Thyroid hormone receptor alpha can control action potential duration in mouse ventricular myocytes through the KCNE1 ion channel subunit.

AIMS: The reduced heart rate and prolonged QT(end) duration in mice deficient in thyroid hormone receptor (TR) alpha1 may involve aberrant expression of the K(+) channel alpha-subunit KCNQ1 and its regulatory beta-subunit KCNE1. Here we focus on KCNE1 and study whether increased KCNE1 expression can explain changes in cardiac function observed in TRalpha1-deficient mice. METHODS: TR-deficient, KCNE1-overexpressing and their respective wildtype (wt) mice were used. mRNA and protein expression were assessed with Northern and Western blot respectively. Telemetry was used to record electrocardiogram and temperature in freely moving mice. Patch-clamp was used to measure action potentials (APs) in isolated cardiomyocytes and ion currents in Chinese hamster ovary (CHO) cells. RESULTS: KCNE1 was four to 10-fold overexpressed in mice deficient in TRalpha1. Overexpression of KCNE1 with a heart-specific promoter in transgenic mice resulted in a cardiac phenotype similar to that in TRalpha1-deficient mice, including a lower heart rate and prolonged QT(end) time. Cardiomyocytes from KCNE1-overexpressing mice displayed increased AP duration. CHO cells transfected with expression plasmids for KCNQ1 and KCNE1 showed an outward rectifying current that was maximal at equimolar plasmids for KCNQ1-KCNE1 and decreased at higher KCNE1 levels. CONCLUSION: The bradycardia and prolonged QT(end) time in hypothyroid states can be explained by altered K(+) channel function due to decreased TRalpha1-dependent repression of KCNE1 expression.

Receptor-mediated activation of gastric vagal afferents by glucagon-like peptide-1 in the rat.

The vagus nerve plays a role in mediating effects of the two glucagon-like peptides GLP-1 and GLP-2 on gastrointestinal growth, functions and eating behaviour. To obtain electrophysiological and molecular evidence for the contribution of afferent pathways in chemoreception from the gastrointestinal tract, afferent mass activity in the ventral gastric branch of the vagus nerve and gene expression of GLP-1 receptors and GLP-2 receptors in the nodose ganglion were examined in Sprague-Dawley rats. Intravenous administration of GLP-1 (30-1000 pmol kg(-1)), reaching high physiological plasma concentrations, increased vagal afferent mass activity peaking (13-52% above basal level, P < 0.05) 3-5 min after injection. Repeated administration of GLP-1 (1000 pmol kg(-1); five times, 15 min intervals) elicited similar responses. Pretreatment with GLP-1 receptor antagonist exendin(9-39)amide (500 pmol kg(-1)) abolished the GLP-1 response to doses 30-300 pmol kg(-1) but had no effect on the vagal response to gastric distension. For comparison, GLP-2 (1000 pmol kg(-1)) had no effect on vagal afferent activity. Vagal chemoreception of GLP-1 is supported by expression of the GLP-1 receptor gene in the nodose ganglion. However, the GLP-2 receptor was also expressed. To conclude, our results show that peripherally administered GLP-1, differently from GLP-2, activates vagal afferents, with no evidence of desensitisation. The GLP-1 effect was blocked by exendin(9-39)amide, suggesting that GLP-1 receptors on vagal afferent nerves mediate sensory input from the gastrointestinal tract or pancreas; either directly or indirectly via the release of another mediator. GLP-2 receptors appear not be functionally expressed on vagal afferents.

Hypotonic stress activates an intermediate conductance K+ channel in human colonic crypt cells.

