Voltage-gated K+ channels in chemoreceptor sensory neurons of rat petrosal ganglion.

A subpopulation of sensory neurons in the petrosal ganglion transmits information between peripheral chemoreceptors (glomus cells) in the carotid body and relay neurons in the nucleus of the solitary tract. Expression of voltage-gated K+ channels in these neurons was characterized by immunohistochemical localization. Five members of the Kv1 family, Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 and members of two other families, Kv2.1 and Kv4.3, were identified in over 90% of the chemoreceptor neurons. Although the presence of these channel proteins was consistent throughout the population, individual neurons showed considerable variation in K+ current profiles.

Brain-derived neurotrophic factor acutely inhibits AMPA-mediated currents in developing sensory relay neurons.

Brain-derived neurotrophic factor (BDNF) is expressed by many primary sensory neurons that no longer require neurotrophins for survival, indicating that BDNF may be used as a signaling molecule by the afferents themselves. Because many primary afferents also express glutamate, we investigated the possibility that BDNF modulates glutamatergic AMPA responses of newborn second-order sensory relay neurons. Perforated-patch, voltage-clamp recordings were made from dissociated neurons of the brainstem nucleus tractus solitarius (nTS), a region that receives massive primary afferent input from BDNF-containing neurons in the nodose and petrosal cranial sensory ganglia. Electrophysiological analysis was combined in some experiments with anterograde labeling of primary afferent terminals to specifically analyze responses of identified second-order neurons. Our data demonstrate that BDNF strongly inhibits AMPA-mediated currents in a large subset of nTS cells. Specifically, AMPA responses were either completely abolished or markedly inhibited by BDNF in 73% of postnatal day (P0) cells and in 82% of identified P5 second-order sensory relay neurons. This effect of BDNF is mimicked by NT-4, but not NGF, and blocked by the Trk tyrosine kinase inhibitor K252a, consistent with a requirement for TrkB receptor activation. Moreover, analysis of TrkB expression in culture revealed a close correlation between the percentage of nTS neurons in which BDNF inhibits AMPA currents and the percentage of neurons that exhibit TrkB immunoreactivity. These data document a previously undefined mechanism of acute modulation of AMPA responses by BDNF and indicate that BDNF may regulate glutamatergic transmission at primary afferent synapses.

Opioid modulation of calcium current in cultured sensory neurons: mu-modulation of baroreceptor input.

We used the whole cell open-patch or perforated-patch technique to characterize mu-opioid modulation of Ca(2+) current (I(Ca)) in nodose sensory neurons and in a specific subpopulation of nodose cells, aortic baroreceptor neurons. The mu-opiate receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol enkephalin (DAGO) inhibited I(Ca) in 95% of neonatal [postnatal day (P)1-P3] nodose neurons. To the contrary, only 64% of juvenile cells (P20-P35) and 61% of adult cells (P60-P110) responded to DAGO. DAGO-mediated inhibition of I(Ca) was naloxone sensitive, irreversible in the presence of guanosine 5'-O-(3-thiotriphosphate), absent with guanosine 5'-O-(2-thiodiphosphate), and eliminated with pertussis toxin; DAGO's inhibition of I(Ca) was G protein mediated. Incubation of neurons with omega-conotoxin GVIA eliminated the effect of DAGO in neonatal but not in juvenile cells. In the latter, DAGO reduced 37% of the current remaining in the presence of omega-conotoxin. In the subset of nodose neurons, aortic baroafferents, the effect of DAGO was concentration dependent, with an IC(50) of 1.82 x 10(-8) M. DAGO slowed activation of I(Ca), but activation curves constructed from tail currents were the same with and without DAGO (100 nM). In summary, mu-opiate modulation of I(Ca) in nodose neurons was demonstrated in three age groups, including specifically labeled baroafferents. The demonstration of a mechanism of action of mu-opioids on baroreceptor afferents provides a basis for the attenuation of the baroreflex that occurs at the level of the nucleus tractus solitarii.

Molecular characterization of the human CRT-1 creatine transporter expressed in Xenopus oocytes.

