Block of receptor response in the stretch receptor neuron of the crayfish by gadolinium

The trivalent lanthanide gadolinium was found to block the mechanotransducer response in the stretch receptor neuron of the crayfish. At normal calcium concentration (13.5 mm) a 50 per cent block of the receptor current was found at 395 ± 59 (mean ± SD), μM gadolinium. At a calcium concentration of 1.35 mm a 50 per cent block of the receptor current was obtained at 103 ± 14 (mean ± SD) μM gadolinium. The potential activated potassium current was also affected by gadolinium. At 200 μM the amplitude of the peak outward current as a result of a 90 mV positive potential step was decreased by about 40 per cent. The fast inward sodium current was decreased less than 10 per cent by gadolinium. It is concluded that in the crayfish stretch receptor gadolinium blocks the receptor current, reflecting block of stretch-activated channels, but at higher concentrations than have been described for other stretch-activated channels. In addition the outward rectifier potassium current is also blocked reflecting a block of potassium channels.     

Block of potassium outward currents in the crayfish stretch receptor neurons by 4-aminopyridine, tetraethylammonium chloride and some other chemical substances.

The effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the outward potassium currents in the rapidly and slowly adapting stretch receptor neurons (SRNs) of the crayfish (Pacifastacus leniusculus) were studied using a two micro-electrode voltage-clamp technique. The leakage current was not affected by either 4-AP or TEA. External 4-AP blocked the peak outward current in a dose-dependent manner (1:1 stoichiometry) with an apparent dissociation constant (Kd) of 2.3 +/- 0.2 mM (mean +/- SEM) in the slowly and 1.4 +/- 0.2 mM in the rapidly adapting SRN, the block being voltage dependent. External application of TEA resulted in a block of the steady state current enhancing the transient characteristics of the current response. The block appeared to deviate from a 1:1 stoichiometry and the apparent Kd for TEA was 9.6 +/- 3.4 mM with a cooperativity factor n = 0.43 +/- 0.03 in the slowly adapting SRN and 34.5 +/- 9.2 mM and 0.37 +/- 0.03 respectively in the rapidly adapting SRN. Low Ca2+, apamin and charybdotoxin, which are known to block Ca(2+)-dependent K-currents, had no effects on the outward current as was also the case with catechol. It is concluded that the different effects of TEA and 4-AP on the outward current in the two types of SRNs can be explained by the presence of at least two, probably heteromultimeric, channel populations having similar sensitivity to 4-AP but different sensitivity to TEA. One channel has a high affinity (Kd = 0.8-1.6 mM) for TEA and the other a low affinity (Kd = 173-213 mM) for TEA. The low-affinity channel seems to dominate in the slowly adapting SRN while both channels are equally common in the rapidly adapting SRN. Further, the present results do not support the existence of a macroscopic Ca(2+)-dependent K+ current in the SRNs.     

Potential-dependent potassium currents in the slowly adapting stretch receptor neuron of the crayfish

The outward current in the stretch receptor neuron of the crayfish Pacifastacus leniusculus was analysed using a two-micropipette potential-clamp technique. The outward current was shown to be carried by K+. When the sodium-dependent inward current was blocked by tetrodotoxin a fast-activating potassium current was revealed, resembling the delayed rectifier. The time-course of activation (Tau n) was dependent on potential and had a mean value of I ms at potential steps of to mV. The activation followed a second-order process according to the Hodgkin-Huxley model. The potential dependence of activation (n infinity) followed a sigmoid curve, n infinity = I/(I + exp [(E-En)/a]) with half-maximal activation potential En = -31 mV and a = -13 mV. When long pulses were applied, the potassium current showed marked inactivation with a fast time constant of 0.5 s that was potential independent and a slow component that was slightly potential dependent. The minimum value for the slow time constant was 4 s for steps to about 0 mV. The potential dependence of inactivation followed a sigmoid function k infinity = I/(I + exp [(E-Ek)/a]) with Ek = -39 mV and a = II mV. No transient potassium outward current (IA) was found in the crayfish stretch receptor neuron. In experiments on tail currents after depolarizing potential steps of different duration, it was found that the reversal potential changed in the positive direction when the duration of the pre-pulse increased. This could be due to K- accumulation in a space close to the neuronal membrane. The potassium current during depolarizing potential steps in the crayfish stretch receptor is similar to the delayed current found in other cells, for example the frog myelinated nerve, but different from many other invertebrate neurons.     