AIM: To investigate the effect of hypotonic stress on human colonic crypts cells in terms of ion channel activity and intracellular Ca2+ concentration. METHODS: Single crypts were isolated from biopsies taken during colonoscopy. The patch clamp technique was used (in the cell-attached mode) to observe the activity of ion channels during hypotonic stress. Calcium measurements were made using the fluophores Fluo 3 or 4. RESULTS: The intermediate conductance (29 pS), Ca2+ -sensitive, K+ channel (also known as KCNN4) previously described (Sandle et al. 1994) was seen in 54 of 149 patches (36%) when the crypts were bathed in normal extracellular solution (290 mOsm kg(-1)). Forty-one patches could be used for further analysis. Activation of one or several 29 pS channels was seen in 15 of 41 patches (39%) after 30 s to 4 min of exposure to hypotonic solution (160 mOsm kg(-1)). The open probability increased from 0.0043 in control solution to 0.44 at 5 min of hypotonic stress. When the crypts were exposed to hypotonic solution, an increase in intracellular Ca2+ could be seen. The increase in intracellular Ca2+ emanates mainly from intracellular stores. CONCLUSION: The 29 pS K+ channel takes part in volume regulation in human colonic crypt cells. The activation of this channel is mediated through an increase in intracellular Ca2+.

Characterization of a delayed rectifier potassium channel in the slowly adapting stretch receptor neuron of crayfish.

Single channel recordings were performed on enzyme-cleaned slowly adapting sensory neurons of crayfish, in cell-attached configuration, with a physiological K(+) gradient across the neuronal membrane. An outward rectifying, voltage-gated K(+) channel with a slope conductance of 13 pS and a K(+) ion permeability of P(K)=6.5 x 10(-14) cm(3)/s was characterized. This 13 pS K(+) channel started to be activated at around 20 mV depolarization. Its open probability increased upon depolarization with V(0.5)= -25.3 mV and P(max)=0.83. The averaged currents showed a delay following the onset of depolarization. The activation time constant was voltage-dependent. The maximal value was 17.0 ms at -25 mV and at +35 mV the time constant was 1.7 ms. Little inactivation was observed throughout the 80- or 1500-ms long depolarization pulses. A sum of two exponentials provided the optimal fit for open time and closed time distribution. At 80-mV depolarization, the open time constants were 0.4 and 10.4 ms; the close time constants were 0.4 and 2.3 ms. The first-latency distribution suggested that at least two closed states preceded two open states. This 13 pS delayed rectifier plays a minor role in the maintenance of the resting membrane potential but contributes to the action potential repolarization. It may also modify the stretch-induced receptor potential and affect the adaptation behaviours in this neuron.

Inhibition of mechanotransducer currents in crayfish sensory neuron by CGS 9343B, a calmodulin antagonist.

The effects of CGS 9343B (zaldaride maleate), a calmodulin antagonist, on mechanosensitive channels were examined in crayfish slowly adapting sensory neurons using the two-electrode voltage clamp technique. In addition to its inhibition of voltage-gated Na(+) and K(+) currents, CGS 9343B (<30 microM) blocked reversibly the receptor current in a dose-dependent and voltage-dependent manner with a dissociation constant (K(d)) of 26.8 microM. The time course of the block was 265 s. Within the extension range of 3-30%, the reduction in receptor current was stimulus-independent and the gating mechanisms were not affected. Extracellular Ca(2+) was not necessary for its blocking effects. No changes in passive muscle tension were observed in the presence of 20 microM CGS 9343B. These results suggest that CGS 9343B, as a calmodulin antagonist, can also block mechanosensitive channels, possibly by being incorporated into the lipid membrane and/or interacting with the channel protein.

Macrocurrents of voltage gated Na+ and K+ channels from the crayfish stretch receptor neuronal soma.

Currents from the slowly adapting stretch receptor neuron of the crayfish (Pacifastacus leniusculus) were studied in a cell attached configuration using patch pipettes with an opening diameter of 2-10 microm. The neuronal membrane was enzymatically freed from the glial layer. The voltage gated Na+ and K+ channels seemed to be more concentrated in the lower part of soma close to the axon hillock. The Na+ and K+ currents could be analysed by fitting the currents to a fourth-order exponential function for Na+ current and a second-order exponential function for the K+ current. The macropatch recordings of enzymatically treated neurons are superior to two electrode voltage clamp recordings when analyzing voltage gated Na+ and K+ currents.