The protein sequence encoded by a creatine transporter cDNA cloned from a human heart library was identical to that cloned from a human kidney library (Nash et al., Receptors Channels 2, 165-174, 1994), except that at position 285 the former contained an Ala residue and the latter contained a Pro residue. Expression of this human heart cDNA clone in Xenopus laevis oocytes induced a Na+- and Cl--dependent creatine uptake activity that saturated with a Km of approximately 20 microM for creatine. The induced uptake was inhibited by beta-guanidinopropionic acid (IC50 approximately 44.4 microM), 2-amino-1-imidazolidineacetic acid (cyclocreatine; IC50 approximately 369.8 microM), gamma-guanidinobutyric acid (IC50 approximately 697.9 microM), gamma-aminobutyric acid (IC50 approximately 6.47 mM), and amiloride (IC50 approximately 2.46 mM). The inhibitors beta-guanidinopropionic acid, cyclocreatine, and gamma-guanidinobutyric acid also inhibited the uptake activity of the Ala285 to Pro285 (A285P) mutant as effectively as that of the wild type. In contrast, guanidinoethane sulfonic acid, a potent inhibitor of taurine transport, inhibited the uptake activity of the A285P mutant approx. two times more effectively than that of the wild type. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), but not its inactive analog, 4alpha-phorbol 12, 13-didecanoate, inhibited the creatine uptake, and the inhibitory effect of PMA was both time and concentration dependent. The protein kinase A activator 8-bromo-cyclic AMP, however, had no effect on the creatine uptake. The rate of uptake increased hyperbolically with the increasing concentration of the external Cl- (equilibrium constant KCl- approximately 5 mM) and sigmoidally with the increasing concentration of the external Na+ (equilibrium constant KNa+ approximately 56 mM). Further analyses of the Na+ and Cl- concentration dependence data suggested that at least two Na+ and one Cl- were required to transport one creatine molecule via the creatine transporter.

Contribution of the hyperpolarization-activated current to the resting membrane potential of rat nodose sensory neurons.

1. The voltage- and time-dependent characteristics of the hyperpolarization-activated current (IH) and its contribution to the resting membrane potential of neonatal rat nodose sensory neurons were investigated using the whole-cell tight seal method of voltage and current clamp recording. 2. IH was found in all neonatal nodose neurons in vitro, contrary to previous reports where its presence was particular for A-type neurons. We used the presence of both tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents to distinguish C- from A-type neurons (TTX-S only). We obtained further support for the presence of IH in C-type neurons with experiments in which IH was demonstrated in a subset of neurons sensitive to capsaicin. 3. In both groups IH activated at potentials negative to -50 mV, developed slowly with time and was inhibited by 1-5 mM extracellular caesium. At -120 mV, IH activated with a fast time constant of 73 +/- 3 ms in A-type neurons and 163 +/- 37 ms in C-type neurons (P < 0.05). A second, slower time constant of 682 +/- 83 ms was observed in A-type neurons and 957 +/- 122 ms in C-type neurons. 4. A- and C-type neurons differed in the amplitude of IH. The mean magnitude of IH at -110 mV was -2338 +/- 258 pA in A-type neurons but only -241 +/- 40 pA (P < 0.001) in C-type neurons. This disparity persisted when currents were normalized for capacitance. The reversal potentials for IH were -39 +/- 4 mV for A-type neurons and -37 +/- 5 mV for C-type neurons (P > 0.05). 5. During current clamp recording IH caused time-dependent rectification in response to hyperpolarizing current injections from the resting membrane potential. CsCl abolished the rectification and hyperpolarized the resting potential of A-type neurons from -55 +/- 3 mV to -61 +/- 4 mV and C-type neurons from -62 +/- 2 mV to -71 +/- 3 mV. Taken together, the results in these studies indicate that IH contributes to the resting membrane potential in all nodose neurons.

Analysis of a Ca2+-activated K+ channel that mediates hyperpolarization via the thrombin receptor pathway.

