Biomedical subjects

V Vlachová

Publications and source records attributed to V Vlachová.

At least 19 recordsLinked to original sources

PubMed2004

Activation and modulation of ligand-gated ion channels.

Ligand-gated ionic channels are integral membrane proteins that enable rapid and selective ion fluxes across biological membranes. In excitable cells, their role is crucial for generation and propagation of electrical signals. This survey describes recent results from studies performed in the Department of Cellular Neurophysiology, Institute of Physiology ASCR, aimed at exploring the conformational dynamics of the acetylcholine, glutamate and vanilloid receptors during their activation, inactivation and desensitization. Distinct families of ion channels were selected to illustrate a rich complexity of the functional states and conformational transitions these proteins undergo. Particular attention is focused on structure-function studies and allosteric modulation of their activity. Comprehension of the fundamental principles of mechanisms involved in the operation of ligand-gated ion channels at the cellular and molecular level is an essential prerequisite for gaining an insight into the pathogenesis of many psychiatric and neurological disorders and for efficient development of novel specifically targeted drugs.

Allosteric Regulation↗

PubMed2002

The effects of excessive heat on heat-activated membrane currents in cultured dorsal root ganglia neurons from neonatal rat.

The effects of high temperature (53-61 degrees C) on membrane currents (I(heat)) or depolarization (V(heat)) induced by noxious heat were studied in cultured dorsal root ganglia neurons from neonatal rats using the whole cell patch clamp technique. I(heat) or V(heat) produced by 3 s ramps of increasing temperature between 43 and 50 degrees C exhibited a fast slope (Q10>10) that was similar both during rising and falling temperature (n=85). Temperatures exceeding 52 degrees C resulted in slowdown in the recovery of I(heat), and the threshold for inducing I(heat) was shifted to lower temperatures in successive trials. These high temperatures (54-60 degrees C) caused a linear and incomplete recovery of I(heat) (Q10 decreased to <5; 4.5 +/- 0.4; n=17) and in successive trials the threshold of I(heat) decreased to temperatures close to that in the bath. The neurons, however, remained sensitive to capsaicin and to decreased extracellular pH. It is suggested that exposure of nociceptive neurons to excessive noxious heat results in an irreversible decrease of the energy barrier between the resting and activated state of the protein structures responsible for generation of I(heat). This may explain the sensitization of nociceptors after heat injury.

Animals↗

PubMed2002

Reducing agent dithiothreitol facilitates activity of the capsaicin receptor VR-1.

The vanilloid receptor subtype 1 (VR1) is expressed in a sub-population of small dorsal root ganglion (DRG) neurones in mammals and serves as the common transducer of the pain-producing signals, such as noxious heat, acids and capsaicin [Caterina et al., Nature 389 (1997) 816-824; Tominaga et al., Neuron 21 (1998) 531-543]. On the extracellular side, VR1 has three cysteine residues at positions 616, 621 and 634. Here we report that dithiothreitol (DTT) (2-60 mM), an agent that maintains -SH groups of cysteines in a reduced state, greatly facilitates membrane currents induced by noxious heat or capsaicin (1 microM) in cultured DRG neurones from the rat and in VR1-transfected HEK293 cells. The effects of DTT are concentration-dependent and fully reversible. We suggest that the ratio between free sulfhydryl groups and disulfide bonds of the cysteine residues of VR1 pre-sets sensitivity of primary nociceptors to algogens and may represent a new target for treating some pain states in humans.

Animals↗

PubMed2002

Cellular mechanisms of nociception in the frog.