Different spatial distributions of sodium channels in the slowly and rapidly adapting stretch receptor neuron of the crayfish.

Inward Na+ currents were studied, using a two-microelectrode intracellular voltage-clamp technique, in the slowly adapting (SA) and rapidly adapting (RA) stretch receptor neurons of the crayfish after the axons were cut at different distances from the soma. In the SA neuron, inward Na+ currents were recorded in the soma even when the axon was cut as close as 100 microm from the center of the soma, indicating the presence of Na+ channels in these parts. Also, two populations of Na+ channels seem to exist in the SA neuron. In the RA neuron, only minute Na+ currents were observed if the axon was shorter than 250 microm. The results strongly indicate that the voltage-gated Na+ channels in the SA and RA neurons have different distributions and that the difference in the spatial distribution of Na+ channel types may be important for the difference in firing properties in the two types of neurons.

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.

Action potential and sodium current in the slowly and rapidly adapting stretch receptor neurons of the crayfish (Astacus astacus).

Action potentials (APs) and sodium current from the slowly and the rapidly adapting stretch receptor neurons in the crayfish (Astacus astacus) were recorded with a two microelectrode voltage- and current-clamp technique. In the rapidly adapting neuron the APs had a duration of 3.2 +/- 0.2 ms (means +/- SE) and an amplitude of 55.2 +/- 1.5 mV. In the slowly adapting receptor neuron APs had a duration of 4.1 +/- 0.2 ms and an amplitude 79.9 +/- 2.0 mV. APs in the rapidly adapting neuron had a larger amplitude if they were recorded from the axon. In the rapidly adapting neuron adaptation of the impulse response was prolonged by hyperpolarization or by exposure to scorpion venom. Also, sinusoidal current stimulation added to the current steps prevented impulse adaptation. Block of the potassium currents in the slowly adapting neuron resulted in a rapid adaptation of the impulse response. The maximum sodium current amplitude was 313 +/- 15 nA in slowly adapting neuron and 267 +/- 11 nA in the rapidly adapting neuron. The current-voltage relationship showed a hump most marked in the slowly adapting neuron and abolished when a depolarizing prepulse was given. In the rapidly adapting neuron the inactivation starts at a more negative potential (Eh = -45 mV) and is faster compared with the slowly adapting neuron (Eh = -41 mV). The crude scorpion venom of Leiurus quinquestriatus (ScVLq) shifted hinfinity curve toward more positive potentials and slowed down the rate of inactivation. The results indicate the possible presence of more than one Na+ channel population and that the relative density and the spatial distribution is different in the slowly and rapidly adapting neuron. The difference contributes to the adaptive properties of the two receptor neurons.

Carbachol induces oscillations in membrane potential and intracellular calcium in a colonic tumor cell line, HT-29.

The patch-clamp technique was used to study the effects of carbachol (CCh) on HT-29 cells. During CCh exposure, the cells (n = 23) depolarized close to the equilibrium potential for Cl- (E(Cl-); -48 mV) and the membrane potential then started to oscillate (16/23 cells). In voltage-clamp experiments, similar oscillations in whole cell currents could be demonstrated. The whole cell conductance increased from 225 +/- 25 pS in control solution to 6,728 +/- 1,165 pS (means +/- SE, n = 17). In substitution experiments (22 mM Cl- in bath solution, E(Cl-) = 0 mV), the reversal potential changed from -41.6 +/- 2.2 mV (means +/- SE, n = 9) to -3.2 +/- 2.0 mV (means +/- SE, n = 7). When the cells were loaded with the calcium-sensitive fluorescent dye, fluo 3, and simultaneously patch clamped, CCh caused a synchronous oscillating pattern of fluorescence and membrane potential. In cell-attached patches, the CCh-activated currents reversed at a relative membrane potential of 1.9 +/- 3.7 mV (means +/- SE, n = 11) with control solution in the pipette and at 46.2 +/- 5.3 mV (means +/- SE, n = 10) with a 15 mM Cl- solution in the pipette. High K+ (144 mM) did not change the reversal potential significantly (P < or = 0.05, n = 8). In inside-out patches, calcium-dependent Cl- channels could be demonstrated with a conductance of 19 pS (n = 7). It is concluded that CCh causes oscillations in membrane potential that involve calcium-dependent Cl- channels and a K+ permeability.