Dami human leukemia cells express G protein-coupled thrombin receptors that operate through the phospholipase C pathway. When these receptors are activated by alpha-thrombin or by thrombin receptor-activating peptide, an elevation in cytosolic Ca2+ concentration develops that is accompanied by hyperpolarization of the plasma membrane. This transitory phase of hyperpolarization is primarily mediated by inwardly rectifying, Ca2+-activated K+ channels that have an inward conductance of approximately 24 pS. In cell-attached patches the channels open within seconds after superfusion of the cell with thrombin receptor-activating peptide. In inside-out patches, perfusion of submicromolar Ca2+ onto the cytosolic surface of the membrane is sufficient to activate the channels. In outside-out patches, channel opening can be blocked by nanomolar concentrations of charybdotoxin. The function of these intermediate-sized inwardly rectifying, Ca2+-activated K+ channels has not been established; however, by analogy with other cell systems, they may serve to regulate cell volume during cellular activation or to increase the electromotive drive that sustains Na+ and/or Ca2+ influx through ligand-gated cation channels.

Inwardly rectifying K+ channels in dispersed bovine parathyroid cells.

Excitation-secretion coupling in various endocrine cells is dependent on membrane voltage which is controlled by ion channels. In order to characterize and determine the functional significance of voltage-gated ion channels in the parathyroid cell, the patch clamp technique was used in cell-attached and whole cell configurations to study single channel and whole cell currents in dispersed bovine parathyroid cells. Whole cell voltage clamp recordings from dissociated bovine parathyroid cells were obtained in a physiologic solution containing (in mM): 140 NaCl, 5.4 KCI, 2 CaCl2, and 2 MgCl2. The pipette (intracellular) solution contained (in mM) 145 KAsp, 10(-5) CaCl2, and 2 MgCl2. Currents were recorded in response to 20-mV incremental changes in voltage of 300-ms duration every 3 s from -80 to +40 mV and from -40 to -140 mV. There was a small outward current recorded in response to 300-ms pulses of 20-mV increments from -80 to +40 mV. A large inward current was recorded following hyperpolarization of the parathyroid cell from -40 to -140 mV. The reversal potential for the current was -60 to -65 mV, suggesting that the majority of the current is carried by a channel that is K+ selective. Our results suggest that the whole cell currents of dispersed bovine parathyroid cells in physiologic extracellular solution include an in inwardly rectifying K+ current which is open at low intracellular calcium concentration. This inwardly rectifying K+ channel is likely to play a major role in maintaining negative membrane potential by opposing calcium-induced depolarization of the parathyroid cell and, as a result, may have an important role in regulation of PTH secretion.

A role for L-type calcium channels in developmental regulation of transmitter phenotype in primary sensory neurons.

To examine the influence of activity-dependent cues on differentiation of primary afferent neurons, we investigated the short- and long-term effects of depolarization and calcium influx on expression of transmitter traits in sensory ganglion cell cultures. We focused on expression of tyrosine hydroxylase (TH), a marker for dopaminergic neurons, in developing petrosal ganglion (PG), nodose ganglion, and dorsal root ganglion neurons grown in the presence or absence of depolarizing concentrations of KCl. Exposure to 40 mM KCl increased the proportion of TH-immunoreactive neurons in all three ganglia in a developmentally regulated manner that corresponded to the temporal pattern of dopaminergic expression in vivo. PG neurons, for example, were most responsive to elevated KCl on embryonic day 16.5 (E16.5), the age at which the dopaminergic phenotype is first detectable in vivo. However, KCl was relatively ineffective at increasing TH expression in neonatal PG, indicating a critical period for induction of this phenotype by depolarization. Detailed analysis of TH induction in PG neurons demonstrated that, although N-type calcium channels carried the majority of the high voltage-activated calcium current, only L-type calcium channel blockade inhibited the effect of elevated KCl. Further studies revealed that after removal of high KCl, neurons remained sensitized to subsequent stimulation for >1 week. Specifically, cultures exposed to KCl beginning on E16.5 (the conditioning stimulus), then returned to control medium, and subsequently re-exposed to elevated KCl after 9 d (the test stimulus) contained fourfold more TH-positive neurons than did cultures exposed to the test stimulus alone. Moreover, blockade of L-type calcium channels during the conditioning stimulus completely abolished long-term potentiation of the TH response to elevated KCl. These findings demonstrate a novel role for L-type calcium channels in activity-dependent plasticity of transmitter expression in sensory neurons and indicate that exposure to depolarizing stimuli during early development may alter neuronal response properties at later ages.

