Restoration of the potential-dependent L-phenylalanine-inhibited calcium current by L-tyrosine in cultured hippocampal neurons]

Calcium currents were recorded in cultured (5-7 days) hippocampal neurons isolated from one-day rats. The animals were intraperitoneally injected L-phenylalanine which induced in their brain biochemical changes typical of phenylketonuria. It has been found that in neurons from injected animals the amplitude of the high-threshold calcium current was substantially inhibited and amounted to 40 +/- 30% at Vt = +20 mV (amplitude of calcium currents at Vt = -10 mV taken as 100%). Addition of L-tyrosine to the cultivation medium (50 mumol/l) restored the high-voltage calcium current, its relative amplitude reaching 280 +/- 57%.     

Single channels of low and high-threshold calcium channels of the membrane of sensory neurons in the mouse

Single calcium channels of cultured dorsal root ganglion cells from mouse embryos were studied using patch clamp method in its cell-attached configuration. Two types of activity of unitary calcium channels were found. The first one which arose at membrane potentials near--50-40 mV was characterized by unitary current amplitude of 0.37 +/- 0.04 pA with 40 mmol/l Ca2+ in the pipette solution, mean open time of 0.6 ms and intraburst mean shut time of 1.2 ms. It displayed voltage- and time-dependent inactivation. The corresponding values for the second one which required much more positive depolarization to be activated (approximately 0 mV) and did not express noticeable inactivation were: 0.53 +/- 0.04 pA, 0.8 ms and 0.8 ms. It is concluded that the recorded types of unitary activity are associated respectively with low- and high-threshold calcium currents which have been found earlier while studying whole cell currents.     

Multiple types of high-threshold calcium channels in rabbit sensory neurons: high-affinity block of neuronal L-type by nimodipine.

Whole-cell and cell-attached patch recording have been used to characterize multiple types of voltage-dependent calcium channels in neurons freshly dispersed from rabbit dorsal root ganglia. In whole-cell patch recordings, high-threshold current, strongly resistant to inactivation by depolarized holding potentials (L-type; V1/2 = -27.2 mV), was potently inhibited by nimodipine. Assuming 1:1 binding, the dissociation constant for nimodipine binding to the inactivated state of the L-type calcium channel (KI) was 5.3 nM (n = 8). In contrast, a second type of high-threshold current less resistant to inactivation by depolarized holding potentials (N-type; V1/2 = -56.9 mV) was not blocked by nimodipine. Nimodipine-resistant N-type calcium current was inhibited by omega-conotoxin (5 microM). Cell-attached patch recordings of single calcium channel currents demonstrated the existence of three different unitary conductances; 7.4 pS, 13.1 pS, and 24.1 pS. The 24.1 pS high-threshold channel was enhanced by (-) BAY K 8644 and inhibited by nimodipine in a concentration- and voltage-dependent manner. Hyperpolarization reversed this block. These results demonstrate that, as in cardiac and smooth muscle, there is a component of neuronal high-threshold current corresponding to the L-type calcium channel that can be blocked with high affinity by nimodipine.     

Action of omega-conotoxin on calcium currents of the rat pituitary GH3 cell line]

Whole-cell modification of the patch clamp method was used to examine the action of omega-CgTX on calcium currents in GH3 pituitary cells. Two quite distinct components of inward calcium currents were observed in the presence of 15 mmol/l of calcium in the external solution. One was activated from the holding potential -80 mV by testing pulses more positive than -50 mV. The shift of the holding potential to -40 mV resulted in the stationary inactivation of this low voltage activated current component. It was found that omega-CgTX activated both low-threshold and high-threshold calcium currents at the first moment of application, but low-threshold current component increased more significantly. Full effect was developed for less than 30 s. Then time decay of currents was comparable with that of the "wash-out" process. Incubation of cells in the growth medium that contained 5 mumol/l omega-CgTX during 2 hour induced an increase in density of both types of calcium currents, then it fell after 2 hours of incubation in the same medium.     

Blocking effect of intraperitoneal injection of phenylalanine on high-threshold calcium currents in rat hippocampal neurones.

