Calcium currents in squid giant axon.

Voltage-clamp experiments were carried out on intracellularly perfused squid giant axons in a Na-free solution of 100 mM CaCl2+sucrose. The internal solution was 25 mM CsF+sucrose or 100 mM RbF+50mM tetraethylammonium chloride+sucrose. Depolarizing voltage clamp steps produced small inward currents; at large depolarizations the inward current reversed into an outward current. Tetrodotoxin completely blocked the inward current and part of the outward current. No inward current was seen with 100 mM MgCl2+sucrose as internal solution. It is concluded that the inward current is carried by Ca ions moving through the sodium channel. The reversal potential of the tetrodotoxin-sensitive current was +54mV with 25 mM CsF+sucrose inside and +10 mV with 100 mM RbF+50 mM tetraethylammonium chloride+sucrose inside. From the reversal potentials measured with varying external and internal solutions the relative permeabilities of the sodium channel for Ca, Cs and Na were calculated by means of the constant field equations. The results of the voltage-clamp experiments are compared with measurements of the Ca entry in intact axons.     

Transmembrane outward hydrogen current in intracellularly perfused neurones of the snail Helix pomatia

The ionic nature and pharmacological properties of the outward current activated by membrane depolarization were studied on isolated neurones of the snail Helix pomatia, placed in Na+- and Ca2+-free extracellular solutions and intracellularly perfused with K+-free solution ("nonspecific outward current"). It was shown that the amplitude and reversal potential of this current (estimated from instantaneous current-voltage characteristics) are determined mainly by the transmembrane gradient for H+ ions. Lowering of pHi induced an increase in the current amplitude and a shift of the reversal potential to more negative values; the shift magnitude was comparable with that predicted for the hydrogen electrode. Raising pHi, as well as lowering pHo, induced a decrease in the current amplitude and a displacement of the current activation curve to more positive potentials. Addition of EGTA (8 mmol/l) to the intracellular perfusate did not affect the current amplitude. Extracellular 4-aminopyridine (10 mmol/l), verapamil (0.25 mmol/l) or Cd2+ (0.5 mmol/l) blocked the current. It is concluded that the current studied is carried mainly by H+ ions. In the same neurones the nature of the fast decay of the calcium inward current was also studied (in the presence of extracellular Ca2+ ions). This decay considerably slowed when pHi was raised or pHo was lowered, and it became less pronounced upon extracellular application of 4-aminopyridine or upon intracellular introduction of phenobarbital (4 mmol/l) and tolbutamide (3 mmol/l). It is suggested that the fast decay of the calcium inward current is due to activation of a Ca-sensitive component of the hydrogen current which depends on accumulation of Ca2+ ions. The possible physiological role of the transmembrane hydrogen currents is discussed.     

Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane properties of isolated mudpuppy taste cells

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.     

Role of Na-Ca exchange in the action potential changes caused by drive in cardiac myocytes exposed to different Ca2+ loads.

The role of Na-Ca exchange in the membrane potential changes caused by repetitive activity ("drive") was studied in guinea pig single ventricular myocytes exposed to different [Ca2+]o. The following results were obtained. (i) In 5.4 mM [Ca2+]o, the action potentials (APs) gradually shortened during drive, and the outward current during a train of depolarizing voltage clamp steps gradually increased. (ii) The APs shortened more and were followed by a decaying voltage tail during drive in the presence of 5 mM caffeine; the outward current became larger and there was an inward tail current on repolarization during a train of depolarizing steps. (iii) These effects outlasted drive so that immediately after a train of APs, currents were already bigger and, after a train of steps, APs were already shorter. (iv) In 0.54 mM [Ca2+]o, the above effects were much smaller. (v) In high [Ca2+]o APs were shorter and outward currents larger than in low [Ca2+]o. (vi) In 10.8 mM [Ca2+]o, both outward and inward currents during long steps were exaggerated by prior drive, even with steps (+80 and +120 mV) at which there was no apparent inward current identifiable as I(Ca). (vii) In 0.54 mM [Ca2+]o, the time-dependent outward current was small and prior drive slightly increased it. (viii) During long steps, caffeine markedly increased outward and inward tail currents, and these effects were greatly decreased by low [Ca2+]o. (ix) After drive in the presence of caffeine, Ni2+ decreased the outward and inward tail currents. It is concluded that in the presence of high [Ca2+]o drive activates outward and inward Na-Ca exchange currents. During drive, the outward current participates in the plateau shortening and the inward tail current in the voltage tail after the action potential.     