Properties of single Drosophila Trpl channels expressed in Sf9 insect cells.

The transient receptor potential (trp)-like (trpl) gene is thought to encode an ion channel important for signal transduction in Drosophila photoreceptor cells. Consistent with this hypothesis, heterologous expression of the trpl-encoded protein (Trpl) is associated with the appearance of an outwardly rectifying, nonselective cation current. In the present study, single channels were recorded in cell-attached, inside-out, and outside-out membrane patches from Sf9 insect cells infected with recombinant baculovirus-containing trpl cDNA under control of the polyhedrin promoter. The single-channel current-voltage relationship was linear from -100 to +80 mV with a slope conductance of 89-110 pS. The probability of opening was voltage sensitive, increasing at positive potentials contributing to the outwardly rectifying properties of the whole cell currents. The single channels 1) were never observed in Sf9 cells infected with recombinant baculovirus containing the B2 bradykinin receptor cDNA or in noninfected Sf9 cells; 2) appear at the same time postinfection as the Trpl whole cell current; 3) were nonselective with respect to Na+, Ca2+, and Ba2+; 4) were blocked by 1-2 mM La3+ and Gd3+ (but not 10 microM); and 5) were blocked by 4-8 mM Mg2+. The single Trpl channel activity increased spontaneously with time after patch formation, and the activity was further increased by application of bradykinin to cells expressing both the B2 bradykinin receptor and the Trpl protein. These results suggest that this single-channel activity reflects expression of the Trpl protein and provides conclusive evidence that trpl encodes a nonselective cation channel consistent with its proposed role in Drosophila phototransduction.

Experimental and modeling study of Na+ current heterogeneity in rat nodose neurons and its impact on neuronal discharge.

This paper is a combined experimental and modeling study of two fundamental questions surrounding the functional characteristics of Na+ currents in nodose sensory neurons. First, when distinctly different classes of Na+ currents are expressed in the same neuron, is there a significant difference in the intrinsic biological variability associated with the voltage- and time-dependent properties of these currents? Second, in what manner can such variability in functional properties impact the discharge characteristics of these neurons? Here, we recorded the whole cell Na+ currents in acutely dissociated rat nodose sensory neurons using the patch-clamp technique. Two general populations of neurons were observed. A-type neurons (n = 20) expressed a single rapidly inactivating tetrodotoxin-sensitive (TTX-S) Na+ current. C-type neurons (n = 87) coexpressed this TTX-S current along with a slowly inactivating TTX-resistant (TTX-R) Na+ current. The TTX-S currents in both cell types had submillisecond rates of activation at room temperature with thresholds near -50 mV. The TTX-R current exhibited about the same rates of activation but required potentials 20-30 mV more depolarized to reach threshold. Over the same clamp voltages the rates of inactivation for the TTX-R current were three to nine times slower than those for the TTX-S current. However, the TTX-R current recovered from complete inactivation at a rate 10-20 times faster than the TTX-S current (10 ms as compared with 100-200 ms). Across the population of neurons studied the TTX-S data formed a relatively tight statistical distribution, exhibiting low standard deviations across all measured voltage- and time-dependent properties. In contrast, the same pooled measurements on the TTX-R data exhibited standard deviations that were 3-10 times larger. The statistical profiles of the voltage- and time-dependent properties of these currents then were used as a physiological guide to adjust the relevant parameters of a mathematical model of nodose sensory neurons previously developed by our group (). Here, we show how the relative expression of TTX-S and TTX-R Na+ currents and the differences in their apparent biological variability can shape the regenerative discharge characteristics and action potential waveshapes of sensory neurons. We propose that the spectrum of variability robust reactivation characteristics of the TTX-R current are important determinants in establishing the heterogeneous stimulus-response characteristics often observed across the general population of C-type sensory neurons.

