Tyrosine kinases enhance the function of glycine receptors in rat hippocampal neurons and human α1β glycine receptors

Glycine receptors (GlyRs) are transmitter-gated channels that mediate fast inhibitory neurotransmission in the spinal cord and brain. The GlyR β subunit contains a putative tyrosine phosphorylation site whose functional role has not been determined. To examine if protein tyrosine kinases (PTKs) regulate the function of GlyRs, we analysed whole-cell currents activated by applications of glycine to CA1 hippocampal neurons and spinal neurons. The role of a putative site for tyrosine phosphorylation at position 413 of the β subunit was examined using site-directed mutagenesis and expression of recombinant (α₁β^Y413F ) receptors in human embryonic kidney (HEK 293) cells. Lavendustin A, an inhibitor of PTKs, depressed glycine-evoked currents ( I _Gly) in CA1 neurons and spinal neurons by 31 % and 40 %, respectively. In contrast, the intracellular application of the exogenous tyrosine kinase, cSrc, enhanced I _Gly in CA1 neurons by 56 %. cSrc also accelerated GlyR desensitization and increased the potency of glycine 2-fold (control EC₅₀= 143 μ m ; cSrc EC₅₀= 74 μ m ). Exogenous cSrc, applied intracellularly, upregulated heteromeric α₁β receptors but not homomeric α₁ receptors. Substitution mutation of the tyrosine to phenylalanine at position β-413 prevented this enhancement. Furthermore, a selective inhibitor of the Src family kinases, PP2, down-regulated wild-type α₁β but not α₁β^Y413F receptors. Together, these findings indicate that GlyR function is upregulated by PTKs and this modulation is dependent on the tyrosine-413 residue of the β subunit.     

Functional properties of spontaneous IPSCs and glycine receptors in rod amacrine (AII) cells in the rat retina

AII amacrine cells play a crucial role in retinal signal transmission under scotopic conditions. We have used rat retinal slices to investigate the functional properties of inhibitory glycine receptors on AII cells by recording spontaneous IPSCs (spIPSCs) in whole cells and glycine-evoked responses in outside-out patches. Glycinergic spIPSCs displayed fast kinetics with an average 10–90% rise time of ∼500 μs, and a decay phase best fitted by a double-exponential function with τ_fast∼ 4.8 ms (97.5% amplitude contribution) and τ_slow∼ 33 ms. Decay kinetics were voltage dependent. Ultrafast application of brief (∼2–5 ms) pulses of glycine (3 m m ) to patches, evoked responses with fast deactivation kinetics best fitted with a double-exponential function with τ_fast∼ 4.6 ms (85% amplitude contribution) and τ_slow∼ 17 ms. Double-pulse experiments indicated recovery from desensitization after a 100-ms pulse of glycine with a double-exponential time course  (τ_fast∼ 71 ms and τ_slow∼ 1713 ms)  . Non-stationary noise analysis of spIPSCs and patch responses, and directly observed channel gating yielded similar single-channel conductances (∼41 to ∼47 pS). In addition, single-channel gating occurred at ∼83 pS. These results suggest that the fast glycinergic spIPSCs in AII cells are probably mediated by α1β heteromeric receptors with a contribution from α1 homomeric receptors. We hypothesize that glycinergic synaptic input may target the arboreal dendrites of AII cells, and could serve to shunt excitatory input from rod bipolar cells and transiently uncouple the transcellular current through electrical synapses between AII cells and between AII cells and ON-cone bipolar cells.     

Functional roles of presynaptic GABAA receptors on glycinergic nerve terminals in the rat spinal cord

GABA_A receptor-mediated presynaptic depolarization is believed to induce presynaptic inhibition of excitatory synaptic transmission. We report here the functional roles of presynaptic GABA_A receptors in glycinergic transmission of the rat spinal cord. In mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with native glycinergic and GABAergic nerve terminals, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were isolated from a mixture of both glycinergic and GABAergic sIPSCs by perfusing the SDCN nerve cell body with ATP-free internal solution. Under such experimental conditions, exogenously applied muscimol (0.5 μM) depolarized glycinergic presynaptic nerve terminals and significantly increased glycinergic sIPSC frequency to 542.7 ± 47.3 % of the control without affecting the mean current amplitude. The facilitatory effect of muscimol on sIPSC frequency was completely blocked by bicuculline (10 μM) or SR95531 (10 μM), selective GABA_A receptor antagonists. This muscimol-induced presynaptic depolarization was due to a higher intraterminal Cl⁻ concentration, which is maintained by a bumetanide-sensitive Na-K-Cl cotransporter. On the contrary, when electrically evoked, this muscimol-induced presynaptic depolarization was found to decrease the action potential-dependent glycine release evoked by focal stimulation of a single terminal. The results suggest that GABA_A receptor-mediated presynaptic depolarization has two functional roles: (1) presynaptic inhibition of action potential-driven glycinergic transmission, and (2) presynaptic facilitation of spontaneous glycinergic transmission.     