Electrical properties and transmembrane ion currents of single smooth-muscle cells

Current and voltage clamp investigations of freshly isolated smooth muscle cells from guinea-pig ileum and taenia coli were performed using single suction micropipette technique. Specific membrane capacity of smooth muscle cells was calculated and accounted for 1.6 microF/cm2, with specific resistance varying from 50 to 150 k omega X cm2. Transmembrane currents consisted of two inward components, inactivating and noninactivating ones, carried by Ca2+ ions, overlapping with early activated potassium outward current. Time constant of inward current activation was not only voltage-sensitive but also ion-dependent. When Ca2+ ions in Krebs solution were replaced by Ba2+, both the rate of activation and inactivation of inward current were significantly reduced. Estimation of intracellular Ca2+ concentration increase has indicated that inward calcium current transports enough Ca2+ for direct contraction activation.     

Inactivation of calcium current in the somatic membrane of snail neurons

The decline of calcium inward currents evoked by a long-lasting membrane depolarization was studied on isolated snail neurons internally perfused with a K+-free solution. Two exponential components superimposed on a steady inward current could be distinguished, a slow decline with a time constant of several hundreds of milliseconds, observed at all the testing potentials used, and a fast one with a time constant of several dozens of milliseconds, which appeared at depolarizations to about -10 mV and above. When the calcium current was blocked by extracellular Cd2+ or verapamil, an outward current could be recorded at the same depolarizations. Subtraction of the latter current from the total current, recorded prior to the blockage, largely reduced the fast component of the decline of the total current. An increase in pHi from 7.3 to 8.1 led to the elimination of both the outward current and the fast component of the calcium current decline. The slow component remained practically unchanged, with its rate depending upon the current amplitude. It was slowed following intracellular administration of EDTA, and after equimolar substitution of Ba2+ for Ca2+. It is concluded that the fast component of the calcium inward current decline is mainly due to the superposition of the outward current produced by low selective channels. Only the slow component represents an actual decline of the inward current through calcium channels; it is due to ion accumulation at the inner surface of the cell membrane.     

Conversion of beating to bursting pacemaker activity: Action of quinidine

External quinidine converts the pacemaker neurone L-11, found in the Aplysia abdominal ganglion, from spontaneously "beating" to "bursting" discharge activity. Quinidine-induced bursting ceased when entry of Ca2+ ions into the cells was blocked in a Ca2+-free, Co2+-containing solution or if internal Ca2+ accumulation was prevented by the injection of EGTA. The analysis of membrane currents from voltage clamp experiments showed that quinidine blocks the Ca2+ inward current in a dose- and time-dependent manner. In addition, the currents were displaced to the left on the voltage axis, causing an increase of the inward current at negative membrane potentials. External quinidine suppresses the Ca2+-activated K+ current induced by intracellular Ca2+ injections and acts to prolong its decay phase. The slowing of the decay phase of the Ca2+-activated K+ current by quinidine was prevented after intracellular injection of EGTA, indicating that Ca2+ removal is impaired by the drug. It is suggested that the increase of Ca2+ inward current at negative potentials and the prolonged activation of the Ca2+-activated K+ current play a major role in causing the bursting discharge behavior in normally beating cells.     