Cellular mechanisms underlying defense reactions induced by noxious heat and acids were studied in frogs (Rana pipiens) by measuring whole cell membrane currents in cultured dorsal root ganglion (DRG) neurons. Seventy-eight of 82 DRG neurons exposed to 3-s ramps of increasing temperature to 48 degrees C exhibited an inward current (I(HEAT)) of 490 +/- 70 pA at -70 mV. I(HEAT) exhibited reversal at approximately 10 mV with a pronounced outward rectification, suggesting opening of nonselective cation channels. In frogs, in contrast to mammals, I(HEAT) was not influenced by capsaicin (5 microM), capsazepine (10 microM), or ruthenium red (10 microM). In a large proportion (approximately 80%) of heat-sensitive DRG neurons, acids produced a large slowly inactivating sodium carried current (I(ACID)) with average pH(50) 5.7. I(ACID) was blocked by 1 mM amiloride (to 22%) and was absent if extracellular Na(+) was substituted by Cs(+). Elevating temperature to 38 degrees C increased I(ACID), whereas temperatures >40 degrees C profoundly inhibited it (by 82 +/- 2%; n = 42). The inhibition was long-lasting (>30 s) but fully reversible. Phorbol ester myristate acetate (PMA, 1 microM) and forskolin (1 microM) inhibited I(ACID) to 37 +/- 5% (n = 5) and 78 +/- 8% (n = 4), respectively. It is suggested that I(HEAT) in frog DRG neurons is carried through capsaicin-insensitive nonselective cation channels distinct from vanilloid receptor in mammals, whereas I(ACID) is carried through amiloride-sensitive sodium channels that are strongly inhibited by noxious heat, possibly due to activation of the intracellular messenger systems.

Acids↗

PubMed2002

Suramin affects capsaicin responses and capsaicin-noxious heat interactions in rat dorsal root ganglia neurones.

The effect of suramin, an inhibitor of G protein regulated signalling, was studied on the membrane currents induced by noxious heat and by capsaicin in cultured dorsal root ganglia neurones isolated from neonatal rats. Whole-cell responses induced by a heat ramp (24-52 degrees C) were little affected by suramin. The noxious heat-activated currents were synergistically facilitated in the presence of 0.3 microM capsaicin 13.2-fold and 6.3-fold at 40 degrees C and 50 degrees C, respectively. In 65% of neurones, the capsaicin-induced facilitation was inhibited by 10 microM suramin to 35 +/- 6% and 53 +/- 6% of control at 40 degrees C and 50 degrees C (S.E.M., n = 15). Suramin 30 microM caused a significant increase in the membrane current produced by a nearly maximal dose (1 microM) of capsaicin over the whole recorded temperature range (2.4-fold at 25 degrees C and 1.2-fold at 48 degrees C). The results demonstrate that suramin differentially affects the interaction between capsaicin and noxious heat in DRG neurones and thus suggest that distinct transduction pathways may participate in vanilloid receptor activation mechanisms.

Animals↗

PubMed2001

The effects of capsaicin and acidity on currents generated by noxious heat in cultured neonatal rat dorsal root ganglion neurones.

1. The effects of capsaicin, acidic pH, ATP, kainate and GABA on currents generated by noxious heat were studied in cultured dorsal root ganglion (DRG) neurones (< 20 microm in diameter) isolated from neonatal rats. The patch clamp technique was used to record membrane currents or changes of membrane potential. 2. In agreement with previous results, inward membrane currents (I(heat)) induced by a 3 s ramp of increasing temperature from room temperature (approximately 23 degrees C) to over 42 degrees C varied greatly between cells (-100 pA to -2.4 nA at 48 degrees C) and had a temperature coefficient (Q(10)) > 10 over the range of 43-52 degrees C. 3. Capsaicin potentiated the heat-induced current even when capsaicin, at room temperature, had little or no effect on its own. In cells in which capsaicin induced no or very small membrane current at room temperature (< 50 pA), I(heat) exhibited detectable activation above 40 degrees C and increased 5.1 +/- 1.1 (n = 37) and 6.3 +/- 2.0 (n = 18) times at 0.3 and 1 microM capsaicin, respectively. 4. A rapid decrease in extracellular pH from 7.3 to 6.8, 6.3 or 6.1 produced an inward current which inactivated in ~5 s either completely (pH 6.8 or 6.3) or leaving a small current (approximately 50 pA) for more than 2 min (pH 6.1). After inactivation of the initial low pH-induced current, I(heat) at 48 degrees C increased 2.3 +/- 0.4 times at pH 6.8, 4.0 +/- 0.6 times at pH 6.3 and 4.8 +/- 0.8 times at pH 6.1 with a Q(10) > 10 (n = 16). 5. ATP (n = 22), kainate (n = 7) and GABA (n = 8) at 100 microM, produced an inactivating inward current in all heat-sensitive DRG neurones tested. During inactivation and in the presence of the drug, I(heat) was increased slightly with ATP and unaffected with kainate and GABA. These agents apparently do not directly affect the noxious heat receptor. 6. The results indicate a novel class of capsaicin-sensitive cells, in which capsaicin evokes no or very small inward current but nevertheless increases sensitivity to noxious heat.