Calcium currents were recorded in cultured (5-7 days) hippocampal neurones isolated from one-day-old rats. The animals obtained intraperitoneal injections of L-phenylalanine which induces in the brain biochemical changes characteristic of phenylketonuria. It has been found that the amplitude of the low-threshold calcium current in L-phenylalanine-affected neurones was not appreciably changed compared with that in neurones from control (non-injected) animals. However, the amplitude of the high-threshold calcium current was essentially decreased. Its relative amplitude at Vt = +20 mV became 40 +/- 30% as contrasted to 416 +/- 130% in neurones from control animals (the amplitude of the calcium currents at Vt = -10 mV taken as 100%). The decrease remained during the whole time of culturing. Addition of L-tyrosine to the cultivation medium (50 microM) restored the high-voltage calcium current, its relative amplitude reaching 280 +/- 57%. The data are discussed in conjunction with the previously obtained results about antagonistic modulatory action of tyrosine and phenylalanine on the functioning of high-threshold calcium channels and possible mechanisms of brain dysfunction during phenylketonuria.     

Evidence for the involvement of more than one mRNA species in controlling the inactivation process of rat and rabbit brain Na channels expressed in Xenopus oocytes

The properties of rat and rabbit brain sodium (Na) channels expressed in Xenopus oocytes following either unfractionated or high-molecular-weight mRNA injections were compared to assess the relative contribution of different size messages to channel function. RNA was size-fractionated on a sucrose gradient and a high-molecular-weight fraction (7-10 kilobase) encoding the alpha-subunit gave rise to functional voltage-dependent Na channels in the oocyte membrane. Single-channel conductance, mean open time, and time to first opening were all similar to the values for channels following injection of unfractionated RNA. In contrast, inactivation properties were markedly different; Na currents from high-molecular-weight RNA inactivated with a several-fold smaller macroscopic inactivation rate and showed a steady-state voltage dependence that was shifted in the depolarizing direction by at least 10 mV relative to that for unfractionated RNA. Single-channel recording revealed that the kinetic difference arose from a greater probability for high-molecular-weight RNA induced channels to reopen during a depolarizing voltage step. Pooling all gradient fractions and injecting this RNA into oocytes led to the appearance of Na channels with inactivation properties indistinguishable from those following injection of unfractionated RNA. These results suggest that mRNA species not present in the high-molecular-weight fraction can influence the inactivation process of rat brain Na channels expressed in Xenopus oocytes. This mRNA may encode beta-subunits or other proteins that are involved in posttranslational processing of voltage-dependent Na channels.     

2 components of the high-threshold calcium current possessing different sensitivities to omega-conotoxin in the membrane of pheochromocytoma cells

Blockade of the high-threshold calcium currents by omega-conotoxin (CgTx) was investigated by patch-clamp recordings of whole-cell currents in cultured cells of pheochromocytoma PC12. CgTx (10 mumol/l) preferentially blocked only a steady-state component of the calcium current. Inactivated component was insensitive to CgTx (up to 50 mumol/l concentration). The two component nature of the high-threshold calcium current in pheochromocytoma PC12 cells was suggested.     

The potential dependence of calcium current deactivation via the somatic membrane of sensory neurons in the mouse

Potential dependence of calcium inward current deactivation kinetics was studied in the somatic membrane of mouse dorsal root ganglion neurons by intracellular dialysis technique. The decay of the high-threshold calcium current upon repolarization was reasonably described by single-exponential process with the time constant tau less than or equal to 130 microseconds (V = = -80 mV), when the intracellular solution contained tris-PO4, and by two-exponential process (tau congruent to 0.1 and tau = 0.8 divided by ms, V = -80 mV), when the intracellular solution contained Cs-aspartate and EGTA. Both time constants were strongly voltage dependent. The amplitude of the fast component of the tail current had sigmoidal voltage dependence, and the slow component had V-shaped voltage dependence. The low-threshold calcium current deactivation occurs more slowly with high voltage dependent kinetics (tau = 1.1 divided by 1.2 ms, V = -160 mV). A dependence of low-threshold current deactivation time constant on the type of penetrating cation was observed. A kinetic model of calcium current deactivation was proposed considering three types of calcium channels presented in the somatic membrane of the neurons studied.     

2 calcium currents in the somatic membrane of neurons of Helix pomatia

Two different calcium currents were revealed in the somatic membrane of Helix pomatia neurons. In addition to the main current described in literature, depolarizing the membrane from the holding potential level (-120 divided by -100 mV) an additional calcium current was observed. It was activated at depolarizations to -80 divided by -40 mV. Contrary to the main calcium current it did not deteriorate during intracellular perfusion by solutions containing fluoride. Time-dependence of this current could be described in the framework of the Hodgkin-Huxley model with time constants for activation and inactivation equal to tau m = 6-8 ms and tau h = 300-600 ms, respectively. The amplitude of this current increased with increase of extracellular Ca2+ concentration and decreased after addition of Co2+, Ni2+, Cd2+, nifedipine and verapamil. Dissociation constants of these substances with corresponding channels determined for the maximum of current-voltage relationship were 2 (Ca2+), 3 (Co2+), 0.06 (nifedipine) and 0.2 mmol/l (verapamil). Properties of the fluoride-insensitive calcium current and data obtained for other calcium channels are compared. Its possible functional role is also discussed.     

