Glycine-activated Channels Research Articles

NR3A Modulates the Outer Vestibule of the “NMDA” Receptor Channel

Classical NMDA receptors (NMDARs), activated by glycine and glutamate, are heteromultimers comprised of NR1 and NR2 subunits. Coexpression of the novel NR3 family of NMDAR subunits decreases the magnitude of NR1/NR2 receptor-mediated currents or forms glycine-activated channels with the NR1 subunit alone. The second (M2) and third (M3) membrane segments of NR1 and NR2 subunits of classical NMDARs form the core of the channel permeation pathway. Structural information regarding NR1/NR3 channels remains unknown. Using the Xenopus oocyte expression system and the SCAM (substituted cysteine accessibility method), we found that M3 segments of both NR1 and NR3A form a narrow constriction in the outer vestibule of the channel, which prevents passage of externally applied sulfhydryl-specific agents. The most internal reactive residue in each M3 segment is the threonine in the conserved SYTANLAAF motif. These threonines appear to be symmetrically aligned. Several NR3A M3 mutations change the behavior of NR1/NR3A channels. Unlike NR1, however, the M3 segment of NR3A does not undergo extensive molecular rearrangement during channel gating by added glycine. Additionally, in the M2 segment, our data suggest that the amino acid at the asparagine (N) site of NR1, but not NR3A, contributes to the selectivity filter of NR1/3A channels. We therefore conclude that NR3A modulates the NR1/NR3A permeation pathway via a novel mechanism of forming a narrow constriction at the outer channel vestibule. This modified channel vestibule may also explain the dominant-negative effect of the NR3 subunit on channel behavior when coexpressed with NR1 and NR2 subunits.

Voltage dependence of the glycine receptor-channel kinetics in the zebrafish hindbrain.

Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were made on patches derived from the Mauthner cells of the 50-h-old zebrafish larva. As for glycinergic miniature inhibitory postsynaptic currents (mIPSCs), depolarizing the patch produced a broadening of the transient outside-out current evoked by short applications (1 ms) of a saturating concentration of glycine (3 mM). When the outside-out patch was depolarized from -50 to +20 mV, the peak current varied linearly with voltage. A 1-ms application of 3 mM glycine evoked currents that activated rapidly and deactivated biexponentially with time constants of approximately 5 and approximately 30 ms (holding potential of -50 mV). These two decay time constants were increased by depolarization. The fast deactivation time constant increased e-fold per 95 mV. The relative amplitude of the two decay components did not significantly vary with voltage. The fast component represented 64.2 +/- 2.8% of the total current at -50 mV and 54.1 +/- 10% at +20 mV. The 20-80% rise time of these responses did not show any voltage dependence, suggesting that the opening rate constant is insensitive to voltage. The 20-80% rise time was 0.2 ms at -70 mV and 0.22 ms at +20 mV. Responses evoked by 100-200 ms application of a low concentration of glycine (0.1 mM) had a biphasic rising phase reflecting the complex gating behavior of the glycine receptor. The time constant of these two components and their relative amplitude did not change with voltage, suggesting that modal shifts in the glycine-activated channel gating mode are not sensitive to the membrane potential. Using a Markov model to simulate glycine receptor gating behavior, we were able to mimic the voltage-dependent change in the deactivation time course of the responses evoked by 1-ms application of 3 mM glycine. This kinetics model incorporates voltage-dependent closing rate constants. It provides a good description of the time course of the onset of responses evoked by the application of a low concentration of glycine at all membrane potentials tested.

α2-adrenoceptor-mediated enhancement of glycine response in rat sacral dorsal commissural neurons