Local anaesthetics potentiate GABA-mediated Cl- currents by inhibiting GABA uptake.

A two-electrode voltage clamp was used to examine the effects of the local anaesthetics (LAs) lidocaine, tetracaine and bupivacaine, and the meta-isomer of lidocaine (LL33), on the gamma-aminobutyric acid (GABA)-gated Cl- channel in the crayfish stretch receptor neurone. The voltage-induced current changes were recorded while exposing the neurone to GABA in saline containing different LAs. All LAs enhanced the voltage-induced Cl- current equivalent to a shift of the conductance vs GABA curve towards lower concentrations. The mechanism of the facilitation of GABA-induced conductance was found to be mainly a block of the GABA uptake in the stretch receptor neurone. Tetracaine, and to a lesser extent LL33, also seem to affect directly the GABA/receptor Cl-/channel complex resulting in an increased conductance of the channel.

A mathematical model of the crustacean stretch receptor neuron. Biomechanics of the receptor muscle, mechanosensitive ion channels, and macrotransducer properties.

1. A mathematical model of the primary transduction process in a mechanoreceptor, the slowly adapting stretch receptor organ of the crayfish, has been developed taking into account the viscoelastic properties of the accessory structures of the receptor, i.e., the receptor muscle, the biophysical properties of the mechanosensitive channels (MSCs) and the passive electrical properties of the neuronal membrane (leak conductance and capacitative properties). The work is part of an effort to identify and characterize the mechanical and ionic mechanisms in a complex mechanoreceptor. The parameters of the model are based mainly on results of our own experiments and to some extent on results from other studies. The performance of the model has been compared with the performance of the slowly adapting receptor. 2. The model resulted in nonlinear differential equations that were solved by an iterative, fourth order Range-Kutta method. For the calculations of potential, the cell was treated as an idealized spherical body. The extension of the receptor muscle was 0-30%, which is within the physiological limits for this receptor. 3. The mechanical properties of the receptor muscle were modeled by a simple Voigt element (a spring in parallel with a dashpot) in series with a nonlinear spring. This element can describe resonably well the tension development in the receptor muscle at least for large extensions (> 12%). However, for small extensions (< 12%), the muscle seems to be more stiff than for large extensions. 4. The receptor current at different extensions of the receptor was computed using typical viscoelastic parameters for a receptor muscle together with a transformation of muscle tension to tension in the neuronal dendrites and finally the properties of the mechanosensitive channels. The model fit was satisfactory in the high extension range whereas in the low extension range the deviation from the experimental results could be explained partly by insufficient modeling of the nonlinear viscoelastic properties. The voltage dependence of the receptor current was also well predicted by the model. 5. If the parameters of the viscoelastic model were adjusted for each extension so that each tension response closely resembled the experimental values, the fit of the current responses was improved but still deviated from the experimental currents. One factor that might explain the difference is the possibility that the MSCs in the stretch receptor neuron might have intrinsic adaptive properties. Introducing an exponential adaptive behavior of individual MSCs increased the ability of the model to predict the receptor current. 6. The receptor potential was calculated by modeling the neuronal membrane by a lumped leak conductance and capacitance The calculated receptor potential was higher than the experimental receptor potential. However, the fit of the receptor potential was improved substantially by introducing an adaptation of the MSCs as outlined in the preceding paragraph. the remaining discrepancy might be explained by insufficient blocking of K+ channels in the experiment. 7. The model can predict a wide range of experimental data from the slowly adapting stretch receptor neuron including the mechanical response of the receptor muscle, the receptor current and its voltage dependence, and the receptor potential. It also describes accurately the passive electrical properties of the neuronal membrane.

Visco-elastic properties of the rapidly adapting stretch receptor muscle of the crayfish.