Stimulus-response properties of the slowly adapting stretch receptor neuron of the crayfish

The receptor potential and receptor current in response to ramp-and-hold extensions were measured in the slowly adapting stretch receptor of the crayfish, using potential clamp technique. The stimulus-response relationship for the peak amplitude of the receptor current showed a linear behaviour for extensions less than 2% and a nonlinear behaviour for extensions larger than 5%. Using the Stevens power law, R = k(S--S0)n, where R is response, S is stimulus, S0 is threshold stimulus and n the power coefficient, n was found to be 3 for extensions between 5 and 15%. The receptor current saturated at extensions above 20-25% of the zero length of the muscle, resulting in a lower n value. However, the n value is difficult to define in this region due to the saturation. The stimulus-response relation for the receptor current can be explained by the properties of the stretch-activated channels for which the open probability is exponentially dependent on the square of the membrane tension, as suggested by recent findings. The receptor potential, using tetrodotoxin, in response to identical ramp-and-hold extensions as those used to record current responses showed a more complex time-course, indicating involvement of potential-dependent channels, potassium channels being the most probable candidate. This was supported by a mathematical model which takes into account the viscoelastic properties of the receptor muscle, the properties of the stretch-activated channels and a potential dependent K+ current.     

Effects of a toxic phospholipase A2 (AgTx) from the venom of the Pit viper (Agkistrodon halys (Pallas)) on the crayfish stretch receptor neuron

Neurotoxicity of an isolated fraction (V) of the venom from the snake Agkistrodon halys (Pallas) was studied on the crayfish stretch receptor using a two-electrode voltage-clamp technique. The toxin was previously shown to have less phospholipase A2 activity but to be more toxic in mice compared with bee venom phospholipase A2. At 5 micrograms ml-1 (0.35 microM) the AgTx had very little effects on the electrophysiological properties of the receptor neuron whereas at 50 micrograms ml-1 (3.5 microM) the leak conductance was increased about three times and the net outward (K) current was reduced to 70% of control. The net inward (Na) current was not affected except for a small (+ 10 mV) shift in the I-V relationship. The resting membrane potential and the membrane capacitance were not changed by AgTx. These effects of AgTx were similar to those seen after exposure to bee venom phospholipase A2 at concentrations 10-20 times lower. At concentrations of AgTx which presynaptically affect the frog neuromuscular transmission (5-10 micrograms ml-1) the effects on ion channels in the stretch receptor neuron are very small and probably reflect the low phospholipase A2 activity compared with that of bee venom phospholipase A2.     

Potential-dependent potassium currents in the rapidly adapting stretch receptor neuron of the crayfish

The outward current was analysed in the rapidly adapting stretch receptor neuron of the crayfish Pacifastacus leniusculus with a two-micropipette potential clamp technique and K(+)-selective microelectrodes in an attempt to establish if the properties of this current could explain the difference in adaptive behaviour compared to the slowly adapting receptor. A fast activating outward current carried by K+ was revealed. The time constant of activation(tau n) was dependent on potential and had a mean value of 0.5 ms at potential steps to 0 mV. Activation followed a second-order process according to the Hodgkin-Huxley model. The potential dependence of activation (n infinity) followed by a sigmoid curve n infinity = 1/(1 + exp/[(E - En)/a]) with a half maximal activation potential En = -44 mV and a = -13 mV. When long pulses were applied the outward potassium current decreased with two time constants, one that was potential independent (0.2 s) and one that was potential dependent (2-8 s). The latter could be explained by accumulation of K+ in the extracellular space of the neuron. The potential dependence of inactivation followed a sigmoid function infinity = 1/(1 + exp[(E - Ek)/+a]) with Ek = -36 mV and a = 13 mV. The inactivation properties are different from those of the classical fast transient (IA) current. The transport system for the outward potassium current during depolarizing potential steps in the rapidly adapting stretch receptor is similar to the current found in the slowly adapting receptor neuron. However, the activation is faster and seems to occur at potentials more negative than in the slowly adapting receptor. These differences can contribute to but not entirely explain the difference in adaptive behaviour between the slowly and rapidly adapting receptor.     

