The effect of leu-enkephalin on membrane potential and activity of the rat respiratory center neurons in vitro

In frontal brainstem slices of Wistar rats, the whole-cell patch-clamp recordings showed the effect of opioid peptide leu-enkephalin (10 nM-1 microM) on membrane potential and spontaneous activity pattern of neurons in two divisions of the respiratory center, ventro-lateral area of the solitary tract nucleus, and the pre-B?tzinger complex. Leu-enkephalin induced a membrane hyperpolarization of the respiratory centre neurons and reduction of the spike activity level in spontaneously active units. After administration of leu-enkephalin, a decrease in frequency of bursts was found in bursting cells of the pro-B?tzinger complex; in two cases, there was a transition of bursting activity to tonic one. The data suggest that the mechanism of the central respiratory activity of leu-enkephalin is based on its direct action at the level of membrane of the respiratory centre neurons.     

Thyroliberin blocks potassium A-current in neurons of the respiratory center of adult rats in vitro

TRH is a well-known respiratory active neuropeptide. To study neuronal mechanisms of its activity, we have tested the effects of TRH on the potassium A-current in neurons of the ventrolateral solitary tract nucleus and pre-Botzinger complex in voltage-clamp experiments on adult rat brain slices. A-current was present in the neurons and it was partially and reversibly blocked by administration of THR (10(-8) M) to the bath solution. The significant decrease in amplitude of A-current was accompanied by the increase in inactivation constant (t). The effect of TRH on A-current amplitude was simulated by 5 mM 4-aminopyridine. The results presented here indicate that the stimulatory effects of TRH on neurons of the respiratory centre can be at least partially explained by its ability to block the potassium A-current.     

Changes in voltage-dependent currents and membrane area during maturation of starfish oocytes: species differences and similarities

Full grown starfish oocytes are arrested at meiotic prophase I in the ovary. The natural hormone 1-methyladenine triggers oocyte maturation which involves meiosis reinitiation along with a variety of morphological, biochemical, and electrical changes. In studying oocytes of two species, Henricia leviuscula and Asterina miniata, using the voltage-clamp technique, we found interesting differences and similarities in the electrophysiological changes which occurred during maturation. Oocytes of both species have three major voltage-dependent currents: an inward Ca2+ current, an inwardly rectifying K+ current, and a transient outward K+ current (A-current). The Ca2+ current and the A-current were similar in the two species but the inward rectifier in Henricia had activation kinetics that were more than 10-fold slower than in Asterina. Nonetheless, all three currents were affected similarly during maturation: the inward Ca2+ currents remained constant in both species, while the two K+ currents decreased in amplitude. In Henricia the membrane surface area decreased substantially during maturation, while in Asterina it remained constant. This may be explained by the more highly infolded state of the membrane in the immature Henricia oocyte. The selective loss of K+ current followed the time course of the area decrease in Henricia, but the same percentage decrease in current occurred in Asterina without a net membrane loss.     

Ionic conductances in two types of sensory neurons in the leech, Macrobdella decora

1. Ion conductances were investigated in two kinds of leech sensory neurons (PM and N1 cells) which differ in their membrane excitability and action potential. 2. In the PM cell body excitable membrane behavior is dominated by only two currents, a sodium current (INa) and a delayed rectifier (IK). 3. In contrast, in the N1 cell INa and IK is supplemented with the presence of a transient potassium current, IA. 4. A comparison between INa and IK in the two cell types did not reveal any significant difference in activation and inactivation kinetics of either current between neurons. 5. Thus, the properties and presence of the A-current in the N1 cell and not in the PM cell may account for the functional difference in excitability of the two kinds of neurons.     