The effect of noradrenaline on the glycine response was investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus using nystatin perforated patch recording configuration under voltage-clamp conditions. Noradrenaline reversibly potentiated the 10 −5M glycine-induced Cl − current in a concentration-dependent manner. Single channel recordings in a cell-attached mode revealed that noradrenaline decreased the closing time of the glycine-activated channel activity. Noradrenaline neither changed the reversal potential of the glycine response nor affected the affinity of glycine to its receptor. Clonidine mimicked and yohimbine blocked the noradrenaline action on glycine response. N-[2(methylamino)ethyl]-5-misoquinoline sulfonamide dihydrochloride, protein kinase A inhibitor, mimicked the effect of noradrenaline on glycine response. Noradrenaline failed to affect the glycine response in the presence of these intracellular cyclic AMP and protein kinase A modulators. However, noradrenaline further enhanced the glycine response even in the presence of phorbol-12-myristate-13-acetate and chelerythrine, a protein kinase C inhibitor. Pertussis toxin treatment for 6–8 h blocked the noradrenaline facilitatory effect on the glycine response. In addition, noradrenaline potentiated the strychnine-sensitive postsynaptic currents evoked in a slice preparation of sacral dorsal commissural nucleus. These results suggest that the activation of α2-adrenoceptor by noradrenaline coupled with pertussis toxin-sensitive G-proteins reduces intracellular cyclic AMP formation through the inhibition of adenyl cyclase. The reduction of cyclic AMP decreases the protein kinase A activity, thus resulting in the potentiation of the glycinergic inputs to the sacral dorsal commissural neurons. It is thus feasible that the noradrenergic input to the sacral dorsal commissural nucleus modulates such nociceptive signals as pain by intracellular enhancing the glycine response.

Halothane increases the open probability of glycine-activated channel current in rat central neurones

Glycine-activated single-channel currents in rat central neurones were recorded using the outside-out mode of the patch-clamp technique. The unitary conductance of the current was 21 pS. The current evoked by 10 microM glycine had a mean burst duration of 47.8 (2.6) ms and open probability of 0.09 (0.016). Halothane (1 mM) increased the open probability to 0.19 (0.03) without changing either unitary conductance or burst duration. These results suggest that halothane increased the open probability via an increase in the affinity of the receptor for the agonist. Potentiation of the glycine response may reduce excitability of postsynaptic neurones and may contribute to general anaesthesia produced by volatile agents.

Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum.

1. Patch-clamp methods have been used to characterize GABA-and glycine-activated channels and spontaneous synaptic currents in granule cells in thin cerebellar slices from 7- to 20-day-old rats. 2. All granule cells responded to 10 microM GABA, while approximately 60% responded to 100 microM glycine. With repeated against application, whole-cell responses to GABA, but not those to glycine, declined over a period of minutes unless the pipette solution contained Mg-ATP. 3. Whole-cell concentration-response curves gave EC50 values at 45.2 and 99.6 microM and Hill slopes of 0.94 and 2.6 for GABA and glycine, respectively. At saturating concentrations, currents evoked by GABA were fivefold larger than those evoked by glycine. 4. Whole-cell current-voltage (I-V) relationships of GABA- and glycine-activated currents reversed close to the predicted Cl- equilibrium potential. Partial replacement of intracellular Cl- with F- shifted the GABA reversal potential to a more negative value. 'Instantaneous' I-V relationships produced by ionophoretic application of GABA were linear, while 'steady-state' I-V relationships produced by ramp changes in potential showed outward rectification. For glycine, 'steady-state' I-V plots were linear. 5. Responses to GABA were blocked by the GABAA receptor antagonists bicuculline (15 microM), SR-95531 (10 microM) and picrotoxinin (100 microM) while responses to glycine were selectively blocked by strychnine (200 nM), indicating the presence of two separate receptor types. 6. In outside-out membrane patches, GABA opened channels with conductances of 16 and 28 pS. The proportion of openings to each of the conductances varied between patches, possibly indicating the activation of two distinct channel types. Glycine-activated single-channel currents had conductances of 32, 55 and 104 pS. Single-channel I-V relationships were linear. 7. Spontaneous synaptic currents with a rapid rise time and biexponential decay were present in more than half of the cells examined. These currents were eliminated by bicuculline (15 microM) or SR-95331 (10 microM) and were greatly reduced in frequency by tetrodotoxin (TTX; 300 nM), suggesting that they were mediated by GABA and arose from spontaneous activity in Golgi interneurones. In granule cells where this spontaneous synaptic activity was apparent, glycine and low concentrations of GABA increased the frequency of the synaptic currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage dependence of conductance changes evoked by glycine release in the zebrafish brain.