The mechanotransduction of the crayfish stretch receptor neurone can be differentially activated or inactivated by local anaesthetics.

The effect of the local anaesthetics lidocaine, its meta-isomer, LL33, bupivacaine, tetracaine and procaine on the transducer properties of the stretch receptor neurone of the crayfish Pacifastacus leniusculus was investigated using a two microelectrode voltage clamp. Lidocaine increased the receptor current whereas LL33, bupivacaine and tetracaine reduced the receptor current in a reversible dose-dependent way. Procaine did not affect the receptor responses. The onset of the effect was generally slow in the order of minutes. Lidocaine increased the conductance of the mechanotransducer 50 +/- 7% (mean +/- SD, n = 4) and changed the reversal potential -8 +/- 1 mV (mean +/- SEM, n = 8), which indicates a major K+ conductance increase through the mechanosensitive channels. The other local anaesthetics increase the K+ conductance of the mechanotransducer without increasing the total conductance, which suggests that only P(Na)/P(K) is changed. These substances seem to have a Ca2+ dependent effect on the gating properties of the mechanosensitive channels in addition to their effect on the permeability through the channels as compared with lidocaine. All local anaesthetics investigated decreased the leak conductance of the receptor neurone. The effects of local anaesthetics on the mechanosensitive channels whether activating or blocking is correlated to the oil:water distribution coefficients and their relative hydrophobicity/hydrophilicity ratio. The results are consistent with the hypothesis that the local anaesthetic effect is mediated by changes in the lipid phase of the membrane.     

K+ currents generated by NMDA receptor activation in rat hippocampal pyramidal neurons

Long lasting outward currents mediated by Ca2+-activated K+ channels can be induced by Ca2+ influx through N-methyl-D-aspartate (NMDA)-receptor channels in voltage-clamped hippocampal pyramidal neurons. Using specific inhibitors, we have attempted to identify the channels that underlie these outward currents. At a holding potential of -50 mV, applications of 1 mM NMDA to the soma of cultured hippocampal pyramidal neurons induced the expected inward currents. In 44% of cells tested, these were followed by outward currents (average amplitude 60 +/- 7 pA) that peaked 2.5 s after the initiation of the inward NMDA currents and decayed with a time constant of 1.4 s. In 43% of those cells exhibiting an outward current, SK channel inhibitors, UCL 1848 (100 nM) and apamin (100 nM) abolished the outward current. In the remainder of the cells, the outward currents were either insensitive or only partly inhibited (44 +/- 4%) by 100 nM UCL 1848. In these cells, the outward currents were reduced by the slow afterhyperpolarization (sAHP) inhibitors, muscarine (3 microM; 43 +/- 9%), UCL 1880 (3 microM; 34 +/- 10%), and UCL 2027 (3 microM; 57 +/- 6%). Neither the BK channel inhibitor, charybdotoxin (100 nM), nor the Na+/K+ ATPase inhibitor, ouabain (100 microM), reduced these outward currents. Irrespective of the pharmacology, the time course of the outward current did not differ. Interestingly, no correlation was observed between the presence of a slow apamin-insensitive afterhyperpolarization and an outward current insensitive to SK channel blockers following NMDA-receptor activation. It is concluded that an NMDA-mediated rise in [Ca2+]i can result in the activation of apamin-sensitive SK channels and of the channels that underlie the sAHP. The activation of these channels may, however, depend on their location relative to NMDA receptors as well as on the spatial Ca2+ buffering within individual neurons.     