Single-channel study of the spasmodic mutation α1A52S in recombinant rat glycine receptors

Inherited defects in glycine receptors lead to hyperekplexia, or startle disease. A mutant mouse, spasmodic , that has a startle phenotype, has a point mutation (A52S) in the glycine receptor α1 subunit. This mutation reduces the sensitivity of the receptor to glycine, but the mechanism by which this occurs is not known. We investigated the properties of A52S recombinant receptors by cell-attached patch-clamp recording of single-channel currents elicited by 30–10000 μ m glycine. We used heteromeric receptors, which resemble those found at adult inhibitory synapses. Activation mechanisms were fitted directly to single channel data using the HJCFIT method, which includes an exact correction for missed events. In common with wild-type receptors, only mechanisms with three binding sites and extra shut states could describe the observations. The most physically plausible of these, the 'flip' mechanism, suggests that preopening isomerization to the flipped conformation that follows binding is less favoured in mutant than in wild-type receptors, and, especially, that the flipped conformation has a 100-fold lower affinity for glycine than in wild-type receptors. In contrast, the efficacy of the gating reaction was similar to that of wild-type heteromeric receptors. The reduction in affinity for the flipped conformation accounts for the reduction in apparent cooperativity seen in the mutant receptor (without having to postulate interaction between the binding sites) and it accounts for the increased EC ₅₀ for responses to glycine that is seen in mutant receptors. This mechanism also predicts accurately the faster decay of synaptic currents that is observed in spasmodic mice.     

Molecular determinants of glycine receptor αβ subunit sensitivities to Zn2+-mediated inhibition

Glycine receptors exhibit a biphasic sensitivity profile in response to Zn²⁺-mediated modulation, with low Zn²⁺ concentrations potentiating (< 10 μ m ), and higher Zn²⁺ concentrations inhibiting submaximal responses to glycine. Here, a substantial 30-fold increase in sensitivity to Zn²⁺-mediated inhibition was apparent for the homomeric glycine receptor (GlyR) α1 subunit compared to either GlyR α2 or α3 subtypes. Swapping the divergent histidine (H107) residue in GlyR α1, which together with the conserved H109 forms part of an intersubunit Zn²⁺-binding site, for the equivalent asparagine residue present in GlyR α2 and α3, reversed this phenotype. Co-expression of heteromeric GlyR α1 or α2 with the ancillary β subunit yielded receptors that maintained their distinctive sensitivities to Zn²⁺ inhibition. However, GlyR α2β heteromers were consistently 2-fold more sensitive to inhibition compared to the GlyR α2 homomer. Comparative studies to elucidate the specific residue in the β subunit responsible for this differential sensitivity revealed instead threonine 133 in the α1 subunit as a new vital component for Zn²⁺-mediated inhibition. Further studies on heteromeric receptors demonstrated that a mutated β subunit could indeed affect Zn²⁺-mediated inhibition but only from one side of the intersubunit Zn²⁺-binding site, equivalent to the GlyR α1 H107 face. This strongly suggests that the α subunit is responsible for Zn²⁺-mediated inhibition and that this is effectively transduced, asymmetrically, from the side of the Zn²⁺-binding site where H109 and T133 are located.     

Temperature dependence of NR1/NR2B NMDA receptor channels

N-methyl-D-aspartate (NMDA) receptors are highly expressed in the CNS, mediate the slow component of excitatory transmission and play key roles in synaptic plasticity and excitotoxicity. These ligand-gated ion channels are heteromultimers composed of NR1 and NR2 subunits activated by glycine and glutamate. In this study, patch-clamp recordings were used to study the temperature sensitivity of recombinant NR1/NR2B receptors expressed in human embryonic kidney (HEK) 293 cells. Rate constants were assessed by fitting a six-state kinetic scheme to time courses of transient macroscopic currents induced by glutamate at 21.9-46.5 degrees C. Arrhenius transformation of the rate constants characterizing NMDA receptor channel activity indicates that the most sensitive were the rate constants of desensitization (temperature coefficient Q(10)=10.3), resensitization (Q(10)=4.6) and unbinding (Q(10)=3.6). Other rate constants and the amplitude of single-channel currents were less temperature sensitive. Deactivation of responses mediated by NR1/NR2B receptors after a brief application of glutamate was best fit by a double exponential function (tau(fast): Q(10)=3.7; tau(slow): Q(10)=2.7). From these data, we conclude that desensitization/resensitization of the NMDA receptor and glutamate unbinding are especially temperature sensitive and imply that at physiological temperatures the channel kinetics play an important role in determining amplitude and time course of NMDA receptor-mediated postsynaptic currents and these receptors mediated synaptic plasticity.     