ANEPIII, a new recombinant neurotoxic polypeptide derived from scorpion peptide, inhibits delayed rectifier, but not A-type potassium currents in rat primary cultured hippocampal and cortical neurons

A new recombinant neurotoxic polypeptide ANEPIII (BmK ANEPIII) derived from Scorpion peptide, which was demonstrated with antineuroexcitation properties in animal models, was examined for its action on K+ currents in primary cultured rat hippocampal and cortical neurons using the patch clamp technique in the whole-cell configuration. The delayed rectifier K+ current (I(k)) was inhibited by externally applied recombinant BmK ANEPIII, while the transient A-current (I(A)) remained virtually unaffected. BmK ANEPIII 3 microM, reduced the delayed rectifier current by 28.2% and 23.6% in cultured rat hippocampal and cortical neurons, respectively. The concentration of half-maximal block was 155.1 nM for hippocampal neurons and 227.2 nM for cortical neurons, respectively. These results suggest that BmK ANEPIII affect K+ currents, which may lead to a reduction in neuronal excitability.     

Sodium Metabisulfite Modulation of Potassium Channels in Pain-Sensing Dorsal Root Ganglion Neurons

The effects of sodium metabisulfite (SMB), a general food preservative, on potassium currents in rat dorsal root ganglion (DRG) neurons were investigated using the whole-cell patch-clamp technique. SMB increased the amplitudes of both transient outward potassium currents and delayed rectifier potassium current in concentration- and voltage-dependent manner. The transient outward potassium currents (TOCs) include a fast inactivating (A-current or I _A) current and a slow inactivating (D-current or I _D) current. SMB majorly increased I_A, and I_D was little affected. SMB did not affect the activation process of transient outward currents (TOCs), but the inactivation curve of TOCs was shifted to more positive potentials. The inactivation time constants of TOCs were also increased by SMB. For delayed rectifier potassium current (I _K), SMB shifted the activation curve to hyperpolarizing direction. SMB differently affected TOCs and I _K, its effects major on A-type K⁺ channels, which play a role in adjusting pain sensitivity in response to peripheral redox conditions. SMB did not increase TOCs and I _K when adding DTT in pipette solution. These results suggested that SMB might oxidize potassium channels, which relate to adjusting pain sensitivity in pain-sensing DRG neurons.     

Effect of sotalol on transmembrane ionic currents responsible for repolarization in cardiac ventricular myocytes from rabbit and guinea pig

The effects of sotalol, a β-adrenoceptor blocker and class III antiarrhythmic agent, on transmembrane ionic currents were examined in single rabbit and guinea pig ventricular myocytes using whole-cell voltage-clamp techniques. In neither of these species did 60 μM sotalol appreciably effect the inward rectifier, the transient outward or the inward calcium currents. In addition, sotalol did not elicit a slowly inactivating component of the sodium current as did 1 μg/ml veratrine. In guinea pig ventricular myocytes, sotalol also significantly depressed the outward delayed rectifier current. An outward delayed rectifier current was not observed in rabbit ventricular myocytes examined at room temperature; and, under these conditions sotalol did not lengthen action potential duration. Sotalol induced lengthening of cardiac action potential duration can, therefore, be explained by depression the outward delayed rectifier current.     

External [K+] and the block of the K+ inward rectifier by external Cs+ in frog skeletal muscle.

Frog skeletal muscle has a K+ channel called the inward rectifier, which passes inward current more readily than outward current. Gay and Stanfield (1977) described a voltage-dependent block of inward K+ currents through the inward rectifier by external Cs+ in frog muscle. Here, frog single muscle fibers were voltage clamped using the vaseline-gap voltage-clamp technique to study the effect of external [K+] on the voltage-dependent block of inward K+ currents through the inward rectifier by external Cs+. The block of inward K+ currents through the channel by external Cs+ was found to depend on external [K+], such that increasing the external concentration of the permeant ion K+ potentiated the block produced by the impermeant external Cs+. These findings are not consistent with a one-ion channel model for the inward rectifier. The Eyring rate theory formalism for channels, viewed as single-file multi-ion pores (Hille and Schwarz, 1978), was used to develop a two-site multi-ion model for the inward rectifier. This model successfully reproduced the experimentally observed potentiation of the Cs+ block of the channel by external K+, thus lending further support to the view of the inward rectifier as a multi-ion channel.     