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons possess both "big" and "small" types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM omega-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 microM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM omega-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 microM omega-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM omega-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly. The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Viscoelastic properties of the slowly adapting stretch receptor muscle of the crayfish

The viscoelastic properties of the muscle associated with the slowly adapting stretch receptor organ of the crayfish (Astacus astacus) were studied by recording the tension response to various length changes. When steady-state length changes were applied to the muscle, the tension developed in a non-linear way, increasing slowly for small extensions and rapidly when extension increased. Muscle tension responses to ramp-and-hold extensions were characterized by a transient peak followed by a gradual decline in tension. At the onset of the ramp the tension increased rapidly, similar to the response seen in resting skeletal muscle. The relation between peak dynamic tension and extension was non-linear. In a log-log plot the relation was linear with a mean slope of 1.4. At small extensions (less than 5%) the slope seemed to be lower. The experimental results have been analysed in relation to a viscoelastic model consisting of a Voigt element in series with a non-linear spring. The model could describe both the static length-tension relation and the dynamic response, but different parameters for the springs had to be used for the two cases. When the measured tension response was transformed by an exponential function of the squared tension, in accord with recent findings on stretch-activated channels, a good agreement was obtained with the time course of the receptor currents. Adaptation is thus likely to be caused by both the mechanical properties of the receptor muscle and the characteristics of stretch-activated channels of the neuron.     

Stimulation-induced changes in the intracellular sodium activity of the crayfish stretch receptor

Changes in intracellular Na+ activity (aiNa) caused by mechanical stimulation in the slowly adapting stretch receptor of the crayfish were examined using Na+-selective microelectrodes. A series of brief stretches (each giving rise to a brief receptor potential and a single action potential) induced a reversible increase in aiNa which was proportional to the stimulation frequency in the range examined, 0-9.5 Hz. At 9.5 Hz, aiNa increased by 4-5 mM from the resting value of 7-10 mM. Tetrodotoxin (TTX) reduced, but did not abolish the stimulation-dependent increase in aiNa indicating the involvement of a Na+-influx pathway in addition to the potential-dependent, TTX-sensitive sodium channels of the neuronal plasma membrane. A likely candidate for this TTX-resistant pathway are the cationic transducer channels of the dendrites.     

Effects of halothane on the transducer and potential activated currents of the crustacean stretch receptor

Halothane was applied to the stretch receptor neuron of the crayfish (Astacus astacus) and the effects on the transducer properties and the potential activated currents were studied with potential clamp technique using two microelectrodes. Exposure to halothane reduced the frequency of action potentials during stretch. This was shown to be due to effects both on the action potential generating currents and the transducer current. Halothane partially blocked the TTX sensitive fast inward current in a dose-dependent manner (Apparent KD = 3 mM). Halothane also reduced the outward current produced by a positive potential step. Both the fast and the slow component were affected, although the fast outward current seemed to be most sensitive. There was little or no change in the currents resulting from negative potential steps. The peak of the receptor potential and the receptor current were very little affected by halothane. The amplitude of the static phase of the receptor potential was reduced to a greater degree than the static phase of the receptor current (cells treated with TTX). A change in reversal potential of about--13 mV was observed for the peak and the static phase of the receptor current in four cells indicating an increased cord conductance for the transducer channel.     

Potassium currents contributing to action potential repolarization and the afterhyperpolarization in rat vagal motoneurons.