Oxidized glutathione mediates cation channel activation in calf vascular endothelial cells during oxidant stress.

1. The oxidant, tert-butylhydroperoxide (tBuOOH) depolarizes calf pulmonary artery endothelial cells by activating a non-selective cation channel. To identify the molecular mediator of channel activation during oxidant stress, the patch-clamp technique was used to compare tBuOOH-induced changes in membrane potential and channel activity with those induced by oxidized glutathione (GSSG), a cytosolic product of oxidant metabolism. 2. When recording pipettes contained GSSG (2 mM), whole-cell zero-current potential measured immediately following pipette break-in was not different from control values (-57 mV). However, within 20 min of break-in, zero-current potential was depolarized to -7 mV. The time course of depolarization was dependent on the concentration of GSSG and was accelerated by inhibition of GSSG metabolism. 3. In excised membrane patches, channels were activated by internal GSSG, but not by internal tBuOOH, reduced glutathione (GSH), or external GSSG. Channels were equal in size (28 pS) and in ionic selectivity to those activated by incubation of intact cells with tBuOOH. As little as 20 microM GSSG was sufficient to maximally activate channels. However, the time course of channel activation was concentration dependent between 20 microM and 2 mM GSSG. 4. Channel activation by GSSG was reversed by GSH and by increasing the [GSH]:[GSSG] ratio. Likewise, channel activation by pre-incubation of intact cells with tBuOOH was reversed by GSH applied after patch excision. 5. These results strongly suggest that GSSG is an endogenous intracellular mediator of channel activation and depolarization during oxidant stress.

Oxidant stress activates a non-selective cation channel responsible for membrane depolarization in calf vascular endothelial cells.

1. In vascular endothelial cells, oxidant stress increases cell Na+ content and inhibits the agonist-stimulated influx of external Ca2+. Further, oxidant stress increases uptake of Ca2+ into otherwise quiescent endothelial cells. To determine the mechanism responsible for altered Na+ and Ca2+ homeostasis, the present study examined the effect of oxidant stress on ionic current and channel activity in calf pulmonary artery endothelial cells. 2. Voltage-clamped control cells had a zero-current potential of -60 mV. Incubation of cells with the oxidant tert-butylhydroperoxide (tBuOOH; 0.4 mM, 1 h) caused depolarization to -4 mV and activation of ionic current equally selective for Na+ and K+. 3. Cell-attached membrane patches made on tBuOOH-treated cells contained ion channels that had a bidirectional conductance of 30 pS and that were not present in patches from control cells. Inside-out patches excised from oxidant-treated cells showed the channel to be equally selective for Na+ and K+ and to allow inward Ca2+ current. 4. Oxidant-activated channels were observed to display two gating modalities that were further evident during analysis of single-channel open probability. Neither modality was significantly affected by altering internal [Ca2+] (1 microM-10 nM). 5. Activation of non-selective channels provides a possible mechanism by which oxidants may increase endothelial cell Na+ content. Channel permeability to Ca2+ may account in part for the elevation of cytosolic free [Ca2+] that occurs in oxidant-treated cells. 6. Channel activation is associated with membrane depolarization, a mechanism that may contribute to oxidant inhibition of the agonist-stimulated Ca2+ influx pathway.

The COOH-terminal domain of Drosophila TRP channels confers thapsigargin sensitivity.