Acetylcholine reduces inward rectification on thymus-derived macrophage cells in culture

Cultures prepared from dissociated rat thymus were examined 1-2 weeks after plating. Macrophage cells were identified by their adherence, morphological appearance, and ability to phagocytize carbon particles or heat-inactivated Staphylococcus aureus. Whole cell current recordings from macrophage cells revealed an inward current at potentials more negative than the equilibrium potential for potassium and an outward current at potentials more positive than -40 mV in normal recording solution. Acetylcholine or muscarine caused a reduction in inward current but did not alter the outward current. The inward current and acetylcholine effect were seen at less negative potentials by decreasing the potassium equilibrium potential and both were blocked by the addition of cesium to the external recording solution. These results indicated that the inward current was mediated by potassium through the inward or anomalous rectifier. Physiologically, the action of acetylcholine on the inward rectifier of these macrophage cells may be mediated by cholinergic innervation of the thymus.     

Lysosome mediated Kir2.1 breakdown directly influences inward rectifier current density

The inward rectifier current generated by Kir2.1 ion channel proteins is primarily responsible for the stable resting membrane potential in various excitable cell types, like neurons and myocytes. Tight regulation of Kir2.1 functioning prevents premature action potential formation and ensures optimal repolarization times. While Kir2.1 forward trafficking has been addressed in a number of studies, its degradation pathways are thus far unknown. Using three different lysosomal inhibitors, NH₄Cl, chloroquine and leupeptin, we now demonstrate involvement of the lysosomal degradation pathway in Kir2.1 breakdown. Upon application of the inhibitors, increased steady state protein levels are detectable within few hours coinciding with intracellular granular Kir2.1 accumulation. Treatment for 24 h with either chloroquine or leupeptin results in increased plasmamembrane originating inward rectifier current densities, while current-voltage characteristics remain unaltered. We conclude that the lysosomal degradation pathway contributes to Kir2.1 mediated inward rectifier current regulation.     

Slow components of potassium tail currents in rat skeletal muscle

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.     

Elevated potassium shortens action potential duration by altering outward currents in chick dorsal root ganglia neurons

Dissociated embryonic chick dorsal root ganglion (DRG) neurons maintained in culture exhibit a mixed Na+/Ca2+ action potential. The characteristic "shoulder" on the repolarizing phase is due to the relatively prolonged inward Ca2+ current. DRG neurons grown in an elevated K+ medium (25 versus. 5 mM) lack the plateau phase of the action potential. Voltage-clamp analysis showed that this plastic change in action potential duration is not due to the loss of the inward Ca2+ current but is partly due to the appearance of a Ca2(+)-dependent, 4-aminopyridine-(4-AP)-sensitive transient outward current. Faster activation of the purely voltage-dependent delayed rectifier outward current also contributes to the rapid repolarization observed in neurons cultured in elevated K+ medium.     

Osmolality influences bistability of membrane potential under hypokalemic conditions in mouse skeletal muscle: an experimental and theoretical study

The membrane potential in mouse skeletal muscle depends on both extracellular osmolality and potassium concentration. These dependencies have been related to two membrane transporters, Na+/K+/2Cl- co-transporter and the inward potassium rectifier channel. To investigate the relation of the Na+/K+/2Cl- co-transporter and the inward potassium rectifier channel in a qualitative way, a combined electrophysiological and modelling approach was used. The experimental results show that the bistability of the membrane potential, which is related to the conductive state of the inward potassium rectifier channel, is shifted to higher extracellular potassium values when medium osmolality is increased. These results are confirmed by the computer simulation calculations for increased co-transporter flux. The combined results indicate that the co-transporter is capable of modulating the conductive state of the inward potassium rectifier channel.     