Characterization of maintained voltage-dependent K(+)-channels induced in Xenopus oocytes by rat brain mRNA.

The voltage-dependent K+ currents encoded by rat brain mRNA were studied in Xenopus oocytes after the voltage-dependent Na+ currents and the Ca(2+)-activated Cl- currents were eliminated pharmacologically. This paper describes the maintained K+ currents (IK), defined primarily by resistance to inactivation for 1 s at a holding potential of -40 mV. IK activates at potentials more positive than -60 to -70 mV and consists of both low-threshold and high-threshold components. IK is partially blocked by both tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which appear to be blocking the same component. Long depolarizing pulses result in incomplete inactivation of IK; the inactivating component is inhibited by TEA. Sucrose density gradient fractionation partially resolves the RNA encoding the several components of IK; most IK arises from size classes between 3.8 and 9.5 kb. The study gives further evidence for the existence of numerous distinct RNA populations that encode brain K+ channels different from previously reported cloned K+ channels that have been expressed in Xenopus oocytes.     

Low- and high-threshold calcium current in the membrane of cultured neurons of Helix pomatia

Two components of the inward calcium current were observed in the membrane of cultured Helix pomatia neurons by the patch clamp method in its whole cell configuration. The first one was activated at high-negative membrane potentials (-70- -60 mV), had low amplitude (200 pA at 15 mmol/l of Ca2+ ions as a charge carrier) and displayed time-dependent inactivation. A second, larger current component (2 nA, the same conditions) appeared at more positive potentials (-20 mV). Its time-dependent inactivation was much less expressed. The tail currents of the low amplitude component were slower than those associated with the high-amplitude component.     

Ionic currents determining the membrane characteristics of type I spiral ganglion neurons of the guinea pig

Enzymatically isolated type I spiral ganglion neurons of the guinea pig have been investigated in the present study. The identity of the cells was confirmed by using anti-neuron-specific enolase immunostaining. The presence and shredding of the myelin sheath was also documented by employing anti-S100 immunoreaction. The membrane characteristics of the cells were studied by using the whole-cell patch-clamp technique. The whole-cell capacitance of the cells was 9 +/- 2 pF (n = 51), while the resting membrane potential of the cells was -62 +/- 9 mV (n = 19). When suprathreshold depolarizing stimuli were applied, the neurons fired a single action potential at the beginning of the stimulation. It was confirmed in this study that type I spiral ganglion cells possess a hyperpolarization-activated nonspecific cationic current (Ih). The major characteristics of this current component were unaffected by the enzyme treatment. Type I spiral ganglion cells also expressed various depolarization-activated K+ current components. A high-threshold outward current was sensitive to 1-10 mm TEA+ application. The ganglion cells also expressed a relatively small, but nevertheless present, transient outward current component which was less sensitive to TEA+ but could be inhibited by 100 micro m 4-aminopyridine. A DTX-I-sensitive current was responsible for some 30% of the total outward current (at 0 mV), showed rapid activation at membrane potentials positive to -50 mV and demonstrated very little inactivation. However, inhibition of the highly 4-AP- or DTX-I-sensitive component did not alter the rapidly inactivating nature of the firing pattern of the cells.     

Three types of voltage-dependent calcium current in cultured rat hippocampal neurons

Voltage-dependent calcium (Ca2+) currents in cultured rat hippocampal neurons were studied with the whole-cell recording mode of the patch-clamp technique. On the basis of the voltage-dependence of activation, kinetics of inactivation and pharmacology, 3 types of Ca2+ currents were distinguished. The low-threshold Ca2+ current (Il) was activated at -60 mV, and completely inactivated during a 100-ms depolarization to -40 mV (time constant: tau = 16 +/- 1 ms). The high-threshold currents (Ih), which were activated at -20 mV, could be separated into two types. The high-threshold, fast inactivating current (Ih,f) decayed quickly during a maintained depolarization (tau = 33 +/- 3 ms at 0 mV), whereas the high-threshold, slowly inactivating current (Ih,s) decayed with a much slower time constant (tau = 505 +/- 42 ms at 0 mV). The inactivations of Ih,f and Ih,s exhibited different time- and voltage-dependencies. Nickel ions (Ni2+, 25 microM) markedly suppressed Il, but little affected Ih. Cadmium ions (Cd2+, 10 microM) almost completely suppressed Ih, but left a small amount of Il. Lanthanum ions (La3+, 10 microM) almost completely suppressed both Il and Ih. Ih,s was sensitive to block by the dihydropyridine antagonist nicardipine (10 microM).     