Previous studies have shown that the Drosophila cation channels designated Trp and Trpl can be functionally expressed in Sf9 insect cells using baculovirus expression vectors. The trp gene encodes a Ca2+-permeable channel that is activated by thapsigargin, blocked by low micromolar Gd3+, and is relatively selective for Ca2+ versus Na+ and Ba2+. In contrast, trpl encodes a Ca2+-permeable cation channel that is constitutively active, not affected by thapsigargin, blocked by high micromolar Gd3+, and non-selective with respect to Ca2+, Na+, and Ba2+. The region of lowest sequence identity between Trp and Trpl occurs in the COOH-terminal domain. To test the hypothesis that this region is responsible for the differential sensitivity of these channels to thapsigargin, chimeric constructs of Trp and Trpl were created in which the COOH-terminal tail region of each protein was exchanged. The Trp construct with the Trpl COOH-tail was constitutively active, insensitive to thapsigargin, but retained selectivity for Ca2+ over Na+ and Ba2+. In contrast, the Trpl construct with the Trp COOH-tail was not constitutively active, could be activated by thapsigargin, but remained non-selective with respect to Ca2+, Ba2+, and Na+. These results suggest that the COOH-terminal domain of Trpl plays an important role in determining constitutive activity, whereas the COOH-terminal region of Trp contains the structural features necessary for activation by thapsigargin.

Thrombin receptors activate potassium and chloride channels.

We used DAMI human megakaryocytic leukemia cells to study transmembrane ion currents activated through the G-protein-coupled thrombin receptor pathway. When the cells were stimulated by thrombin receptor-activating peptide, an increase in cytosolic Ca2+ ([Ca2+]i) developed as predicted by the known effect that thrombin exerts in the platelet. We then monitored the membrane potentials of individual DAMI cells during this response and observed complex, triphasic changes that could not be accounted for by Ca2+ fluxes alone. These consisted of rapid hyperpolarization, followed by depolarization to values more positive than the resting potential and then by slow repolarization. For the purpose of this study, we focused on the hyperpolarizing current that developed immediately after thrombin receptor activation. This proved to be composed of (1) a Ca(2+)-independent, outwardly rectifying Cl- current and (2) a strongly hyperpolarizing, inwardly rectifying, Ba(2+)-sensitive K+ current that required an increase of [Ca2+]i for activation. By analogy with their functions in other cell systems, it is logical to conclude that these prominent K+ and Cl- conductances may serve to regulate the complex volume changes that accompany thrombin receptor activation and/or to increase the electromotive drive that supports Ca2+ influx under these conditions through hyperpolarization of the cell membrane.

Ins(1,4,5)P3 activates Drosophila cation channel Trpl in recombinant baculovirus-infected Sf9 insect cells.

The trp-like (trpl) gene product (Trpl) is thought to form a nonselective cation channel important for signal transduction in Drosophila photoreceptor cells. This channel may be the insect homologue of mammalian channels involved in Ca2+ signal transduction. To determine the mechanism of receptor-mediated activation of Trpl, whole cell membrane currents were examined in Sf9 insect cells after infection with recombinant baculovirus. Stimulation by bradykinin increased whole cell Trpl currents three- to fivefold. Similar activation of Trpl was observed by inclusion of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the pipette solution during whole cell recordings. These currents were 1) not seen in noninfected cells or in cells expressing only the B2 receptor, 2) mimicked by D-myo-inositol 2,4,5-trisphosphate, and 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate, 3) not seen with D-myo-inositol 1,4-bisphosphate or D-myo-inositol 1,3,4,5-tetrakisphosphate, and 4) blocked by heparin, but not by de-N-sulfated heparin. In contrast, Trpl currents were unaffected by thapsigargin. These results demonstrate that the Trpl cation channel is activated by Ins(1,4,5)P3 in a heparin-sensitive fashion. Regulation of channel activity by Ins(1,4,5)P3 may occur by a number of mechanisms, including direct binding of Ins(1,4,5)P3 to the Trpl channel or direct physical interaction between the Ins(1,4,5)P3 receptor/Ca(2+)-release channel of the endoplasmic reticulum and the Trpl protein.

Afferent synaptic drive of rat medial nucleus tractus solitarius neurons: dynamic simulation of graded vesicular mobilization, release, and non-NMDA receptor kinetics.

