine. The features of the yohimbine-induced block suggest an interaction of the drug with open Na channels. The current-dependence of yohimbine block indicates an electrostatic interaction between Na+ ion and charged (protonated) form of yohimbine within the channel lumen and suggests the localization of the receptor at the inner mouth of the channel. Binding of yohimbine to the channel receptor promotes the inactivation of this channel. Comparison of the effects of yohimbine on NA and gating currents with those of local anesthetics leads us to suggest that these drugs share a common receptor.     

Zinc modulation of ionic currents in the horizontal limb of the diagonal band of Broca

We examined modulation of ionic currents by Zn2+ in acutely dissociated neurons from the rat's horizontal limb of the diagonal band of Broca using the whole-cell patch-clamp technique. Application of 50 microM Zn2+ increased the peak amplitude of the transiently activated potassium current, I(A) (at + 30 mV), from 2.20+/-0.08 to 2.57+/-0.11 nA (n = 27). This response was reversible and could be repeated in 0 Ca2+/1 microM tetrodotoxin (n = 15). Zn2+ shifted the inactivation curve to the right, resulting in a shift in the half-inactivation voltage from 76.4+/-2.2 to -53.4+/-2.0 mV (n = 11), with no effect on the voltage dependence of activation gating (n = 15). There was no significant difference in the time to peak under control conditions (7.43+/-0.35 ms, n = 14) and in the presence of Zn2+ (8.20+/-0.57 ms, n = 14). Similarly, the time constant of decay of I(A) (tau(d)) at + 30 mV showed no difference (control: 38.68+/-3.68 ms, n = 15; Zn2+: 38.48+/-2.85 ms, n = 15). I(A) was blocked by 0.5-1 mM 4-aminopyridine. In contrast to its effects on I(A), Zn2+ reduced the amplitude of the delayed rectifier potassium current (I(K)). The reduction of outward K+ currents was reproducible when cells were perfused with 1 microM tetrodotoxin in a 0 Ca2+ external solution. The amplitude of the steady-state outward currents at +30 mV under these conditions was reduced from 6.40+/-0.23 (control) to 5.76+/-0.18 nA in the presence of Zn2+ (n = 16). The amplitudes of peak sodium currents (INa) were not significantly influenced (n = 10), whereas barium currents (I(Ba)) passing through calcium channels were potently modulated. Zn2+ reversibly reduced I(Ba) at -10 mV by approximately 85% from -2.06+/-0.14 nA under control conditions to -0.30+/-0.10 nA in the presence of Zn2+ (n = 14). Further analyses of Zn2+ effects on specific calcium channels reveals that it suppresses all types of high-voltage-activated Ca2+ currents. Under current-clamp conditions, application of Zn2+ resulted in an increase in excitability and loss of accommodation (n = 13), which appears to be mediated through its effects on Ca2+-dependent conductances.     

Voltage-gated potassium currents in rat vas deferens smooth muscle cells

Voltage-gated components of the outward current in single smooth muscle cells isolated from the epididymal part of the rat vas deferens were studied using amphotericin B perforated patch-clamp techniques. The complex kinetics of the net outward current elicited by positive voltage steps from -80 mV to +40 mV suggested the presence of several components. Bath application of 200 nM charybdotoxin, a potent blocker of large-conductance, Ca(2+)-dependent K(+) channels (BK(Ca)), reduced the current amplitude significantly. When BK(Ca) channels were suppressed, fast-inactivating (I(K,f)) and delayed rectifying (I(K,dr)) components of the outward current were identified. I(K,f) was characterized by fast kinetics of current decay, negative steady-state activation and inactivation dependencies and sensitivity to 4-aminopyridine with an apparent K(d) of 0.32 mM, properties similar to those of the A-type K(+) current. In contrast, I(K,dr) activated and inactivated at more positive potentials. The time constant of activation of I(K,dr) was voltage dependent with an e-fold decrease per 21 mV depolarization. I(K,dr) was inhibited by clofilium, a blocker of voltage-gated K(+) channels, with an IC(50) of 12 micro M and was not blocked by 5 mM 4-aminopyridine. The possible significance of the voltage-gated currents is discussed.     