Inactivation of calcium channels in rat pituitary intermediate lobe cells

The voltage-dependent inactivation of Ca currents was explored in dissociated intermediate lobe (IL) cells from the rat pituitary. On the basis of current-voltage relations two main inward currents could be identified in this cell, a transient current, (I-t), and a sustained current, (I-s). Inactivation was explored either by changing the holding potential and testing the change in the inward currents during a brief test pulse, or, by depolarizing the membrane and following the decay of the evoked inward current. Three current decay rates were identified, each with a characteristic dependence on membrane potential. The fastest decay rate (tau 1), was attributed to the inactivation of the I-t current and had a value of 57 ms at -40 mV, decreasing to 10 ms at -10 mV (extrapolated value of 6 ms at 0 mV). The other two decay rates, tau 2 and tau 3, decreased monotonically with depolarization of the membrane potential and reflected the inactivation of the I-s current with values of 1.8 and 20 s at 0 mV. I-s inactivation and reactivation was found to occur even in the normal resting potential range of this cell. These properties of the calcium channels can explain the voltage-dependent inactivation of secretion that has been observed previously in this and other secretory cells. In addition, they suggest that calcium currents, and hence secretion, may be modulated by external factors that cause small, but sustained, changes in the resting potential of the IL cell.     

Maitotoxin-induced membrane current in neuroblastoma cells

Maitotoxin (MTX) is a potent marine toxin isolated from the toxic dinoflagellate, Gambierdiscus toxicus. We have examined the possibility of MTX activating calcium channels using cultured neuroblastoma cells (N1E-115). MTX (10 ng/ml) produced a depolarization of the membrane, which was prevented by the removal of Ca2+ from the external medium. Under voltage clamp conditions, membrane currents were recorded with 50 mM Ba2+ as a charge carrier through calcium channels. After application of MTX (1 ng/ml), an inward current necessary to hold the membrane at -90 mV increased progressively. This was followed by a gradual decrease of the transient inward Ba2+ current through type I calcium channels recorded at -30 mV which was eventually abolished. A similar tendency was observed in the long-lasting inward Ba2+ current through type II calcium channels, which was recorded at +10 mV. The MTX action was antagonized by calcium channel blockers such as verapamil (100 microM) and La3+ (1 mM). A high concentration of verapamil (500 microM) blocked both types of calcium channels persistently. After washout of verapamil but while the calcium channels were still blocked, MTX (1 ng/ml) induced a steady-state current. The MTX-induced current showed an inward-rectifying property with a reversal potential of approximately -30 mV. The results suggest that the MTX-induced current does not flow through calcium channels. Thus, MTX may create a pore in the membrane with pharmacological properties similar to those of calcium channels.     

Two components of calcium channel current in embryonic chick skeletal muscle cells developing in culture

The properties of the Ca channel currents in chick skeletal muscle cells (myoballs) in culture were studied using a suction pipette technique which allows internal perfusion and voltage clamp. The Ca channel currents as carried by Ba ions were recorded, after suppression of currents through ordinary Na, K and Cl channels by absence of Na, K and Cl ions, by external TEA, by internal EGTA and by observing the Ba currents instead of the Ca currents. Two components of Ba current could be distinguished. One was present only if the myoballs were held at relatively negative holding potentials below -50 mV. This component first became detectable at clamp potentials of about -50 mV and reached a maximum between -10 and -20 mV. During long clamp steps, it became inactivated completely. The inactivation process of this component at a clamp potential of -30 mV was well fitted to a single exponential with a time constant of about -20 ms. Half-maximal steady-state inactivation was observed at -63 mV. The other component persisted even at relatively positive holding potentials above -40 mV, was observed during clamp pulses to -20 mV and above, and reached a maximum between +10 and +20 mV. This component inactivated very little; a substantial fraction of this component remained at the end of clamp pulses lasting 1 s. The inactivation process of this component at a clamp potential of -10 mV apparently followed a single exponential with a time constant of about 1 s. Half-maximal steady-state inactivation was attained at -33 mV. Both components of Ba current were blocked by Co ions, but organic Ca channel blocker D600 preferentially blocked the high-threshold, slowly inactivating component. The relationship between the current amplitude and the concentration of the external Ba ions was different between the two components. Furthermore, the two components of Ba current also differed in their developmental profile. These findings demonstrate the existence of two distinct types of Ca channels in the early stages of chick muscle cell development.     