Essential role of rho kinase in the ca2+ Sensitization of Prostaglandin F2α-Induced Contraction of Rabbit Aortae

Although the prostate gland is a rich source of α1-adreno- (α1-AR) and m1-cholino receptors (m1-AChR), the membrane processes associated with their activation in glandular epithelial cells is poorly understood. We used the whole-cell patch-clamp technique to show that the agonists of the respective receptors, phenylephrine (PHE) and carbachol (CCh), activate cationic membrane currents in lymph node carcinoma of the prostate (LNCaP) human prostate cancer epithelial cells, which are not dependent on the filling status of intracellular IP₃-sensitive Ca²⁺ stores, but directly gated by diacylglycerol (DAG), as evidenced by the ability of its membrane permeable analogue, OAG, to mimic the effects of the agonists. The underlying cationic channels are characterized by the weak field-strength Eisenman IV permeability sequence for monovalent cations ( P _K(25) > P _Cs(4.6) > P _Li(1.4) > P _Na(1.0)), and the following permeability sequence for divalent cations: P _Ca(1.0) > P _Mg(0.74) > P _Ba(0.6) > P _Sr(0.36) > P _Mn(0.3). They are 4.3 times more permeable to Ca²⁺ than Na⁺ and more sensitive to the inhibitor 2-APB than SK&F 96365. RT-PCR analysis shows that DAG-gated members of the transient receptor potential (TRP) channel family, including TRPC1 and TRPC3, are present in LNCaP cells. We conclude that, in prostate cancer epithelial cells, α1-ARs and m1-AChRs are functionally coupled to Ca²⁺-permeable DAG-gated cationic channels, for which TRPC1 and TRPC3 are the most likely candidates.     

Reversible inhibition of GABAA receptors by α7-containing nicotinic receptors on the vertebrate postsynaptic neurons

Nicotinic acetylcholine receptors (nAChRs) are expressed throughout the central nervous system and influence a variety of higher order functions including learning and memory. While the effects of presynaptic nAChRs on transmitter release have been well documented, little is known about possible postsynaptic actions. A major species of neuronal nAChRs contains the α7 gene product and has a high relative permeability to calcium. Both on rodent hippocampal interneurons and on chick ciliary ganglion neurons these α7-nAChRs are often closely juxtaposed to GABA_A receptors. We show here that in both cases activation of α7-nAChRs on the postsynaptic neuron acutely down-regulates GABA-induced currents. Nicotine application to dissociated ciliary ganglion neurons diminished subsequent GABA_A receptor responses to GABA. The effect was blocked by α7-nAChR antagonists, by chelation of intracellular Ca²⁺ with BAPTA, and by inhibition of both Ca²⁺–calmodulin-dependent protein kinase II and mitogen-activated protein kinase. A similar outcome was obtained in the hippocampus where electrical stimulation to activate cholinergic fibres reduced the amplitude of subsequent GABA_A receptor-mediated inhibitory postsynaptic currents. The reduction showed the same calcium and kinase dependence seen in ciliary ganglion neurons and was absent in hippocampal slices from α7-nAChR knockout mice. Moreover, α7-nAChR blockade in hippocampal slices reduced rundown of GABA_A receptor-mediated whole-cell responses, indicating ongoing endogenous modulation. The results demonstrate regulation of GABA_A receptors by α7-nAChRs on the postsynaptic neuron and identify a new mechanism by which nicotinic cholinergic signalling influences nervous system function.     

Proton modulation of recombinant GABAA receptors: influence of GABA concentration and the β subunit TM2–TM3 domain

Regulation of GABA_A receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABA_A receptors at alkaline pH has remained elusive. We report here that the GABA-activated current can be potentiated at pH 8.4 for both αβ and αβγ subunit-containing receptors, but only at GABA concentrations below the EC₄₀. Site-specific mutagenesis revealed that a single lysine residue, K279 in the β subunit TM2–TM3 linker, was critically important for alkaline pH to modulate the function of both α1β2 and α1β2γ2 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABA_A receptors was also examined at acidic pH. At pH 6.4, GABA activation of αβγ receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the β subunit. Decreasing the pH further to 5.4 inhibited GABA responses via αβγ receptors, whereas those responses recorded from αβ receptors were potentiated. Inserting homologous β subunit residues into the γ2 subunit to recreate, in αβγ receptors, the proton modulatory profile of αβ receptors, established that in the presence of β2^H267, the mutation γ2^T294K was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the β subunit and suggests that a lysine at this position is an important residue for mediating the allosteric effects of both acidic and alkaline pH changes, rather than forming a direct site for protonation within the GABA_A receptor.     