Acids↗

PubMed2000

Axotomy-induced change in the properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor channels in rat motoneurons.

Properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels were studied in fluorescence-labelled control and axotomized motoneurons in spinal cord slices using a patch-clamp technique. Axotomy performed on the third postnatal day resulted in motoneuron death. Application of AMPA or kainate induced large whole-cell currents, but outside-out patches isolated from control motoneurons were either unresponsive or displayed only single-channel activity in response to rapid application of AMPA. Measurement of AMPA receptor channel openings in outside-out patches revealed multiple single-channel conductance levels: 12.2+/-1.0, 21. 9+/-1.5 and 32.6+/-3.2pS. In control motoneurons dialysed with spermine, the current-voltage relationship of responses induced by activation of AMPA receptor channels exhibited various degrees of inward rectification. The rectification index, the ratio of responses at +40 and -60mV, was used to compare the degree of inward rectification. The mean values of rectification index of responses to focal application of AMPA and AMPA receptor-mediated excitatory postsynaptic currents induced by focal electric stimulation were 0. 64+/-0.17 and 0.50+/-0.27, respectively. In axotomized motoneurons, the degree of rectification was significantly less for both responses induced by application of AMPA and for excitatory postsynaptic currents (0.91+/-0.09 and 0.95+/-0.12, respectively). Deactivation of AMPA receptors assessed from motoneuron excitatory postsynaptic currents at -70 mV was independent of postnatal age, with tau(fast)=0.88+/-0.35ms (A(fast)=78.2+/-11.8%) and tau(slow)=6. 3+/-3.2ms. In axotomized motoneurons, the decay time constants of excitatory postsynaptic currents were similar, tau(fast)=0.91+/-0. 42ms (A(fast)=85.8+/-12.6%) and tau(slow)=5.9+/-3.4ms. However, the mean amplitude of excitatory postsynaptic currents was only 43% of the amplitude recorded in control motoneurons. The results show that the current induced by activation of AMPA receptors in neonatal motoneurons is mediated by opening of both Ca(2+)-permeable and Ca(2+)-impermeable channels. As a result of axotomy, an experimental model of neurodegeneration, AMPA receptor channels in injured motoneurons destined to die become predominantly Ca(2+) impermeable. These findings suggest phenotypic control of AMPA receptor channel properties, presumably by affecting their subunit composition.

Animals↗

PubMed2000

Molecular and functional properties of synaptically activated NMDA receptors in neonatal motoneurons in rat spinal cord slices.