Predicting the activity phase of a follower neuron with A-current in an inhibitory network

The transient potassium A-current is present in most neurons and plays an important role in determining the timing of action potentials. We examine the role of the A-current in the activity phase of a follower neuron in a rhythmic feed-forward inhibitory network with a reduced three-variable model and conduct experiments to verify the usefulness of our model. Using geometric analysis of dynamical systems, we explore the factors that determine the onset of activity in a follower neuron following release from inhibition. We first analyze the behavior of the follower neuron in a single cycle and find that the phase plane structure of the model can be used to predict the potential behaviors of the follower neuron following release from inhibition. We show that, depending on the relative scales of the inactivation time constant of the A-current and the time constant of the recovery variable, the follower neuron may or may not reach its active state following inhibition. Our simple model is used to derive a recursive set of equations to predict the contribution of the A-current parameters in determining the activity phase of a follower neuron as a function of the duration and frequency of the inhibitory input it receives. These equations can be used to demonstrate the dependence of activity phase on the period and duty cycle of the periodic inhibition, as seen by comparing the predictions of the model with the activity of the pyloric constrictor (PY) neurons in the crustacean pyloric network.     

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)     

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.     

Potassium inward rectifier and acetylcholine receptor channels in embryonic Xenopus muscle cells in culture

Embryonic muscle cells of the frog Xenopus laevis were isolated and grown in culture and single-channel recordings of potassium inward rectifier and acetylcholine (ACh) receptor currents were obtained from cell-attached membrane patches. Two classes of inward rectifier channels, which differed in conductance, were apparent. With 140 mM potassium chloride in the electrode, one channel class had a conductance of 28.8 ± 3.4 pS (n = 21), and, much more infrequently, a smaller channel class with a conductance of 8.6 ± 3.6 pS (n = 7) was recorded. Both channel classes had relatively long mean channel open times, which decreased with membrane hyperpolarization. The probability of finding a patch of membrane with an inward rectifier channel was high (66%) and many membrane patches contained more than one inward rectifier channel. The open state probability (with no applied potential) was high for both inward rectifier channel classes so that 70% of the time there was a channel open. Seventy-three percent of the membrane patches with ACh receptor channels (n = 11) also had at least one inward rectifier channel present when the patch electrode contained 0.1 μM ACh. Inward rectifier channels were also found at 71% of the sites of high ACh receptor density (n = 14), which were identified with rhodamine-conjugated α-bungarotoxin. The results indicate that the density of inward rectifier channels in this embryonic skeletal muscle membrane was relatively high and includes sites of membrane that have synaptic specializations. © 1996 John Wiley & Sons, Inc.     

Blockers of potassium current and resting membrane potential in rat muscle fibers.

Rat diaphragm fibers were equilibrated for several hours in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 17 min. This repolarization was blocked by 5 mM BaCl2, a blocker of the inward rectifier K channel. On the other hand, 0.1 mM apamin and 0.02 mM glibenclamide which block the Ca-dependent and ATP sensitive K channels, respectively, and 0.1 mM 9-AC a blocker of the Cl- channel did not affect the repolarization. 5 mM barium decreased the K conductance measured under current-clamp conditions in diaphragm muscle fibers. The possible role of the inward rectifier system in the repolarization following return to normal [K]o is discussed.     

Significance of the corpus callosum in the paired activity of the respiratory center

The asymmetrical reactions of respiratory neurons of the right and left halves of the respiratory center and varied changes in bioelectrical activity of external intercostal muscles on both sides of the chest were discovered in experiments on anesthetized cats in response to successive electrical stimulation of the symmetrical cortical areas of the right and left cerebral hemispheres before and after callosotomy. It was demonstrated that callosotomy increased on both sides of the respiratory center the quantity of neurons responsive to ipsilateral cortical stimulation and determined the character of the asymmetrical reactions of right and left respiratory neurons and intercostal muscles. On the basis of the data obtained it is concluded that the corpus callosum contributes to the functional integration of both halves of the respiratory center.