The visco-elastic properties of the receptor muscle associated with the rapidly adapting stretch receptor organ of crayfish (Pacifastacus Leniusculus) were studied by recording the tension responses to various length changes. Steady-state length changes resulted in a non-linear tension development in the receptor muscle. The tension increased slowly for small extensions and more rapidly when extension increased. Muscle tension responses to ramp-and-hold extension were characterized by a transient peak followed by a gradual non-exponentional decline in tension. At the onset of the ramp the tension increased rapidly, similar to what has been observed in the muscle of the slowly adapting receptor (SM). The steeper rise in tension during the first part of the ramp indicating higher initial stiffness, resulted in a 'hump' when large extensions (> 15%) were applied. The results show that the rapidly adapting receptor muscle has a more pronounced dynamic component; the ratio between the amplitude of the peak and the steady state response was larger in the rapidly than in the slowly adapting receptor muscle. Accordingly, different values for the elements of a visco-elastic model of the muscle had to be set for the two types of receptors. The different properties of the rapidly and slowly adapting receptor muscles are in line with the differences in the overall adaptive behaviour of the organ and give further support to the idea that mechanical factors contribute to the adaptive properties.

Transducer properties of the rapidly adapting stretch receptor neurone in the crayfish (Pacifastacus leniusculus).

1. The transducer properties of the rapidly adapting stretch receptor neurone of the crayfish (Pacifastacus leniusculus) were studied using a two-microelectrode voltage clamp technique. 2. The impulse response to ramp-and-hold extensions of the receptor muscle typically consisted of a high frequency burst followed by cessation of impulses within a relatively short time depending on the amplitude of extension. The type of adaptation was consistent with earlier studies. The stimulus-response relationship for the impulse frequency was non-linear and had a slope in a log-log plot of 2.9. 3. When impulse generation was blocked by tetrodotoxin (TTX), (block of Na+ channels) the receptor potential was extension dependent and similar to that found in the slowly adapting receptor. For small extensions there was an initial peak followed by a fall to a steady potential level. For large extensions the potential response during the ramp phase consisted of a peak followed by a constant potential level lasting to the end of the ramp. When the extension changed to the hold phase the potential fell towards a steady state. The relation between extension and amplitude of receptor potential was non-linear and saturated at -40 to -30 mV (extensions > 15% of zero length, lo). 4. When potassium channels were blocked by TEA (50 mM) and 4-aminopyridine (4-AP, 5 mM) (and Na+ channels blocked by TTX) the shape of the generator potential become less complex with an increased amplitude for large extensions. 5. When the receptor neurone was voltage clamped at the resting potential, extension of the receptor muscle produced an inwardly directed receptor current, the stretch-induced current (SIC). The response consisted of a fast transient phase which decayed towards a steady state. The SIC peak amplitude was dependent on extension in a sigmoidal fashion and saturated at 190 nA (extensions > 25% of lo). The slope of the steepest part of the stimulus-response relation (between 10 and 20% extension) was 4.7 +/- 0.25 (mean +/- S.E.M.) in a log-log plot. 6. The peak amplitude of the SIC increased with increasing extension speed (ramp steepness), the relation between the slope of the ramp and current amplitude being a first order (hyperbolic) function. The amplitude of the receptor current was voltage dependent and had a reversal potential of +16.2 +/- 1.8 mV (mean +/- S.E.M., 32 cells). From the reversal potential the permeability ratio, PNa/PK, of the transducer permeability system was calculated to be 1.5. The I-V curve of SIC was non-linear.(ABSTRACT TRUNCATED AT 400 WORDS)

A light emitting diode microspectrophotometer: intracellular Ca2+ measurements in isolated stretch receptor.