1. Intracellular recordings were made from neurons in the dorsal motor nucleus of the vagus (DMV) in transverse slices of rat medulla maintained in vitro at 30 degrees C. Neurons had a resting potential of -59.8 +/- 1.4 (SE) mV (n = 39) and input resistance of 293 +/- 23 M omega (n = 44). 2. Depolarization elicited overshooting action potentials that were blocked by tetrodotoxin (TTX; 1 microM). In the presence of TTX, two types of action potentials having low and high thresholds could be elicited. The action potentials were blocked by cobalt (2 mM) indicating they were mediated by calcium currents. 3. Under voltage clamp, depolarization of the cell from membrane potentials negative of the resting potential activated a transient potassium current. This current was selectively blocked by 4-aminopyridine (4-AP) (5 mM) and catechol (5 mM) indicating that it is an A-type current. This current inactivated with a time constant of 420 ms and recovered from inactivation with a time constant of 26 ms. 4. When calcium currents were blocked by cadmium or cobalt, the rate of action potential repolarization was slower. In the presence of tetraethylammonium (TEA; 200-400 microM) or charybdotoxin (CTX; 30 nM) a small "hump" appeared on the repolarizing phase of the action potential that was abolished by addition of cadmium. These results indicate that a calcium-activated potassium current (IC) contributes to action potential repolarization. 5. Actions potentials elicited from hyperpolarized membrane potentials repolarized faster than those elicited from resting membrane potential. This effect could be blocked by catechol, indicating that voltage-dependent potassium currents (IA) can also contribute to action-potential repolarization. In the presence of catechol and calcium channel blockers, action potentials still had a significant early afterhyperpolarization suggesting that another calcium independent outward current is also active during repolarization. This fast afterhyperpolarizations (AHP) was not blocked by TEA. 6. Action potentials were followed by prolonged AHPs, which had two phases. The initial part of the AHP was blocked by apamin (100 nM) indicating that it results from activation of SK type calcium-activated potassium channels. The slow phase was selectively blocked by catechol suggesting that it is due to activation of IA. 7. It is concluded that a TTX-sensitive sodium current and two calcium currents contribute to the action potential in rat DMV neurons. At least three different currents contribute to action-potential repolarization: IC, IA, and a third unidentified calcium-insensitive outward current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Noradrenergic modulation of the hyperpolarization-activated cation current (Ih) in dopamine neurons of the ventral tegmental area

Alterations in the state of excitability of midbrain dopamine (DA) neurons from the ventral tegmental area (VTA) may underlie changes in the synaptic plasticity of the mesocorticolimbic system. Here, we investigated norepinephrine's (NE) regulation of VTA DA cell excitability by modulation of the hyperpolarization-activated cation current, Ih, with whole cell recordings in rat brain slices. Current clamp recordings show that NE (40 microM) hyperpolarizes spontaneously firing VTA DA cells (11.23+/-4 mV; n=8). In a voltage clamp, NE (40 microM) induces an outward current (100+/-24 pA; n=8) at -60 mV that reverses at about the Nernst potential for potassium (-106 mV). In addition, NE (40 microM) increases the membrane cord conductance (179+/-42%; n=10) and reduces Ih amplitude (68+/-3% of control at -120 mV; n=10). The noradrenergic alpha-1 antagonist prazosin (40 microM; n=5) or the alpha-2 antagonist yohimbine (40 microM; n=5) did not block NE effects. All NE-evoked events were blocked by the D2 antagonists sulpiride (1 microM) and eticlopride (100 nM) and no significant reduction of Ih took place in the presence of the potassium channel blocker BaCl2 (300 microM). Therefore, it is concluded that NE inhibition of Ih was due to an increase in membrane conductance by a nonspecific activation of D2 receptors that induce an outward potassium current and is not a result of a second messenger system acting on h-channels. The results also suggest that Ih channels are mainly located at dendrites of VTA DA cells and, thus, their inhibition may facilitate the transition from single-spike firing to burst firing and vice versa.     