Changes in the ionic mechanisms of the electro-excitability of the somatic membrane of rat sensory neurons during ontogenesis. Density ratios of inward currents

Correlations between densities of different types of inward currents in the somatic membrane of dorsal root ganglion neurons were studied in three age groups of rats (5-9 days, 45 days and 90 days postnatally). A linear dependence between the densities of high-threshold calcium and slow sodium currents was found. No correlation was observed between the densities of different inward currents in neurons with low-threshold calcium inward current. An inverse dependence was observed between the densities of transmembrane currents in cells having only two types of channels ("fast" sodium and high-threshold calcium ones). Neurons exhibiting slow TTX-resistant sodium and/or low-threshold calcium channels did not show inverse dependence between the densities of "fast" sodium and high-threshold calcium currents.     

Changes in the ionic mechanisms of the electro-excitability of the somatic membrane of rat sensory neurons during ontogenesis. Distribution of ion channels of the inward current

Distribution of different types of ionic channels carrying inward currents was studied in the somatic membrane of rat dorsal root ganglion neurons within three age groups: 5-9 days, 45 days and 90 postnatally. The number of neurons whose membrane contained simultaneously four types of inward current channels ("fast" tetrodotoxin-sensitive and "slow" tetrodotoxin-resistant sodium low- and high-threshold calcium one's) was progressively reduced in successive groups. The first group contained 14.5%, the second 5% and the third group 1% of such neurons. These changes were due to disappearance of "slow" sodium and low-threshold calcium channels from the membrane; the number of neurons whose somatic membrane contained only two types of inward current channels ("fast" sodium and high-threshold calcium) has increased, respectively.     

Properties of endogenous voltage-dependent sodium currents in Xenopus laevis oocytes.

Endogenous voltage-dependent sodium currents were recorded using standard 2-microelectrode techniques in Xenopus laevis oocytes. Maximal inward current occurred at -10 mV with an average amplitude of -279 +/- 17 nA and steady-state inactivation was half-maximal at a voltage of -38 +/- 0.5 mV. Currents were blocked by low concentrations of tetrodotoxin (TTX) with an IC50 value of 6 nM. These properties make the endogenous sodium current in Xenopus oocytes similar to sodium currents expressed following injection of mammalian brain RNA. While endogenous sodium channels have the potential to complicate analysis when using the oocyte expression system, they are only present at significant levels in rare batches of oocytes (less than 5%). Our results do stress the need, however, to reproduce results from exogenous expression studies across several batches of oocytes from different donors.     

The functional properties of the human ether-à-go-go-like (HELK2) K+ channel

The voltage-dependent K+ channels belonging to the ether-à-go-go family (eag, erg, elk) are widely expressed in the mammalian CNS. Their neuronal function, however, is poorly understood. Among the elk clones, elk2 is the most abundantly expressed in the brain. We have characterized the human ELK2 channel (HELK2) expressed in mammalian cell lines. Moreover, we have detected helk2 mRNA and ELK2-like currents in freshly dissociated human astrocytoma cells. HELK2 was inhibited by Cs+ in a voltage-dependent way (Kd was 0.7 mm, at -120 mV). It was not affected by Way 123398 (5 micro m), dofetilide (10 micro m), quinidine (10 micro m), verapamil (20 micro m), haloperidol (2 micro m), astemizole (1 micro m), terfenadine (1 micro m) and hydroxyzine (30 micro m), compounds known to inhibit the biophysically related HERG channel. The crossover of the activation and inactivation curves produced a steady state 'window' current with a peak around -20 mV and considerably broader than it usually is in voltage-dependent channels, including HERG. Similar features were observed in the ELK2 clone from rat, in the same experimental conditions. Thus, ELK2 channels are active within a wide range of membrane potentials, both sub- and suprathreshold. Moreover, the kinetics of channel deactivation and removal of inactivation was about one order of magnitude quicker in HELK2, compared to HERG. Overall, these properties suggest that ELK2 channels are very effective at dampening the neuronal excitability, but less so at producing adaptation of action potential firing frequency. In addition, we suggest experimental ways to recognize HELK2 currents in vivo and raise the issue of the possible function of these channels in astrocytoma.