Sodium and calcium currents in dispersed mammalian septal neurons

Voltage-gated Na+ and Ca2+ conductances of freshly dissociated septal neurons were studied in the whole-cell configuration of the patch-clamp technique. All cells exhibited a large Na+ current with characteristic fast activation and inactivation time courses. Half-time to peak current at -20 mV was 0.44 +/- 0.18 ms and maximal activation of Na+ conductance occurred at 0 mV or more positive membrane potentials. The average value was 91 +/- 32 nS (approximately 11 mS cm-2). At all membrane voltages inactivation was well fitted by a single exponential that had a time constant of 0.44 +/- 0.09 ms at 0 mV. Recovery from inactivation was complete in approximately 900 ms at -80 mV but in only 50 ms at -120 mV. The decay of Na+ tail currents had a single time constant that at -80 mV was faster than 100 microseconds. Depolarization of septal neurons also elicited a Ca2+ current that peaked in approximately 6-8 ms. Maximal peak Ca2+ current was obtained at 20 mV, and with 10 mM external Ca2+ the amplitude was 0.35 +/- 0.22 nA. During a maintained depolarization this current partially inactivated in the course of 200-300 ms. The Ca2+ current was due to the activity of two types of conductances with different deactivation kinetics. At -80 mV the closing time constants of slow (SD) and fast (FD) deactivating channels were, respectively, 1.99 +/- 0.2 and 0.11 +/- 0.03 ms (25 degrees C). The two kinds of channels also differed in their activation voltage, inactivation time course, slope of the conductance-voltage curve, and resistance to intracellular dialysis. The proportion of SD and FD channels varied from cell to cell, which may explain the differential electrophysiological responses of intracellularly recorded septal neurons.     

Action of calcium ions on channels of inward and outward currents in the molluscan neuron membrane

Changes in the characteristics of activity of sodium, calcium, and potassium channels in the surface membrane during variation of the calcium ion concentration in the extracellular and intracellular medium were investigated by the voltage clamp method during intracellular dialysis of isolated neurons of the mollusksLimnea stagnalis andHelix pomatia. Besides their direct role in passage of the current through the membrane, calcium ions were shown to have two actions, differing in their mechanism, on the functional properties of this membrane. The first was caused by the electrostatic action of calcium ions on the outer surface of the membrane and was manifested as a shift of the potential-dependent characteristics of the ion transport channels along the potential axis; the second is determined by closer interaction of calcium ions with the specific structures of the channels. During the action of calcium-chelating agents EGTA and EDTA on the inner side of the membrane the conductivity of the potassium channels is substantially reduced. With an increase in the intracellular free calcium concentration the conductivity is partially restored. The action of EGTA and EDTA on the outer side of the membrane causes a substantial decrease in the ion selectivity of the calcium channels and changes the kinetics of the portal mechanism. These changes are easily abolished by rinsing off the chelating agents or by returning calcium ions to the external medium. A specific blocking action of an increase in the intracellular free calcium concentration on conductivity of the calcium channels was found.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 1, pp. 69–77, January–February, 1977.     

Changes in cytosolic calcium monitored by inward currents during action potentials in guinea-pig ventricular cells

Action potentials were recorded from single cells isolated from guinea-pig ventricular muscle. Contraction was measured with an optical technique. Tail currents thought to be activated by cytosolic calcium were recorded when action potentials were interrupted by application of a voltage-clamp. A family of tail currents was recorded by interrupting the action potential at various times after the upstroke. The envelope of tail current amplitudes was taken as an index of changes in cytosolic calcium. Consistent with this interpretation, tail currents were negligible following intracellular loading with the calcium chelator BAPTA to suppress calcium transients. The cytosolic calcium transient estimated from the envelope of tails reached a peak approximately 50 ms after the upstroke of the action potential, and fell close to diastolic levels before repolarization was complete; 10 mM caffeine delayed the time to peak contraction, and caused a prolongation of the cytosolic calcium transient estimated from the envelope of tail currents. Caffeine also induced the appearance of a distinct late plateau phase of the action potential. Intracellular BAPTA suppressed the late plateau, contraction and tail currents in cells exposed to caffeine. Exposure to caffeine increased the time constant for decay of tail currents (from approximately 25 to 70 ms). When action potentials were greatly abbreviated by interruption with a voltage-clamp, a progressive decline occurred in the subsequent three contractions and tail currents. There was a progressive reversal of these effects over four responses when the full action potential duration was restored. None of these effects was observed in cells exposed to caffeine. Calcium-activated tail currents appear to be a useful qualitative index of changes in cytosolic calcium. The observations are consistent with the suggestion that cytosolic calcium is reduced during the plateau by a combination of calcium extrusion through Na-Ca exchange and calcium uptake into caffeine-sensitive stores. It also appears that reduction of stores loading during abbreviated action potentials reduces subsequent contraction in cells not exposed to caffeine.     