1. The kinetics and mechanisms underlying the voltage dependence of inhibitory postsynaptic currents (IPSCs) recorded in the Mauthner cell (M cell) were investigated in the isolated medulla of 52-h-old zebrafish larvae, with the use of whole cell and outside-out patch-clamp recordings. 2. Spontaneous miniature IPSCs (mIPSCs) were recorded in the presence of 10(-6) M tetrodotoxin (TTX), 10 mM MgCl2, and 0.1 mM [CaCl2]o. Depolarizing the cell from -50 to +50 mV did not evoke any significant change in the distribution of mIPSC amplitudes, whereas synaptic currents were prolonged at positive voltages. The average decay time constant was increased twofold at +50 mV. 3. The voltage dependence of the kinetics of glycine-activated channels was first investigated during whole cell recording experiments. Currents evoked by voltage steps in the presence of glycine (50 microM) were compared with those obtained without glycine. The increase in chloride conductance (gCl-) evoked by glycine was time and voltage dependent. Inactivation and reactivation of the chloride current were observed during voltage pulses from 0 to -50 mV and from -50 to 0 mV, respectively, and they occurred with similar time constants (2-3 s). During glycine application, voltage-ramp analysis revealed a shift in the reversal potential (ECl-) occurring at all [Cl-]i tested. 4. The basis of the voltage sensitivity of glycine-evoked gCl- was first analyzed by measuring the relative changes in the total open probability (NPo) of glycine-activated channels with voltage.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycinergic Inhibitory Synaptic Currents and Related Receptor Channels in the Zebrafish Brain

To extend our study of the inhibitory synaptic network we have developed an isolated whole-brain preparation of the 52-h-old zebrafish (Brachydanio rerio) in which the structural and functional integrity of the brain is preserved. We report the characterization of quantal inhibitory events and the correlation of their properties with those of the underlying activated channels. During whole-cell recordings of the Mauthner cells, applications of 10(-6) M tetrodotoxin greatly reduced the frequency and amplitude of the spontaneously occurring synaptic events, which were dominated by Cl--dependent inhibitory postsynaptic currents (IPSCs). Lowering Ca2+ and adding Mg2+ to tetrodotoxin-containing solutions resulted in a further decrease in amplitude of the recorded synaptic currents, the remaining ones being considered as miniature IPSCs (mIPSCs). Applications of 0.5-1 microM strychnine in the presence of tetrodotoxin eliminated > 90% of the inhibitory currents in the preparation. The amplitude histograms of these mIPSCs exhibited two initial equally spaced peaks, followed by a skewed distribution for higher values. The first two components were well fitted by the sum of two Gaussian curves, giving a mean quantal amplitude of 35.7 pA (at a holding potential of -50 mV) and a coefficient of variation of 0.25 for the first peak. Outside-out recordings showed at least two classes of glycine receptor channels, one having multiple conductance levels with a main state of 81-86 pS and another displaying only one opening level of 41-43 pS. These two mean conductance states had similar mean open times, of 0.6-1 and 4.5-6 ms respectively. In addition, three mean closed times were observed for the 41-43 pS level. The shortest group (0.6-1 ms) was considered as representing gaps within bursts. Burst analysis revealed three mean burst durations, of 0.6, 4 and 35 ms. Comparisons of the amplitude of the first class of mIPSCs and of the open channel conductances indicated that one quantum opens 14-22 channels. Moreover, the correspondence between the mean decay time of mIPSCs and the mean open time or medium burst duration (4-5 ms) suggests that glycine-activated channels open only once in response to a single exocytosis. The pre- and postsynaptic origins of mIPSCs amplitude fluctuations are discussed in the context of multivesicular release versus the hypothesis of postsynaptic receptor saturation.

Protein kinase a-mediated phosphorylation reduces only the fast desensitizing glycine current in acutely dissociated ventromedial hypothalamic neurons

Modulation of glycine receptor-ionophore complex by internally perfused cyclic AMP was investigated and compared to that of GABA in the acutely dissociated ventromedial hypothalamic neurons using whole-cell and outside-out patch-clamp techniques. Cyclic AMP significantly reduced both GABA- and glycine-gated chloride currents. The reduction in glycine-induced chloride current was specific in that only the fast-desensitizing one gated by high concentrations of glycine (30–100 μM) was affected. Cyclic AMP did not modulate the non-desensitizing current induced by lower concentrations (6–10 μM). Addition of N-[-2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride, a protein kinase A inhibitor, did not have a significant effect on its own but prevented the attenuation of fast desensitizing glycine current induced by cyclic AMP. Both the reversal potential and inactivation kinetics of glycine current were not affected by the activation of protein kinase A, suggesting that cyclic AMP-mediated attenuation is not due to an enhancement of desensitization. In outside-out patch studies intracellular perfusion of cyclic AMP reduced the open probability of the 100 μM glycine-activated channels without affecting that of the 6 μM glycine-activated channels. In conclusion, cyclic AMP selectively modulates the channel open frequency of the glycine receptor when activated at higher concentrations through a protein kinase A-mediated phosphorylation.