The functional properties of N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSC) were studied in fluorescence-labelled motoneurons in thin spinal cord slices. The deactivation of NMDA receptor EPSCs in motoneurons voltage-clamped at +40 mV was independent of intensity or location of stimulation and of postnatal age [taufast = 28.5 +/- 4.6 ms (63.6 +/- 8.8%) and tauslow = 165.6 +/- 49.6 ms]. In the presence of 1 mM Mg2+ the amplitude of NMDA receptor EPSCs was voltage-dependent. Boltzmann analysis of the relationship between peak NMDA receptor EPSC amplitude and membrane potential suggested an apparent Kd of Mg2+ (at 0 mV) of 0.87 mM. Nonstationary variance analysis of NMDA receptor EPSCs gave an estimated single-channel conductance of 59 +/- 14 pS. Direct measurement of the NMDA receptor channel openings in outside-out patches isolated from motoneurons indicated the presence of single-channel conductance levels of 21.8 +/- 2.8 pS, 37. 1 +/- 3.2 pS, 49.6 +/- 5.1 pS and 69.6 +/- 4.2 pS. Single-cell RT-PCR analysis of mRNA revealed that NR1, NR2A-D and NR3A transcripts were expressed in motoneurons. These results suggest that specific assembly of NMDA receptor subunits in motoneurons determines the functional and pharmacological properties of the receptors in these cells.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1999

Temperature coefficient of membrane currents induced by noxious heat in sensory neurones in the rat.

1. Membrane currents induced by noxious heat (Iheat) were studied in cultured dorsal root ganglion (DRG) neurones from newborn rats using ramps of increasing temperature of superfusing solutions. 2. Iheat was observed in about 70 % of small (< 25 microm) DRG neurones. At -60 mV, Iheat exhibited a threshold at about 43 C and reached its maximum, sometimes exceeding 1 nA, at 52 C (716 +/- 121 pA; n = 39). 3. Iheat exhibited a strong temperature sensitivity (temperature coefficient over a 10 C temperature range (Q10) = 17.8 +/- 2.1, mean +/- s.d., in the range 47-51 C; n = 41), distinguishing it from the currents induced by capsaicin (1 microM), bradykinin (5 microM) and weak acid (pH 6.1 or 6.3), which exhibited Q10 values of 1.6-2.8 over the whole temperature range (23-52 C). Repeated heat ramps resulted in a decrease of the maximum Iheat and the current was evoked at lower temperatures. 4. A single ramp exceeding 57 C resulted in an irreversible change in Iheat. In a subsequent trial, maximum Iheat was decreased to less than 50 %, its threshold was lowered to a temperature just above that in the bath and its maximum Q10 was markedly lower (5.6 +/- 0.8; n = 8). 5. DRG neurones that exhibited Iheat were sensitive to capsaicin. However, four capsaicin-sensitive neurones out of 41 were insensitive to noxious heat. There was no correlation between the amplitude of capsaicin-induced responses and Iheat. 6. In the absence of extracellular Ca2+, Q10 for Iheat was lowered from 25.3 +/- 7.5 to 4. 2 +/- 0.4 (n = 7) in the range 41-50 C. The tachyphylaxis, however, was still observed. 7. A high Q10 of Iheat suggests a profound, rapid and reversible change in a protein structure in the plasma membrane of heat-sensitive nociceptors. It is hypothesized that this protein complex possesses a high net free energy of stabilization (possibly due to ionic bonds) and undergoes disassembly when exposed to noxious heat. The liberated components activate distinct cationic channels to generate Iheat. Their affinity to form the complex at low temperatures irreversibly decreases after one exposure to excessive heat.

Animals↗

PubMed1999

Procaine excites nociceptors in cultures from dorsal root ganglion of the rat.

Procaine, a classical local anesthetic, produces, at low concentration (2-200 microM), excitation in a distinct population of small sensory neurons isolated from newborn rats (2D) and cultured for 1-5 days. The excitation or inward current (>50 pA) induced by procaine was observed in 59 out of 78 neurons. Nearly all these procaine-sensitive neurons (56 of 59) were also sensitive to capsaicin while 8 procaine-insensitive neurons responded to capsaicin (1 microM). In procaine-sensitive neurons tested for responsiveness to noxious heat, a 10 s temperature ramp from 24 to 48 degrees C induced an inward current of 413 +/- 47 pA (SEM, n = 27) and this current was enhanced, in the presence of procaine, about 3-fold (2.8 +/- 0.4, SEM, n = 27). The responses to procaine were concentration dependent and underwent pronounced tachyphylaxis after repeated applications. The voltage-current relationship exhibited outward rectification and the apparent reversal at 25 +/- 4.2 mV (SEM, n = 9) suggesting that the current is carried by cations including Ca2+. This procaine effect may offer an explanation for toxic consequence of the clinical use of local anesthetics.