Sodium inward currents through calcium channels in mealworm muscle fibers

The contribution of Na⁺ ions to the nonsynaptic electrogenesis was studied in the larval muscle fibers of mealworm, Tenebrio molitor, using currentclamp and voltage-clamp techniques. Na-dependent graded responses were generated by depolarizing current stimuli in Ca²⁺-free solutions. These responses were insensitive to tetrodotoxin and were blocked by Co²⁺. Large inward-going currents were elicited by step depolarizations in Ca²⁺-free solutions under voltage-clamp conditions. The inward currents were totally eliminated by removal of Na⁺ from the bathing solution. These results indicate that the calcium channel of mealworm muscle is permeable to Na⁺.     

Synchronization of calcium channel activity in mollusk neurons after treatment with ferricyanide and barium ions

Using the patch-voltage-clamp method on the isolated membrane patches from molluscan neurons, effects of ferricyanide and barium on fast potassium channels with a priori destroyed synchronism in the transitions between conductance sublevels were studied. Ferricyanide (0.1-0.5 mM) applied at the inner membrane side produced irreversible transformation of occasional transitions of the channel conductance between intermediate states into highly cooperative and potential-dependent process. Barium ions completely or partly reversibly restore synchronism.     

Long-lasting inward current in snail neurons in barium solutions in voltage-clamp conditions

Summary The inward membrane current was recorded under voltage clamp from nonbursting neurons of the snailHelix pomatia in Na-free solutions containing Ba ions but no other divalent cations. The inward current was separated into two components: (i) an early fast inactivating component and (ii) a smaller long-lasting component. Both components were dependent on the external Ba concentration. It is concluded that both components of the inward current are carried by Ba ions. The activation of the early fast inactivating component of the inward current occurred at more positive membrane potential than that of the long-lasting component. The shape of the inactivation curve for the peak value of the inward current was similar to that for the long-lasting component. The potentials of half-inactivation for the peak value of the inward current and for its long-lasting component were –28 and –22 mV, respectively. The blocking effect of Co⁺⁺ on the early fast inactivating component was substantially greater. In some neurons after treatment with 15mm Co⁺⁺ only the long-lasting component was recorded. The activation kinetics of the long-lasting component of the inward current were analyzed using the Hodgkin-Huxley equations. The results could be explained by assuming that two components of the inward current in Na–Ca-free solution with Ba ions flowed through the two different channels. The significance of the long-lasting inward current for the normal spike generation is discussed.     

Membrane currents induced by inflow of sodium ions into mollusk giant neurons

Membrane currents induced by inflow of sodium ions were investigated in giant neurons of the molluskHelix pomatia during tetanic stimulation or prolonged membrane depolarization under voltage clamp conditions. The membrane current thus produced consists of two components, a fast component with a reversal potential close to the potassium equilibrium potential, and a slow component only slightly dependent on membrane potential in the region from −50 to −90 mV. Addition of strophanthin K to the external solution, or replacement of sodium in the external solution by lithium or calcium abolished the slow component of the membrane current and reduced the fast component. It is concluded that the slow component appears as the result of activation of the sodium pump under electrogenic conditions, where-as the fast component arises as the result of an increase in potassium permeability, possibly coupled with intensive activity of this pump.     

Effect of calcium ions on outward currents in the somatic membrane of mollusk giant neurons

Potassium currents through the somatic membrane of giant neurons ofHelix pomatia in normal (10 mM Ca) Ringer's solution and low-calcium (1 mM Ca) solution were studied by the voltage clamp method. With a decrease in the Ca concentration to 1 mM peak potassium conductance versus membrane, potential curves and inactivation curves were shifted along the voltage axis in the negative direction by about 10 mV. Inactivation of the delayed potassium current was slowed in low Ca solution. The effect of a decrease in external calcium concentration on volt-ampere and inactivation characteristics increased with a rise in external pH. These effects of a low Ca concentration on potassium mechanisms of the giant neuron somatic membrane can be attributed to changes in the negative surface potential in the region of the potassium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Biology, Hungarian Academy of Sciences, Tihany. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 400–409, July–August, 1976.     