Differences in mechano-transducer channel kinetics underlie tonotopic distribution of fast adaptation in auditory hair cells

The first step in audition is a deflection of the sensory hair bundle that opens mechanically gated channels, depolarizing the sensory hair cells. Two mechanism of adaptation of mechano-electric transducer (MET) channels have been identified in turtle auditory hair cells. The rate of fast adaptation varies tonotopically and is postulated to underlie a mechanical tuning mechanism in turtle auditory hair cells. Fast adaptation is driven by a calcium-dependent feedback process associated with MET channels. The purpose of this paper is to test the hypothesis that fast adaptation contributes to MET channel kinetics and that variations in channel kinetics underlie the tonotopic distribution of fast adaptation. To test for kinetic differences, the open channel blocker dihydrostreptomycin (DHS) was used. DHS blocked MET currents from low-frequency cells (IC(50) = 14 +/- 2 microM) more effectively than high-frequency cells (IC(50) = 75 +/- 5 microM), suggesting differences in MET channel properties. DHS block showed similar calcium sensitivities at both papilla locations. No difference in calcium permeation or block of the transducer channels was observed, indicating that the DHS effect was not due to differences in the channel pore. Slowing adaptation increased DHS efficacy, and speeding adaptation decreased DHS efficacy, suggesting that adaptation was influencing DHS block. DHS block of MET channels slowed adaptation, most likely by reducing the peak intraciliary calcium concentration achieved, supporting the hypothesis that the rate of adaptation varies with the calcium load per stereocilia. Another channel blocker, amiloride showed similar efficacy for high- and low-frequency cells with an IC(50) of 24.2 +/- 0.5 microM and a Hill coefficient of 2 but appeared to block high-frequency channels faster than low-frequency channels. To further explore MET channel differences between papilla locations, stationary noise analysis was performed. Spectral analysis of the noise gave half power frequencies of 1,185 +/- 148 Hz (n = 6) and 551 +/- 145 Hz (n = 5) for high- and low-frequency cells in 2.8 mM external calcium. The half power frequency showed similar calcium sensitivity to that of adaptation shifting to 768 +/- 205 Hz (n = 4) and 289 +/- 63 Hz (n = 4) for high- and low-frequency cells in 0.25 mM external calcium. Both the pharmacological data and the noise analysis data are consistent with the hypothesis that the tonotopic distribution of fast adaptation is in part due to differences in MET channel kinetics. An increase in the number of MET channels per stereocilia (termed summation) and or intrinsic differences in MET channel kinetics may be the underlying mechanism involved in establishing the gradient.     

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

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

Effects of gadolinium chloride on slowly adapting and rapidly adapting receptors of the rabbit lung

Effects of gadolinium chloride, an inhibitor of stretch-activated channels, on the responses of slowly adapting receptors (SARs) and rapidly adapting receptors (RARs) to hyperinflation were investigated. The increase in activity of RARs resulting from sustained elevations of left atrial pressure (LAP) was also assessed with gadolinium chloride application. Action potentials (AP) of SARs and RARs during hyperinflation were recorded from the vagus nerve of anesthetized New Zealand White rabbits before and after application of gadolinium chloride (20mM) directly on the receptor area of the nerve endings. There was a significant reduction of activity in SARs (n = 9) and RARs (n = 7) after application of gadolinium chloride. Activity of RARs (n = 6) increased when the LAP was elevated by 5 and 10 mmHg. This effect was abolished after gadolinium chloride was applied to receptor endings and the activity was restored when gadolinium chloride was removed. This suggests that stretch-activated channels play a role in SARs and RARs activity.     

Role of calcium influx and buffering in the kinetics of Ca(2+)-activated K+ current in rat vagal motoneurons.