Anesthetics, Local↗

PubMed1999

Properties of NMDA receptors in rat spinal cord motoneurons.

Postnatal development and properties of N-methyl-d-aspartate (NMDA) receptors were studied with whole-cell and outside-out patch-clamp techniques in interneurons and fluorescence-labelled motoneurons in rat spinal cord slices. Both the absolute amplitude of NMDA-induced currents and currents normalized with respect to the motoneuron capacitance increased significantly at postnatal days 10-13 when compared to the responses evoked at postnatal days 2-3. The mean amplitude of the responses to kainate also increased in motoneurons of postnatal days 10-13. Single-channel currents induced by low concentrations of glutamate, exhibited four distinct amplitude levels corresponding to 19.2 +/- 2.4 pS, 38.4 +/- 3.5 pS, 56.3 +/- 2. 4 pS and 69.6 +/- 3.7 pS. In contrast, the conductance of single channels, recorded under identical conditions, in rat spinal cord interneurons was less, 15.3 +/- 3.2 pS, 29.9 +/- 5.4 pS, 46.7 +/- 4. 8 pS and 62.4 +/- 3.9 pS. The high (56/70 pS) conductance single-channel openings in motoneuron patches were sensitive to NMDA receptor inhibitors D-2-amino-5-phosphonovalerate, 7-chlorokynurenic acid and ifenprodil. Whole-cell NMDA-evoked currents were blocked in a voltage-dependent manner by extracellular Mg2+ with an apparent dissociation constant for Mg2+ binding at 0 mV of 1.8 +/- 0.5 mm. The conductance and relative distribution of NMDA receptor channel openings induced by 1 micrometer glutamate in patches isolated from the motoneurons were independent of age from postnatal day 4 to 14. The results suggest that the properties of NMDA receptor channels in motoneurons differ from those in spinal cord interneurons and cells transfected with NR1/NR2 subunits.

2-Amino-5-phosphonovalerate↗

PubMed1998

A technique for fast application of heated solutions of different composition to cultured neurones.

A technique is described that allows the application of fast temperature changes (time constant approximately 300 ms) of solutions superfusing cultured neurones under whole-cell mode of membrane current recording. Its principle is in heating the common outlet of the manifold which consists of 12 tubes connected to barrels containing test solutions of different composition. The outlet is made from a glass capillary (25 mm length, 620/350 microns outer/inner diameter) coated on the outside wall with platinum for a length of 12 mm. The heating element, a platinum layer, is electrically connected to the probe fixed to the micromanipulator used for positioning the manifold. The solutions, driven by gravity, are applied by opening electronic valves controlled either manually or in programmed sequences. The DC current for heating is controlled either manually or by external voltage command. The advantage of the technique is that the same temperature pattern can be applied to 12 different solutions. The technique is used for classifying sensory neurones in culture with respect to their sensitivity to heat and algogens; however, it is applicable to any study of the effects of increased temperature on the activity of ion channels in cultured cells.

Cell Membrane↗

PubMed1998

Inflammatory mediators at acidic pH activate capsaicin receptors in cultured sensory neurons from newborn rats.