Effects of strontium and barium ions on calcium bindings and transport in nerve cells

Absorption of strontium and barium ions by intracellular organelles after loading the cell with these cations together with their effects on Ca release from the intracellular stores were investigated in neurons isolated fromHelix pomatia using fura-2, a Ca-sensitive fluorescent probe. It was found that strontium ions can successively replace intracellular calcium ions in this response, whereas barium ions are not absorbed by the cell; they block calcium channels of the intracellular stores as well as at the surface membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 820–825, November–December, 1989.     

Noise of secretagogue-induced inward currents dependent on extracellular calcium in rat mast cells

We analyzed the noise of the inward currents induced by stimulation of rat peritoneal mast cells with compound 48/80 (48/80), a secretagogue, and examined the role of extracellular Ca2+ in generation of the large noise. In the presence of 2 mM Ca2+ in the external solution, the power density spectra of the 48/80-induced inward currents in most cells were fitted with the sum of two Lorentzian functions. The cut-off frequencies (fc) at -50 mV for the low and high frequency components were 16.3 +/- 7.3 (n = 10) and 180 +/- 95 (n = 9) Hz. Involvement of a cation-selective channel in the large noise was identified in some cells, but the single channel current amplitude estimated from parameters of the noise varied among cells (0.20-2.47 pA at -50 mV), thereby indicating that the currents were mediated by more than two classes of channel. The low frequency component of the 48/80-induced currents was suppressed by lowering the extracellular Ca2+ concentration to 1 microM with the addition of EGTA, without appreciable changes in the high frequency component. When the extracellular Ca2+ was reduced to 1 microM by EGTA 1 min prior to stimulation, 48/80 induced little or no currents in most cells and small currents in some cells. The power density spectra of the small currents were fitted mainly by a single Lorentzian curve with an fc of 150 +/- 5.8 Hz (n = 3). Re-admission of 1.3 mM Ca2+ produced a low frequency part of current noise with an fc of 18.8 (n = 2) Hz.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cadmium ions modulate GABA induced currents in molluscan neurons

The effect of Cd2+, as one of the most widespread toxic environmental pollutants, was studied on gamma-aminobutyric acid (GABA) evoked responses of identified neurons in the central nervous system of the pond snail, LYmnaea stagnalis L. (Gastropoda). In the experiments, the modulation of the action of GABA both on neuronal activity (current clamp recording) and on the a GABA activated membrane Cl- current (voltage clamp studies) has been shown. It was found that: 1. GABA could evoked three different various types of response in GABA sensitive neurons: i) hyperpolarization with strong inhibition of ongoing spike activity, ii) short depolarization with an increase of spike the activity, iii) biphasic respone with a short excitation followed by a more prolonged long inhibition. 2. In low-Cl- solution the inhibitory action of GABA was reduced or eliminated, but the excitatory one was not or only moderately affected. 3. CdCl2 inhibited the GABA evoked hyperpolarization, but left intact or only slightly reduced the excitation evoked by GABA. 4. The inward Cl- current evoked by GABA at a -75 mV holding potential was slightly augmented in the presence of I micromol/l Cd2+, but was reduced or blocked at higher cadmium concentrations. The effect of Cd2+ was concentration and time dependent. 5. Parallel with reducing the GABA evoked current, cadmium increased both the time to peak and the half inactivation time of the current. 6. CdCl2 alone, in 50 micromol/l concentration, induced a 1-2 nA inward current. The blocking effect of cadmium on GABA activated inhibitory processes can be an important component of the neuro-toxic effects of this heavy metal ion.     

Quisqualate receptor-mediated depression of calcium currents in hippocampal neurons

The modulation of Ca2+ currents by the excitatory neurotransmitter glutamate and its analogs was investigated in hippocampal neurons in culture. In the presence of glutamate receptor-gated ion channel antagonists, all of the analogs tested caused either a small reversible depression or had no effect on the Ca2+ current. However, in neurons dialyzed with GTP gamma S, quisqualate and glutamate but not NMDA, kainate, AMPA, or L-APB caused marked and irreversible depressions of the Ca2+ current. This inhibition was only observed if Ca2+ was present in either the internal or external medium. Intracellular H-7, staurosporine, IP3, cAMP, cGMP, or calmodulin inhibitors failed to prevent the quisqualate-induced Ca2+ current inhibition. These observations are consistent with an interaction between a G protein-coupled glutamate receptor and Ca2+ channels.     