Calcium channel currents in isolated guinea-pig ventricular cells superfused with Ca-free EGTA solution

Changes in membrane currents seen in Ca-free, EGTA (1 mM)-containing Tyrode solution (EGTA Tyrode), were studied in isolated guinea-pig ventricular cells, under the voltage clamp performed with a "G omega seal" patch electrode. Application of the EGTA Tyrode (calculated [Ca]0 = 1.3 X 10(-9) M) first eliminated the usual calcium current, but induced an extra inward current within 2 min. The reversal potential of this current, as judged by the direction of the current change, was about +25 mV (without correction of a liquid junction potential of -12 mV), but above this voltage a decaying outward current was observed. The decay of these inward and outward currents during depolarization was slow, but a large, nearly time-independent component was evident. These currents, regardless of their polarity and time course, were reduced by application of verapamil (10(-5) M) and Mg (5 mM), and were inactivated by pre-depolarizations. In Na-free EGTA Tyrode, the inward current disappeared but the outward current persisted at high voltages. These results suggest that in ventricular cells, reduction of external Ca concentrations to a nanomolar range induces a Ca channel current composed of an inward current carried by Na, and an outward current, presumably carried by K ions. Because of the persistence of the apparently non-inactivating Ca channel current, the net membrane current evoked at voltages around 0 mV remained close to zero, or even inward, after the decay of the time-dependent component, which was completed within a few hundreds ms. This characteristic I-V relation was considered to be linked to the development of the long-lasting action potentials, with a plateau maintained at around 0 mV, in EGTA Tyrode.     

Voltage-dependent potassium channels in activated rat microglia.

1. Voltage-dependent currents of untreated (proliferating) and lipopolysaccharide (LPS)-treated rat microglial cells in culture were recorded using the whole-cell patch-clamp technique. 2. Membrane potentials showed prominent peaks at -35 mV and -70 mV. Membrane potentials of LPS-treated cells alternated between the two values. This may be due to a negative slope region of the I-V relation resulting in two zero current potentials. 3. From a holding potential of -70 mV, hyperpolarizing steps evoked an inwardly rectifying current both in proliferating and in LPS-treated cells, while depolarizing steps below -50 mV evoked an outwardly rectifying current only in LPS-treated microglia. The currents were K+ selective, as indicated by their reversal potential of approximately 0 mV in symmetric K+ concentrations (150 mM both intra- and extracellularly) and the reversal potential of the outward tail currents of approximately -90 mV at a normal extracellular K+ concentration (4.5 mM). 4. The activation of the outward current could be fitted by Hodgkin-Huxley-type n4 kinetics. The time constant of activation depended on voltage. 5. The inactivation of the inward and outward currents could be fitted by a single exponential. The time constant of the inward current inactivation was dependent on voltage, whereas the time constant of the outward current inactivation was virtually independent of voltage, except near the threshold of activation. Recovery of the outward from inactivation was slow and could be fitted by two exponentials. Responses to depolarizing steps were stable at 0.125 Hz, but greatly decreased from the first to the second pulse at 1 Hz. 6. The inactivation of the inward, but not of the outward, current disappeared in a low Na(+)-containing medium (5 mM). The inward current was selectively inhibited by extracellular Cs+ and Ba2+. The outward current was selectively inhibited by Cd2+, 4-aminopyridine and charybdotoxin. Replacement of intracellular K+ by an equimolar concentration of Cs+, and the extracellular application of tetraethylammonium and quinine inhibited both currents. 7. An increase of extracellular Ca2+ from 2 to 20 mM resulted in outwardly rectifying K+ channels activating at more positive potentials. Omission of Ca2+ from the extracellular medium had the opposite effect. When the intracellular free Ca2+ was increased from 0.01 to 1 microM, the outward current amplitudes were depressed. The Ca2+ ionophore A23187 had a similar effect. 8. LPS-treated microglial cells possess inwardly and outwardly rectifying K+ channels. The physiological and pharmacological characteristics of these two channel populations are markedly different.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels.

1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.