Whole cell membrane currents induced by the inflammatory mediators, bradykinin, 5-hydroxytryptamine (5-HT) and prostaglandin E2, were investigated in capsaicin-sensitive dorsal root ganglion (DRG) neurons from newborn rats grown on a monolayer of hippocampal glia without nerve growth factor (NGF). When firmly attached to an underlying cell, the neurons survived >14 days without growing extensive processes. A majority of the small diameter neurons ( approximately 80%) exhibited sensitivity to capsaicin (3-6 muM) and this was enhanced in solution of low pH. In acidic extracellular solution (pH 6.1), the combination of bradykinin (10 microM), 5-HT (10 microM) and prostaglandin E2 (1 microM) induced an inward membrane current in all capsaicin-sensitive DRG neurons (n = 43). The current exceeded the sustained, low pH-induced membrane current by 205 +/- 53 (SE) pA. The combination of acidic inflammatory mediators was ineffective in cells that were insensitive to capsaicin. In capsaicin-sensitive neurons, the inflammatory mediators when applied singly or in any combination of two, induced no membrane currents or small current at pH 7.3 and 6.1. Capsazepine (10 microM), the capsaicin antagonist, completely inhibited the facilitatory action of inflammatory mediator combination but not the sustained inward current induced by acidic extracellular solution (pH 6.1 or 5.5). It is suggested that the inflammatory mediators, bradykinin,5-HT, and prostaglandin E2 together act as endogenous mediators at capsaicin receptors to generate an inward current when the ion channel is protonized.

Animals↗

PubMed1997

Spontaneous openings of NMDA receptor channels in cultured rat hippocampal neurons.

Spontaneous and N-methyl-D-aspartate (NMDA)-evoked single-channel currents were studied in outside-out patches isolated from cultured rat hippocampal neurons. Both spontaneous and NMDA-evoked single-channel currents reversed at potentials close to 0 mV and exhibited multiple amplitude levels of similar amplitude. Both spontaneous and NMDA-evoked single-channel currents were inhibited by Mg2+ in a voltage-dependent manner and by 7-chlorokynurenic acid. The activity of spontaneous single-channel currents was reduced by the competitive NMDA receptor antagonists, but by one to three orders of magnitude less than expected assuming that the spontaneous activity is due to an ambient NMDA receptor agonist present in the extracellular solution. Our results suggest that, similar to other ligand-gated ion channels, NMDA receptor channels have a dual mode of activation--spontaneous and agonist induced.

Animals↗

PubMed1996

Copper modulation of NMDA responses in mouse and rat cultured hippocampal neurons.

The effect of Cu2+ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole-cell patch-clamp and a fast perfusion system. Analysis of the Cu2+ concentration-response curve for inhibition of NMDA-induced currents suggests that free Cu2+ directly inhibits NMDA receptors with an IC50 of 0.27 microM. Cu2+ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA-Cu2+ and glycine-Cu2+ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu2+ (10-100 microM) of responses to 10 microM NMDA was essentially voltage-independent. The onset of inhibition by 100 microM Cu2+ of responses to 2 microM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that Cu2+ acts as a non-competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu2+ to NMDA receptors, calculated from the rate of onset of block by Cu2+ of test responses to NMDA, was 19 times slower than in the presence of 30 microM NMDA, suggesting that Cu2+ interacts preferentially with agonist-bound receptors. Our results show that Cu2+ is a potent inhibitor of NMDA receptor-mediated responses.

Animals↗

PubMed1995

G-protein modulation of glycine-resistant NMDA receptor desensitization in rat cultured hippocampal neurons.