1. We have developed a comprehensive mathematical model of an afferent synaptic connection to the soma of a medial nucleus tractus solitarius (mNTS) neuron. Model development is based on numerical fits to quantitative data recorded in our laboratory. This work is part of a continuing collaborative effort aimed at identifying and characterizing the mechanisms responsible for the non-linear integrative properties of this first synapse in the baroreceptor reflex. 2. The complete model consists of three major parts: 1) a Hodgkin-Huxley (HH)-type membrane model of the prejunctional sensory terminal bouton; 2) a multistage model describing vesicular storage, adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca(2+)-dependent mobilization, release and recycling; and 3) a HH-type membrane model of the postjunctional mNTS cell that includes descriptions for a desensitizing non-N-methyl-D-aspartate (NMDA) ionic current that is responsible for the fast excitatory postsynaptic potentials (EPSPs) observed in mNTS cells. The membrane models for both the terminal bouton and the mNTS neuron are coupled to separate lumped fluid compartment models describing intracellular Ca2+ ion concentration dynamics. 3. Our modeling strategy is twofold. The first is to validate model performance by reproducing a wide variety of experimental data both from our laboratory and from the literature. The second is to explore the functional aspects of the model in order to gain a greater appreciation for the balance between presynaptic mechanisms (e.g., terminal membrane properties and vesicular dynamics) and postsynaptic mechanisms (e.g., non-NMDA receptor kinetics and neuronal dynamics) that underlie the afferent synaptic drive of mNTS neurons. 4. The model accurately reproduces EPSP dynamics recorded with the use of a wide range of stimulus protocols. The model can also mirror the unique pattern of graded frequency- and use-dependent reduction in peak EPSP magnitude observed experimentally through 60 s of constant, suprathreshold synaptic activation. We demonstrate how vesicular mobilization, recycling, and receptor kinetics can function synergistically in establishing synaptic transfer. Furthermore, we show that by allowing the aggregate rate of vesicle mobilization to respond in a use-dependent manner, it is possible to compensate for the attenuating affects of desensitization at elevated rates of stimulation. 5. Our simulations indicate that the low-frequency characteristics of this synapse are dominated by vesicular dynamics, whereas the high-frequency properties arise from a combination of Ca(2+)-dependent vesicular mobilization and the kinetics of the non-NMDA receptor. Desensitization can influence the peak magnitude and decay time of the EPSP, thereby affecting synaptic throughput. However, we demonstrate that, as the time course of neurotransmitter in the synaptic cleft decreases, the influence of desensitization should be somewhat diminished. As a result, the effective bandwidth of the synapse increases and becomes limited by the gating characteristics of the non-NMDA channel. 6. The model also includes a neuromodulatory aspect in that the frequency response of the synapse can be modulated by an adenylate cyclase-mediated regulatory mechanism. Although our simulations indicate the behavior of a limited number of possible neuromodulatory agents, the results demonstrate the pivotal role such agents could play in modifying synaptic transfer characteristics presynaptically. 7. Both continuous and burst-mode tract stimulation evoke patterns of action potentials in spontaneously active mNTS neurons that are mimicked very well by our model. Our simulations demonstrate that, as the rate of stimulation increases beyond approximately 20-30 Hz, the inherent low-pass frequency-response characteristics of the synapse limit the overall dynamic range of the mNTS neuron, causing the postsynaptic cell to "entrain" at frequencies within its normal operating range.

IP3-activated Ca2+ channels in the plasma membrane of cultured vascular endothelial cells.

Although it is clear that D-myo-inositol 1,4,5-trisphosphate (IP3) plays an important role in the activation of Ca2+ influx, the mechanisms by which this occurs remain controversial. In an attempt to determine the role of IP3 in the activation of Ca2+ influx, patch-clamp single-channel experiments in the cell-attached, inside-out, and outside-out configurations were performed on cultured bovine aortic endothelial cells (BAEC). The results presented indicate that both IP3 and intracellular Ca2+ can modulate the activity of a Ca(2+)-selective channel found in the plasma membrane of these cells. Addition of 10 microM IP3 increased channel open probability (P(o)) from a control value of 0.12 +/- 0.05 to 0.7 +/- 0.13 at a constant intracellular Ca2+ of 1 nM in excised inside-out patches. D-Myo-inositol 1,3,4,5-tetrakisphosphate at 50 microM was ineffective in altering channel P(o). Channel activity declined after approximately 2 min in the continuous presence of IP3. Three to four minutes after addition of IP3, channel P(o) was reduced from 0.7 +/- 0.2 to 0.2 +/- 0.1, indicating that an additional regulator might be required to maintain channel activity in excised patches. The channel was reversibly blocked by application of 1 microgram/ml heparin to the intracellular side of inside-out patches. This Ca(2+)-selective channel is indistinguishable from the depletion-activated Ca2+ channel we have previously described in BAEC.