Anion permeation in GABA- and glycine-gated channels of mammalian cultured hippocampal neurons.

Single-channel currents through GABA- and glycine-activated chloride channels of post-natal tissue-cultured hippocampal neurons were measured to determine their anion selectivity and their concentration dependence of permeation. Current-voltage relations for both agonists displayed rectification with single-channel conductance increasing at positive potentials. Permeabilities determined from reversal potentials were maximal for anions with a diameter of about 4 A. Larger diameter anions had lower permeabilities, consistent with an approximate pore diameter of 6 A for both agonist-activated channels. The permeability for anions of similar size was greatest for those ions with a more symmetrical charge distribution (e.g. NO3- > Bicarbonate-). The permeability sequence was SCN- > NO3- > I- > Br- > Cl- > Formate- > Acetate- > Bicarbonate- > Gluconate- > F- > Phosphate-, whereas the conductance sequence for anion efflux was Cl- > Br- > NO3- > I- > SCN- > Formate- > Acetate- > Bicarbonate- > Gluconate- > F- > Phosphate-. These results suggest that the ions interact with sites within the channel, with hydration forces contributing an important component to the barrier for ion entry into the channel. The spherically symmetrical halides displayed an exponential relation between relative permeability and hydration energy. Concentration dependence of conductance for Cl- channels in symmetrical Cl- solutions with agonist in the pipette showed an increase at positive potentials and a decrease at negative potentials. GABA- and glycine-activated channels also exhibited anomalous mole-fraction effects in a mixture of Cl- and SCN-. These results suggest that both agonist-activated channels act as multi-ion pathways and have similar permeation characteristics.

Effect of cytoplasmic chloride concentration on open-state preference of glycine-activated chloride channels in cultured spinal cord cells

1. Glycine was bath-applied to outside-out patches taken from cultured mouse spinal cord neurons. Glycine-activated chloride channels had at least six open states with permeabilities of 40 (A), 68 (B), 100 (C), 145 (D), 180 (E), and 250 (F) al/s (1 attoliter = 10(-18) liter). Usually no more than two such states were seen in any given patch. The usual extracellular chloride concentration was 157 mM; chloride concentration in the electrode (on the cytoplasmic face of the patch) was varied between 7 and 170 mM, replaced at lower concentrations with either of the inert anions gluconate or methane sulphonate. 2. With normal (7 mM) chloride concentration in the electrode, openings to state D were the most probable, and the average permeability of all openings was 115 al/s. When the cytoplasmic chloride concentration was 20 mM, the most probable open state was the lower-permeability state (C), and the average permeability was 92 al/s. In symmetrical 170 mM chloride, the most probable open state was B and the mean permeability 77 al/s. 3. The glycine-activated channels had a vanishingly small permeability to fluoride. However, in experiments in which 150 mM gluconate in the pipette was replaced by fluoride, the most probable open state was B, as in the high-chloride solution. Thus the two halides had similar effects on open state preference. 4. With external and internal chloride concentrations of 7 and 157 mM, respectively, single-channel current-voltage relations could be fit with constant field relations appropriate to one or more of the permeability states.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in the regulation of glycine-activated Cl − channels of cultured rat medullary neurons

Glycine-activated currents in 1- to 11-day-old rat medullary neurons were studied using patch clamp techniques. Glycine produced neither repeatable whole-cell current responses nor single-channel activity in the cell-attached mode until cells were in culture for a week or more. However, Cl − channels were present at the early stages because glycine-activated channels were seen in excised, inside-out patches. Furthermore, for cells less than a week in culture, 10 patches which did not exhibit glycine-activated Cl − channels in the cell-attached mode did upon excision. Consequently, the activation properties of these Cl − channels undergo a developmental change in that some cellular factor(s) presumably prevents the Cl − channels from opening in the intact cell during the initial stages in culture.

Caesium ions activate chloride channels in rat cultured spinal cord neurones.