A microspectrophotometer was designed to measure absorbance changes in single cells. The device utilizes sequentially activated light emitting diodes (LED) to provide different wave lengths of light. The instrument has the advantage of relative simplicity and less cost compared to other devices. The spectrophotometer was tested by measuring absorbance changes of the metallochromic Ca2+ indicator Arsenazo III (AIII) injected into the crayfish (Astacus astacus) stretch receptor. Under the conditions described the detection limit of the concentration of AIII was 0.05 mM and absorbance changes of 0.0005 can be reliably determined which correspond to a detection limit of 10-20 nM for free Ca2+ changes assuming a light path length of 0.003 cm and an apparent dissociation constant (KD) of 2 microM for the Ca(2+)-AIII complex. The upper frequency limit of the device is 3000 Hz. The absorbance measurements of AIII injected into the crayfish stretch receptor neurons revealed a Ca(i) of 375 +/- 177 nM (mean +/- SD: 14 cells). LiCl substituted for NaCl increased Ca(i) 45-100 nM in different cells, suggesting that a Na+ gradient is necessary for Ca2+ homeostasis in this receptor.

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.

GABA-gated anion channels in intact crayfish opener muscle fibres and stretch-receptor neurons are neither activated nor desensitized by glutamate.

The influence of glutamate on the GABA-activated Cl- conductance was studied in the slowly adapting stretch-receptor neuron and dactylopodite opener muscle fibre of the crayfish (Astacus astacus) using a two-microelectrode and a three-microelectrode voltage clamp, respectively. Glutamate (0.5-1.0 mM) had no effect on the GABA-activated conductance in either preparation. This indicates that the availability of the inhibitory channels for activation of GABA is not influenced by glutamate. The present results are in sharp contrast to those obtained by Franke et al. (J Comp Physiol A 159:591-609, 1986) in experiments on excised membrane patches, which suggested that glutamate is capable of both activating and desensitizing inhibitory postsynaptic channels in the crayfish opener muscle fibre.

Inward current caused by sodium-dependent uptake of GABA in the crayfish stretch receptor neurone.

A two-microelectrode current-voltage clamp and Cl(-)-selective microelectrodes were used to examine the effects of gamma-aminobutyric acid (GABA) on membrane potential, current and intracellular Cl- activity (aiCl) in the crayfish stretch receptor neurone. All experimental solutions were CO2-HCO3- free. 2. GABA (500 microM) produced a mono- or biphasic depolarization (amplitude < or = 10 mV), often with a prominent initial depolarizing component followed by a transient shift to a more negative level. In some neurones, an additional depolarizing phase was seen upon washout of GABA. Receptor desensitization, being absent, played no role in the multiphasic actions of GABA. 3. The pronounced increase in membrane conductance evoked by GABA (500 microM) was associated with an increase in aiCl which indicates that the depolarizing action was not due to a current carried by Cl- ions. 4. The currents activated by GABA under voltage clamp conditions were inwardly directed when recorded at the level of the resting membrane potential, and they often revealed a biphasic character. The reversal potential of peak currents activated by pulses of 500 microM-GABA (EGABA) was 9-12 mV more positive than the reversal potential of the simultaneously measured net Cl- flux (ECl). ECl was 2-7 mV more negative than the resting membrane potential. 5. EGABA (measured using pulses of 500 microM-GABA) was about 10 mV more positive than the reversal potential of the current activated by 500 microM-muscimol, a GABA agonist that is a poor substrate of the Na(+)-dependent GABA uptake system. 6. In the absence of Na+, the depolarization and inward current caused by 500 microM-GABA were converted to a hyperpolarization and to an outward current. Muscimol produced an immediate outward current both in the presence and absence of Na+. 7. Following block of the inhibitory channels by picrotoxin (100-200 microM), the depolarizing effect of 500 microM-GABA was enhanced and the transient hyperpolarizing shifts were abolished. 8. In the presence of picrotoxin, GABA (> or = 2 microM) produced a concentration-dependent monophasic inward current which had a reversal potential of +30 to +60 mV. This current was inhibited in the absence of Na+ and by the GABA uptake blocker, nipecotic acid. Unlike the channel-mediated current, the picrotoxin-insensitive current was activated without delay also at low (2-10 microM) concentrations of GABA.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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) microM 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) microM gadolinium. The potential activated potassium current was also affected by gadolinium. At 200 microM 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.