Nonselective cationic currents activated by acetylcholine in swine tracheal smooth muscle cells

Membrane currents in isolated swine tracheal smooth muscle cells were investigated using a pipette solution containing BAPTA-Ca2+ buffer and Cs+ as the major cation. With a pipette solution containing 100 nM free Ca2+, acetylcholine (ACh; 1-100 microM), in a concentration-dependent manner, activated a current without inducing shortening of cells, although neither 1 mM histamine nor 1 microM leukotriene D4 activated the current (n = 7, n is the number of cells). The effect of 100 microM ACh was suppressed by pretreatment with 100 microM atropine (n = 6) or intracellular application of preactivated pertussis toxin at a concentration of 0.1 microg x mL(-1) (n = 8). Genistein (0.1-100 microM), in a concentration-dependent manner, suppressed the activation of the inward current by 100 microM ACh, whereas it did not significantly suppress that of the outward current (n = 6-8). With a pipette solution containing 50 nM free Ca2+, outward current, but not inward current, was activated by 100 microM ACh (n = 10). When the pipette solution had free Ca2+ concentrations greater than 50 nM, the inward current together with the outward current was activated. The ratio between the amplitude of the inward and outward currents was significantly increased as the free Ca2+ concentration in the pipette solution increased. The steady-state activation curve of the ACh-activated current with the 50 nM free Ca2+ pipette solution was fitted by a single Boltzmann distribution (Vh = +69.8 mV, k = -11.9 mV, n = 10). The activation time constant became smaller as the membrane potential was more depolarized (164.3+/-5.9 ms at +40 mV to 92.4+/-6.3 ms at +120 mV, n = 10). The reversal potential was not significantly changed by reducing extracellular Cl- concentration to one-tenth of the control (n = 8), suggesting that the current is a nonselective cationic current. These results suggest that ACh activates an outward nonselective cationic current via pertussis toxin-sensitive G-protein(s) coupled with muscarinic receptors. Involvement of genistein-sensitive tyrosine kinase in the activation process of the current is unlikely.     

Voltage-dependent conductances in Limulus ventral photoreceptors

The voltage-dependent conductances of Limulus ventral photoreceptors have been investigated using a voltage-clamp technique. Depolarization in the dark induces inward and outward currents. The inward current is reduced by removing Na+ or Ca2+ and is abolished by removing both ions. These results suggest that both Na+ and Ca2+ carry voltage-dependent inward current. Inward current is insensitive to tetrodotoxin but is blocked by external Ni2+. The outward current has a large transient component that is followed by a smaller maintained component. Intracellular tetraethylammonium preferentially reduces the maintained component, and extracellular 4-amino pyridine preferentially reduces the transient component. Neither component is strongly affected by removal of extracellular Ca2+ or by intracellular injection of EGTA. It is concluded that the photoreceptors contain at least three separate voltage-dependent conductances: 1) a conductance giving rise to inward currents; 2) a delayed rectifier giving rise to maintained outward K+ current; and 3) a rapidly inactivating K+ conductance similar to the A current of molluscan neurons.     

Effects of ionomycin and thapsigargin on ion currents in oocytes of Bufo arenarum

In this study, two electrode voltage clamp technique was used to assess the ionic current of oocytes of the South American toad Bufo arenarum and to study the dependence of these currents on the extracellular and intracellular Ca2+ concentrations. Ca2+ chelators, ionomycin -a calcium ionophore- and thapsigargin, a blocker of the Ca2+ pump of the sarcoplasmic reticulum, were used. The main results were the following: Most oocytes showed a voltage activated rectifying conductance. Ionomycin (1 microM) increased inward and outward currents in control solution. The effect of ionomycin was blocked partially at negative potentials and was blocked completely at positive potentials in absence of extracellular Ca2+. When the oocytes were treated with thapsigargin (2 microM) or BAPTA-am, a membrane-permeant intracellular chelator in control solution (10 microM), ionomycin did not increased either inward nor outward currents. The conclusion of our experiments is that there are two sources of Ca2+ for activation of the current induced by ionomycin, the cytoplasmic stores and the extracellular space. We believe ionomycin directly translocates Ca2+ from the SER into the cytoplasm but not from the extracellular medium. Ca2+ entry probably occurs through store-operated-Ca-channels.     