Inositol-trisphosphate-dependent calcium currents precede cation currents in insect olfactory receptor neurons in vitro

Specialized olfactory receptor neurons in insects respond to species-specific sex pheromones with transient rises in inositol trisphosphate and by opening pheromone-dependent cation channels. These channels resemble cation channels which are directly or indirectly Ca²⁺-dependent. But there appear to be no internal Ca²⁺ stores in the outer dendrite where the olfactory transduction cascade is thought to start. Hence, it remains to be determined whether an influx of external Ca²⁺ precedes pheromone-dependent cation currents. Patch clamp measurements in cultured olfactory receptor neurons from Manduca sexta reveal that a transient inward current precedes pheromone-dependent cation currents. A transient inositol trisphosphate-dependent Ca²⁺ current, also preceding cation currents with the characteristics of pheromone-dependent cation currents, shares properties with the transient pheromone-dependent current. These results match the biochemical measurements with the electrophysiological data obtained in insect olfactory receptor neurons.Abbreviations ORNs Olfactory receptor neurons - IP₃ Inositol-1,4,5-trisphosphate - I_t Transient pheromone-dependent current - I_ir Transient IP₃-dependent current     

Two calcium currents in the somatic membrane of mollusc neurons

Two new types of calcium channels were discovered during research in ionic currents in the somatic membrane ofHelix pomatia neurons, using an intracellular perfusion technique. Apart from the principal calcium current described in the literature with a holding potential of about –110 mV, an additional calcium current was observed activated at depolarizations of –40 to –80 mV and was not reduced when the cell was perfused with solutions containing fluoride anions. The kinetics of this current were well described in the context of the Hodgkin and Huxley model with a time constant of activation of 6–8 msec and of inactivation of 300–600 msec. It increased in amplitude as the Ca⁺⁺ rose in the cellular environment but was reduced by extracellular addition of the Ca⁺⁺ antagonists Co⁺⁺, Ni⁺⁺, and Cd⁺⁺, and the organic blockers nifedipine and verapamil. The association constants of these substances with corresponding channels determined from the maximum of the current-voltage relationship were 2 (Ca⁺⁺), 3 (Co⁺⁺), 0.06 (nifedipine), and 0.2 mM (verapamil). The properties detected in this component of calcium conductance are compared with those of calcium channels in other excitatory formations and its possible functional role is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 627–633, September–October, 1985.     

The influx of calcium ions into human erythrocytes during cold storage

1. When human erythrocytes, suspended in iso-osmotic sucrose containing CaCl(2), are stored at 3 degrees C, Ca(2+) influx into the cells occurs. Simultaneously, efflux of K(+), Na(+), Cl(-) and water takes place and cell volume diminishes. 2. The extent of Ca(2+) influx increases with duration of cold storage and with increasing concentration of Ca(2+) in the suspending medium. 3. Erythrocytes that have been thus loaded with Ca(2+) exhibit Ca(2+) efflux against a concentration gradient when subsequently incubated at 37 degrees C. 4. Ca(2+) influx likewise occurs when the sucrose of the medium is replaced by iso-osmotic solutions of other non-ionized compounds. 5. Replacement of sucrose by iso-osmotic KCl or NaCl greatly diminishes the rate of Ca(2+) influx during cold storage; however, in iso-osmotic choline chloride, Ca(2+) influx is as rapid as in sucrose. 6. Preincubation of erythrocytes in iso-osmotic sucrose at 37 degrees C causes rapid efflux of K(+) and Na(+) and renders the cell membranes highly permeable to Ca(2+) during subsequent cold storage. 7. Preincubation of erythrocytes in iso-osmotic NaCl at 37 degrees C with trypsin or neuraminidase is without effect on the permeability of the membrane towards Ca(2+). 8. The experimental results lead to the conclusion that the main prerequisite for Ca(2+) influx into erythrocytes is the partial depletion of the cells of their univalent cations.     

Effects of diadenosine polyphosphates on inward rectifier potassium currents in rat cardiomyocytes

Diadenosine polyphosphates are now considered a novel class of endogenous paracrine signal compounds. The putative role of these compounds in pathogenesis of myocardial infarction was proposed, since the concentration of diadenosine polyphosphates increases in the cardiac tissue following the ischemic lesion and myocardial necrosis. Therefore, possible effects of diadenosine polyphosphates on cardiac electrical activity and their ionic mechanisms are of considerable interest.     