1. Intracellular recordings were obtained from neurons of the dorsal motor nucleus of the vagus (DMV) in transverse slices of the rat medulla maintained in vitro. These neurons had a resting potential of -59.8 +/- 8.7 (SD) mV. Single action potentials elicited by brief depolarizing current pulses were followed by a prolonged afterhyperpolarization (AHP). Under voltage clamp, the current underlying the AHP was found to be a calcium-activated potassium current. 2. The outward current (GkCa,1) was voltage insensitive and was not blocked by tetraethylammonium (TEA) (10 mM). Unlike the slower time course calcium-activated potassium current recorded in some other neurons, GkCa,1 was blocked by apamin (25-100 nM), indicating that SK type calcium-activated potassium channels underlie this current. 3. GkCa,1 was maximal within 10 ms of the action potential and its decay was well described by a single exponential. After a single action potential the time constant of decay of GkCa,1 was 155 +/- 66 (+/- SD) ms. 4. Calcium influx was increased by adding TEA to the extracellular solution or by firing more than one action potential. As the calcium load was increased, both the peak amplitude and the time constant of decay of GkCa,1 increased. In cells impaled with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-filled electrodes, the time constant of decay of GkCa,1 after a single action current was 71 +/- 19 ms. 5. A simple diffusion-based model that incorporates two intrinsic calcium buffers is developed that accounts for many of the properties of GkCa,1. It is concluded that the decay of GkCa,1 reflects the time course of removal of calcium that has entered the cell during the action potential.     

Block of potassium outward currents in the crayfish stretch receptor neurons by 4-aminopyridine,tetraethylammonium chloride and some other chemical substances

The effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the outward potassium currents in the rapidly and slowly adapting stretch receptor neurons (SRNs) of the crayfish (Pacifastacus leniusculus) were studied using a two micro-electrode voltage-clamp technique. The leakage current was not affected by either 4-AP or TEA. External 4-AP blocked the peak outward current in a dose-dependent manner (1:1 stoichiometry) with an apparent dissociation constant (K_d) of 2.3 ± 0.2 mm (mean ± SEM) in the slowly and 1.4 ± 0.2 mm in the rapidly adapting SRN, the block being voltage dependent. External application of TEA resulted in a block of the steady state current enhancing the transient characteristics of the current response. The block appeared to deviate from a 1: 1 stoichiometry and the apparent K_d for TEA was 9.6 ± 3.4 mm with a cooperativity factor n= 0.43 ± 0.03 in the slowly adapting SRN and 34.5 ± 9.2 mm and 0.37 ± 0.03 respectively in the rapidly adapting SRN. Low Ca²⁺, apamin and charybdotoxin, which are known to block Ca²⁺-dependent K-currents, had no effects on the outward current as was also the case with catechol. It is concluded that the different effects of TEA and 4-AP on the outward current in the two types of SRNs can be explained by the presence of at least two, probably heteromultimeric, channel populations having similar sensitivity to 4-AP but different sensitivity to TEA. One channel has a high affinity (K_d= 0.8–1.6 mm) for TEA and the other a low affinity (K_d= 173–213 mm) for TEA. The low-affinity channel seems to dominate in the slowly adapting SRN while both channels are equally common in the rapidly adapting SRN. Further, the present results do not support the existence of a macroscopic Ca²⁺-dependent K⁺ current in the SRNs.     

Characterization of stretch-activated ion currents in isolated atrial myocytes from human hearts

To explore further the mechanisms that may underlie cardiac arrhythmia, we analysed stretch-activated ion currents in human atrial myocytes. Longitudinal stretch of freshly isolated atrial myocytes prolonged the duration of action potentials, depolarized the resting membrane potential and caused extra action potentials. Under voltage-clamp conditions, the amplitude of stretch-induced transmembrane currents increased reversibly with the intensity of stretch. Stretch-activated currents ( I(SAC)) had a reversal potential of 0 mV and were insensitive to substitution of Cl(-) with aspartate ions in the extracellular fluid. I(SAC) was suppressed by 5 micro M gadolinium (Gd(3+)). Furthermore, mechanical stretch decreased transmembrane ion fluxes through L-type calcium channels (I(Ca,L)). This reduction of I(Ca,L) was inhibited by dialysing the cells for 5 min with 5 mM BAPTA prior to application of stretch. In contrast, both BAPTA and removal of Ca(2+) from the extracellular bathing solution had no significant effect on stretch activation of I(SAC). These findings suggest that non-selective cation channels in human atrial myocytes are sensitive to mechanical stimulation. We propose that activation of transmembrane influx of cations, preferentially Na(+), by local stretch may play a role in cardiac arrhythmia.