A damped oscillation in the intramembranous charge movement and calcium release flux of frog skeletal muscle fibers

Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of frog skeletal muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium-induced and voltage-dependent inactivation of calcium channels in crab muscle fibres

Inactivation of Ca channels was examined in crab muscle fibres using the voltage-clamp method. A satisfactory suppression of outward currents was attempted by the use of K+ blocking agents: TEA, 4AP and Cs ions instead of K+ ions applied extracellularly. The inactivation of Ca current appeared as a bi-exponential process. The faster component had a mean value of the time constant of 50 ms while the second component inactivated at a tenfold slower rate. The extent of inactivation of the faster component increased as the Ca current itself increased in different experimental conditions. Inactivation decreased when ICa was reduced for large applied depolarizations. The time constant of the faster calcium component also depended on the calcium current. Thus the results suggested that Ca2+ entry leads to inactivation of one component of calcium current in crab muscle. Substitution of Ca2+ ions by Sr2+ or Ba2+ ruled out the hypothesis concerning an accumulation process which would explain the decrease of the inward current. The second slower component of Ca current was better described by a voltage-dependent mechanism and its rate was not modified in Ca2+ rich solution or when the inward current was carried by Sr2+ or Ba2+ ions. Thus in crab muscle fibres, inactivation is mediated by both calcium entry and a voltage-gated mechanism.     

Potentiated L-type Ca2+ channels rectify

Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type alpha1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-alpha1C) or repeat III (E3K-alpha1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-alpha1C and E3K-alpha1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (-)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-alpha1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-alpha1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels.     

A Fast Transient Outward Monovalent Current in Rat Saphenous Myocytes Passing Through Ca2+ Channels

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of -100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 +/- 0.8 ms and 23.9 +/- 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at -55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1-5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.     

An amiloride-sensitive and voltage-dependent Na+ channel in an HLA-DR-restricted human T cell clone

We investigated changes in voltage-gated Na+ currents and effects of extracellular Na+ on proliferation in HLA-DR-restricted human CD4+ alphabeta T cells after stimulation with a non-self antigenic peptide, M12p54-68. In the absence of antigenic peptide, neither single (n = 80) nor APC-contacted (n = 71) T cells showed voltage-gated inward currents recording with whole-cell patch-clamp techniques, even with Ca2+ and Na+ ions present in the perfusion solution. However, with the same recording conditions, 31% (26 of 84) of APC-contacted T cells stimulated with the antigenic peptide showed voltage-dependent inward currents that were elicited from -60 mV. The inward currents were not inhibited in extracellular Ca2+-free conditions or in the presence of 1 mM NiCl2. However, they were completely inhibited in extracellular Na+-free conditions, which were made by replacing Na+ with iso-osmotic N-methyl-d -glucamine or choline. The Na+ currents were insensitive to tetrodotoxin, a classical blocker of Na+ channels, but were dose-dependently inhibited by amiloride, a potassium-sparing pyrazine diuretic. Furthermore, the Ag-specific proliferative response of T cells was completely inhibited in Na+-free Tyrode's solution and was suppressed by amiloride in a dose-dependent manner. Our findings suggest that activation of amiloride-sensitive and voltage-gated Na+ channels would be an important step to allow an adequate influx of Na+ and maintain a sustained high Ca2+ level during T cell activation.     