Evidence for multiple calcium response mechanisms in mammalian olfactory receptor neurons

Olfactory receptor neurons employ a diversity of signaling mechanisms for transducing and encoding odorant information. The simultaneous activation of subsets of receptor neurons provides a complex pattern of activation in the olfactory bulb that allows for the rapid discrimination of odorant mixtures. While some transduction elements are conserved among many species, some species-specificity occurs in certain features that may relate to their particular physiology and ecological niche. However, studies of olfactory transduction have been limited to a relatively small number of vertebrate and invertebrate species. To better understand the diversity and evolution of olfactory transduction mechanisms, we studied stimulus-elicited calcium fluxes in olfactory neurons from a previously unstudied mammalian species, the domestic cat. Isolated cells from cat olfactory epithelium were stimulated with odorant mixtures and biochemical agents, and cell responses were measured with calcium imaging techniques. Odorants elicited either increases or decreases in intracellular calcium; odorant-induced calcium increases were mediated either by calcium fluxes through the cell membrane or by mobilization of intracellular stores. Individual cells could employ multiple signaling mechanisms to mediate responses to different odorants. The physiological features of these olfactory neurons suggest greater complexity than previously recognized in the role of peripheral neurons in encoding complex odor stimuli. The investigation of novel and unstudied species is important for understanding the mechanisms of odorant signaling that apply to the olfactory system in general and suggests both broadly conserved and species-specific evolutionary adaptations.     

Amino acid substitutions in the pore of the Ca(V)1.2 calcium channel reduce barium currents without affecting calcium currents

Ba(2+) currents through Ca(V)1.2 Ca(2+) channels are typically twice as large as Ca(2+) currents. Replacing Phe-1144 in the pore-loop of domain III with glycine and lysine, and Tyr-1152 with lysine, reduces whole-cell G(Ba)/G(Ca) from 2.2 (wild-type) to 0.95, 1.21, and 0.90, respectively. Whole-cell and single-channel measurements indicate that reductions in G(Ba)/G(Ca) result specifically from a decrease in Ba(2+) conductance and not changes in V(h) or P(O). Half-maximal block of I(Li) is increased by 3.2-, 3.8-, and 1.6-fold in Ca(2+), and 3.8-, 4.2-, and 1.8-fold in Ba(2+) for F1144G, Y1152K, and F1144K, respectively. High affinity interactions of individual divalent cations to the pore are not important for determining G(Ba)/G(Ca), because the fold increases in IC(50) values for Ba(2+) and Ca(2+) are similar. On the contrary, conductance-concentration curves indicate that G(Ba)/G(Ca) is reduced because the interactions of multiple Ba(2+) ions in the mutant pores are altered. The complexity of these interactions is exemplified by the anomalous mole fraction effect, which is flattened for F1144G and FY/GK but accentuated for F1144K. In summary, the physicochemical properties of the amino acid residues at positions 1144 and 1152 are crucial to the pore's ability to distinguish between multiple Ba(2+) ions and Ca(2+) ions.     

Effects of cAMP on calcium currents in mollusk neurons differing in the sensitivity of their calcium conductance to serotonin action

The effects of cAMP and serotonin (5-HT) on calcium current (I_Ca) were investigated inHelix pomatia neurons using voltage clamp and intracellular perfusion techniques. Three types of neuronal response to extracellular application of 5-HT (1–10 µM) were found: reversible blockage of calcium conductance, absence of response, and increase in I_Ca amplitude. Intracellular application of exogenous cAMP was also found to produce an increase in I_Ca in cells stimulated by 5-HT action. Effects of 5-HT and cAMP were non-additive under these circumstances and were potentiated equally by cyclic nucleotide phosphodiesterase inhibitor. Applying cAMP led to no noticeable increase in I_Ca amplitude in cells with calcium conductance unchanged or blocked by 5-HT. Findings would indicate that the stimulating action of 5-HT is mediated by a rise in intracellular level of cAMP. It is postulated that two types of calcium channels differing in their dependence on cAMP metabolism exist; the presence of cAMP-dependent calcium channels at the neuronal membrane fits in with a certain type of 5-HT receptor also present in the cell, moreover. A new approach is suggested for research on isolated neurons, i.e., that of functional identification.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 605–512, October–September, 1990.