1. Caesium ions (Cs+), applied extracellularly, caused a decrease in the input resistance of cultured spinal cord (s.c.) neurones and depolarized the neurones when they contained 140 mM-CsCl. 2. The reversal potential for Cs+-activated currents shifted 56 mV on average for a 10-fold reduction in the intracellular chloride ion (Cl-) activity, indicating that the Cs+-activated currents were specific to Cl-. 3. The activation of Cl- currents by Cs+ was not due to the depolarization-evoked release of neurotransmitter from presynaptic terminals. We therefore suggest that Cs+ were acting directly on the extracellular surface of the s.c. neurones to activate Cl- currents. 4. Cs+-activated currents showed desensitization in the presence of 140 mM-Cs+. 5. The log-log plot of the dose-response data could be fitted with a straight line with a slope of 1.7 +/- 0.4 (n = 6), indicating that at least 2 Cs+ were needed to activate a single Cl- channel. The KD of the Cs+-induced response was greater than 69 mM. 6. In outside-out patches Cs+ activated single Cl- channels. These channels were not activated by sodium or potassium ions. 7. The Cs+-activated channels displayed a total of five distinct conductance states which had mean conductances of 20, 30, 43, 66 and 92 pS. The 30 and 43 pS states were the most frequently occurring states. 8. The conductance states of the Cs+-activated channel have the same conductances as those reported for gamma-aminobutyric acid (GABA)- and glycine-activated channels in rat s.c. neurones. We therefore conclude that Cs+ activate the same type of Cl- channel as GABA and glycine through an unidentified receptor.

Characteristics of inhibitory post-synaptic currents in brain-stem neurones of the lamprey.

Voltage-clamp techniques were used to record spontaneously occurring inhibitory post-synaptic currents (i.p.s.c.s) from Müller cells in the brain stem of the lamprey. In normal bathing solution, the i.p.s.c.s in most cells had unimodally distributed amplitudes with a mean corresponding to a peak conductance of 107 nS and a coefficient of variation of about 15%. About 20% of the cells displayed, in addition, events of approximately twice the modal amplitude. The falling phase of the i.p.s.c.s was exponential with a mean time constant of about 32 msec. This is the same as the relaxation time constant of glycine-activated channels in these cells under comparable conditions (Gold & Martin, 1983b). When tetrodotoxin (TTX) was added to the bathing solution the spontaneous i.p.s.c.s disappeared, suggesting that they were due to release of transmitter by action potentials in presynaptic terminals. Spontaneous activity was also abolished by removing Ca2+ from the bathing solution. When extracellular Ca2+ was increased, or 4-aminopyridine (4-AP) was added to the bathing solution, the mean amplitude of the i.p.s.c.s increased and the amplitude distribution showed two or more distinct peaks. Analysis of the amplitude distributions suggested that the peaks represented single and multiple quantal events and that the release process obeyed binomial statistics. In TTX-blocked preparations, spontaneous i.p.s.c.s could be induced by raising extracellular K+. These had the same time constant of decay as in normal solution and a unimodal amplitude distribution, with the mean corresponding to a peak conductance of 45 nS. In solutions with raised extracellular K+ and reduced extracellular Cl-, the mean i.p.s.c. amplitude corresponded to a peak conductance of 67 nS. These variations in conductance corresponded closely to variations in conductance with extracellular K+ and Cl- of single glycine-activated channels. It is concluded that the i.p.s.c.s are produced by activation of conductance channels identical to those activated by glycine, and that in normal bathing solution i.p.s.c.s produced by individual presynaptic action potentials are the result of the release of one or, at most, two quanta of transmitter. Each quantum activates approximately 1500 elementary channels.

GABA- and glycine-induced Cl − channels in cultured mouse spinal neurons require the same energy to close

Fluctuation analysis has been used to study the influence of temperature on the conductance and mean open time of Cl − permeable ion channels induced by γ-aminobutyric acid (GABA) and glycine in the membrane of tissue cultured mouse spinal neurons. The conductance of GABA − and glycine-activated channels increased slightly at higher temperatures ( Q 10 ∼ 1.3 ). The mean open times of these channels decreased at higher temperatures ( Q 10 ∼ 3 ). Arrhenius plots for the open times of GABA- and glycine-induced channels were parallel and showed no obvious transition behavior over the temperature range 16–40°C.