Muscarinic activation of transient inward current and contraction in canine colon circular smooth muscle cells

Muscarinic receptor mediated membrane currents and contractions were studied in isolated canine colon circular smooth muscle cells. Carbachol (10(-5) M) evoked a slow transient inward current that was superimposed by a transient outward current at holding potentials greater than -50 mV. Carbachol contracted the cells by 70 +/- 2%. The effects of carbachol were blocked by atropine (10(-6) M), tetraethyl ammonium (20 mM), and BAPTA-AM (25 mM applied for 20 min). The inward current and contraction were not sensitive to diltiazem (10(-5) M), nitrendipine (3 x 10(-7) M), niflumic acid (10(-5) M), or N-phenylanthranilic acid (10(-4) M), but were gradually inhibited after repetitive stimulations in Ca2+ free solution. Ni2+ (2 mM) inhibited the inward current by 67 +/- 4%. The inward current reversed at +15 mV. The outward component could be selectively inhibited by iberiotoxin (20 nM) or by intracellular Cs+. Repeated stimulation in the presence of cyclopiazonic acid (CPA, 3 microM) inhibited the carbachol-induced outward current and partially inhibited contraction. CPA did not inhibit the inward current. In conclusion, muscarinic receptor stimulation evoked a CPA-sensitive calcium release that caused contraction and a CPA-insensitive transient inward current was activated that is primarily carried by Ca2+ ions and is sensitive to Ni2+.     

Extracellular ATP activates receptor-operated cation channels in mouse lacrimal acinar cells to promote calcium influx in the absence of phosphoinositide metabolism.

In exocrine acinar cells a variety of neurotransmitters (e.g. acetylcholine) stimulate phosphatidylinositol 4,5-bisphosphate hydrolysis elevating intracellular calcium to activate calcium-dependent membrane currents (outward K+ and inward Cl-). This study shows that in lacrimal acinar cells extracellular application of ATP is also associated with outward and inward current responses; these, however, are not the result of phosphoinositide metabolism. ATP directly activates receptor-operated cation channels which permit influx of Na+ and Ca+ (the inward current). The elevation in [Ca2+]i which results is sufficient to activate the outward K+ current. ATP thus promotes Ca+ influx in the absence of phosphoinositide metabolism.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polarity of isolated blastomeres from mouse morulae: detection of transcellular ion currents

Eight- to sixteen-cell stage mouse morulae were dissociated with Ca2+-free medium into blastomeres that were labeled with fluoresceinated-succinylated Con A (FS-Con A) to mark their apical-basal axes. The vibrating probe was then used to map their extracellular current patterns. The average current density around normal blastomeres approached the resolution of the probe system (0.2 microA/cm2) and was undetectable in the majority of blastomeres. Since the current density at the measuring point outside the cell is known to increase with cell size in other systems, enlarged blastomeres were created by fusing together blastomeres of 4-cell stage embryos in 45% polyethylene glycol. Enlarged blastomeres were then aggregated with normal blastomeres using phytohemagglutinin and cultured to the 8- to 16-cell stage to allow them to become polarized. Such aggregates were then dissociated with Ca2+-free medium to recover polarized, enlarged blastomeres. The enlarged blastomeres were 30-65 microns in diameter and 70% of them generated a detectable current; currents were detected around 83% of those blastomeres larger than 40 micron in diameter. The current pattern in these most reliable cases was predominantly inward apical (11/16 or 69%) and outward basal (15/16 or 94%), with lateral currents about three-fold smaller in amplitude than these apical-basal currents. Lateral currents were undetectable in 53% of the cases. Preliminary data suggest that the inward current is carried in part by Na+ influx and is independent of the Na+,K+-ATPase over the short term. Transcellular ion currents were detectable as long as 4 hr after dissociation, and the apical-basal current pattern was usually stable during that time. In contrast, the fluorescent cap of FS-Con A faded within 7-30 min at 35 degrees C but remained stable in 0.1% azide or 1.5 micrograms/ml cytochalasin D. The electrical polarity therefore persisted after the apical cap of Con A fluorescence was no longer visible. We propose that these transcellular ion currents may be involved in the establishment of blastomere polarity and describe a mechanism of action in an "ion current polarization" hypothesis.