Activation of recombinant trp by thapsigargin in Sf9 insect cells.

The mammalian protein responsible for Ca2+ release-activated current (Icrac) may be homologous to the Drosophila protein designated trp. Thus the activity of trp, and another Drosophila protein designated trp-like or trpl, may be linked to depletion of the internal Ca2+ store via the so-called capacitative Ca2+ entry mechanism. To test this hypothesis, the effect of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+ pump, on trp- and trpl-induced whole cell membrane current was determined using the baculovirus Sf9 insect cell expression system. The results demonstrate that trp and trpl form Ca(2+)-permeable cation channels. The trpl encodes a nonselective cation channel that is constitutively active under basal nonstimulated conditions and is unaffected by thapsigargin, whereas trp is more selective for Ca2+ than Na+ and is activated by depletion of the internal Ca2+ store. Although evaluation of cation selectivity suggests that trp is not identical to the channel responsible for Icrac, these channels must share some structural feature(s) since both are activated by thapsigargin. A unique proline-rich region in the COOH-terminal tail of trp, which is absent in trpl, may be necessary for capacitative Ca2+ entry.

Dual effects of angiotensin II on calcium currents in neonatal rat nodose neurons.

Angiotensin II (AII) reversibly modulates calcium current in isolated neonatal rat nodose ganglion cells by two different pathways. A maximum inhibitory effect of 43 +/- 6% (n = 25) of the peak calcium current at -10 mV was observed at 10 nM AII. The IC50 of the inhibitory response was 100 pM. Losartan, a specific antagonist for the AT1 type of AII receptor, abolished the AII-induced inhibition, as did preincubation with pertussis toxin (PTX). When omega-conotoxin GVIA (CTX) was added to the bath solution, AII produced no inhibition of the remaining calcium current, indicating that the AII inhibition was mediated through CTX-sensitive calcium channels. Reversible facilitation of calcium current was seen more rarely. The AII-induced facilitation was unaffected by losartan and PTX, indicating that the effect is mediated by a non-AT1 receptor and does not depend upon a PTX-sensitive G-protein. The facilitation is present when the CTX-sensitive current has been blocked and involves activation of a reserve pool of dihydropyridine (DHP)-sensitive channels. In general, a particular neuron exhibited either inhibition or facilitation. However, in some neurons both inhibition and facilitation could be demonstrated in the presence of the appropriate blocking agents.

Depletion of intracellular Ca2+ stores activates a Ca(2+)-selective channel in vascular endothelium.

The present study was designed to identify the channel responsible for Ca2+ influx after depletion of intracellular Ca2+ stores. Different maneuvers that deplete intracellular Ca2+ stores activated a Ca(2+)-selective channel. Superfusion of single bovine aortic endothelial cells with 50 nmol/l bradykinin, 10 mumol/l ATP, or 10 mumol/l 2,5-di(tert-butyl)-1,4-benzohydroquinone produced activation of channels of the same amplitude in cell-attached patches. Channel activity declined within the first minute after patch excision. The channel showed strong inward rectification and a reversal potential of 0 mV in symmetrical sodium sulfate (Na2SO4) solution. Under these conditions, the conductance was 5 pS in the inward direction. Addition of 10 mmol/l Ca2+ to the extracellular solution shifted the reversal potential to +30 +/- 5 mV, and the conductance for inward current was 11 pS. The reversal potential was used to calculate an ion permeability ratio of Ca2+/Na+ > 10:1.