Activation of N-methyl-D-aspartate (NMDA) subtype glutamate receptors increases the excitability of most neurons within the CNS. A common feature of ionotropic glutamate receptors is their ability to undergo desensitization. In the present experiments we have examined the role of guanine nucleotide-binding proteins (G-proteins) in the regulation of NMDA receptor desensitization. Repeated NMDA receptor activation with 2 mM extracellular Ca2+ increased the degree of glycine-resistant NMDA receptor desensitization of subsequent responses to NMDA recorded in the presence of 0.2 mM Ca2+. The recovery of glycine-resistant NMDA receptor desensitization after repeated NMDA receptor activation in the presence of 2 mM Ca2+ was significantly reduced in neurons intracellularly dialysed with guanosine-5'-O-(3-thiotriphosphate), guanosine-5'-triphosphate or AlCl3 and CsF, compounds known to activate G-proteins. Intracellular dialysis with guanosine-5'-O-(2-thiodiphosphate), adenosine triphosphate, adenosine triphosphate, or adenosine-5'-O-(3-thiotriphosphate) was ineffective. The calcium permeability of NMDA receptor-channels was not altered by intracellular dialysis with GTP gamma S. This suggests that modulation of NMDA receptor desensitization by G-proteins represents a novel mechanism for regulation of glutamate-gated ion channel activity.

Animals↗

PubMed1993

Capsaicin-induced membrane currents in cultured sensory neurons of the rat.

Membrane currents induced by capsaicin (CAPS) in cultured sensory neurons from 1- to 2-day-old rats were studied. Responses to CAPS (10 microM) exceeding 1 nA at -50 mV were found in smaller, usually bipolar or tripolar neurons in which GABA (30 microM) induced small or no response. Large, unipolar neurons, which exhibited large responses to GABA, were completely insensitive to CAPS (10 microM). In contrast to GABA, responses to CAPS exhibited a slow rise and slow decay and a marked tachyphylaxis after repeated CAPS applications at high concentrations which made it difficult to study the concentration-response relationship. In partially run-down neurons, which exhibited quasi stable responses, the slope of the ascending phase was concentration-dependent with an apparent association rate constant K1 9 x 10(4) [M-1s-1]. The time constant of the decay was 3.5 s, and was concentration-independent. However, in 5 neurones the EC50 measured from the first series of CAPS applications at increasing concentrations was 0.31 +/- 0.5 microM with a Hill coefficient 1.66 +/- 0.35. The responses to CAPS reversed at +10.4 +/- 2.5 mV suggesting that the current is carried nonselectively by monovalent cations and Ca2+. The channel conductance of CAPS-gated channels at -50 mV calculated from the mean membrane current and variance of the current noise in outside-out patches or measured directly was 28 pS (n = 5). It is suggested that the CAPS-gated channels are either controlled by receptors with a very high affinity or that the channels are controlled by membrane-bound protein(s) which do not depend in their function on the supply of GTP or other intracellular metabolites.

Animals↗

PubMed1992

Membrane currents induced by L-homocysteic acid in mouse cultured hippocampal neurons.

The concentration-response relationship of membrane currents induced by L-homocysteic acid was studied on mouse embryonic hippocampal neurons in culture (n = 56). In the majority of neurons two phases in the dose-response relationship could be distinguished. The first was characterized by responses to 3-100 microM L-homocysteic acid which desensitized with a time-constant greater than 1 s in a concentration-dependent manner and were antagonized by 30 microM D-L-2-amino-5-phosphonovaleric acid indicating activation of the N-methyl-D-aspartate receptors. At higher concentrations of L-homocysteic acid this component was strongly depressed. The second phase was characterized by sustained responses that were concentration-dependent (1 mM L-homocysteic acid maximum concentration tested) and were not blocked by D-L-2-amino-5-phosphonovaleric acid indicating activation of non-N-methyl-D-aspartate receptors. Eight neurons did not exhibit these two-phase characteristics in the concentration-response relationship at the beginning of the recording. The magnitude of responses to L-homocysteic acid was positively related to concentration and the responses were partially blocked by D-L-2-amino-5-phosphonovaleric acid. In these neurons, however, repeated applications of L-homocysteic acid at concentrations 30 microM up to 300 microM resulted in a long-lasting, three- to four-fold increase of the membrane current. This increase was completely blocked by D-L-2-amino-5-phosphonovaleric acid (50-100 microM) suggesting that it was produced by activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Amino-5-phosphonovalerate↗