Heterologous expression of wild-type and mutant β-cardiac myosin changes the contractile kinetics of cultured mouse myotubes

The properties of myosin expressed in muscle are a major determinant of muscle performance. In this study we used a novel approach to examine the functional impact of changes in myosin heavy chain (MHC) isoform expression, as well as the consequences of expressing the mutant MHC implicated in familial hypertrophic cardiomyopathy (FHC). Cultured mouse myoblasts that normally express fast embryonic myosin were untransfected, or stably transfected with a plasmid expressing either wild-type (cWT) or mutant (D778G or G741R) β-cardiac myosin. After differentiation for 5–7 days, cWT or mutant β-cardiac myosin was expressed at 25 % of total myosin in the myotube. We measured time-to-peak shortening (ttp), time for half-relaxation ( t _0.5), the maximum velocity of shortening ( V _max) at 1 Hz stimulation, and the tetanic fusion frequency. Expression of cWT β-cardiac myosin significantly increased ttp and t _0.5 and decreased the fusion frequency compared with untransfected myotubes. However, when we compared myotubes expressing mutant β-cardiac myosin with those expressing cWT β-cardiac myosin, we found that ttp and t _0.5 were significantly decreased, and V _max was increased for the D778G mutant, whereas ttp, t _0.5 and V _max were unchanged for the G741R mutant. The fusion frequency was increased for both mutant myosins. Our data support the conclusion that the impact of the slower myosin isoform dominates when both slow and fast isoforms are present. This work suggests that FHC associated with either D778G or G741R mutation in MHC is an 'energy cost' disease, but that the phenotype of D778G is more severe than that of G741R.     

Pathogenic point mutations in a transmembrane domain of the ε subunit increase the Ca2+ permeability of the human endplate ACh receptor

The ε subunit of the human endplate ACh receptor (AChR) is a key determinant of the large fraction of the ACh-evoked current carried by Ca²⁺ ions ( P _f). Consequently, missense mutations in the ε subunit are potential targets for altering the P _f of human AChR. In this paper we investigate the effects of two pathogenic point mutations in the M2 transmembrane segment AChR ε subunit, εT264P and εV259F, that cause slow-channel syndromes (SCS). When expressed in GH4C1 cells, the mutant receptors subunits raise Ca²⁺ permeability of the receptors ∼1.5 and ∼2-fold above that of wild-type, to attain P _f values of 11.8% (εT264P) and 15.4% (εV259F). The latter value exceeds most P _f values reported to date for ligand-gated ion channels. Consistent with these findings, the biionic Ca²⁺ permeability ratio ( P _Ca/ P _Cs) of the mutant AChRs is also increased. Upon repetitive stimulation with ACh, the mutant receptors show an enhanced current run-down compared with wild-type, leading to a strong reduction of their function. We propose that the enhanced Ca²⁺ permeability of the mutant receptors overrides the protective effect of desensitization and, together with the prolonged opening events of the AChR channel, is an important determinant of the excitotoxic endplate damage in the SCS.     

Caffeine inhibition of ionotropic glycine receptors

We found that caffeine is a structural analogue of strychnine and a competitive antagonist at ionotropic glycine receptors (GlyRs). Docking simulations indicate that caffeine and strychnine may bind to similar sites at the GlyR. The R131A GlyR mutation, which reduces strychnine antagonism without suppressing activation by glycine, also reduces caffeine antagonism. GlyR subtypes have differing caffeine sensitivity. Tested against the EC₅₀ of each GlyR subtype, the order of caffeine potency (IC₅₀) is: α2β (248 ± 32 μ m ) ≈α3β (255 ± 16 μ m ) > α4β (517 ± 50 μ m ) > α1β(837 ± 132 μ m ). However, because the α3β GlyR is more than 3-fold less sensitive to glycine than any of the other GlyR subtypes, this receptor is most effectively blocked by caffeine. The glycine dose–response curves and the effects of caffeine indicate that amphibian retinal ganglion cells do not express a plethora of GlyR subtypes and are dominated by the α1β GlyR. Comparing the effects of caffeine on glycinergic spontaneous and evoked IPSCs indicates that evoked release elevates the glycine concentration at some synapses whereas summation elicits evoked IPSCs at other synapses. Caffeine serves to identify the pharmacophore of strychnine and produces near-complete inhibition of glycine receptors at concentrations commonly employed to stimulate ryanodine receptors.     

Localization and function of ATP and GABAA receptors expressed by nociceptors and other postnatal sensory neurons in rat

The role of endogenous GABA and ATP in regulating transmitter release from primary afferent terminals in the superficial dorsal horn of the spinal cord is still controversial. ATP is co-released with GABA from some inhibitory dorsal horn neurons raising the possibility that ATP could act in concert with GABA to regulate transmitter release from primary afferent terminals if receptors to both transmitters are expressed there. Using electrophysiology together with immunocytochemistry, we have investigated the expression of ATP-gated P2X and GABA_A receptors by identified subpopulations of dorsal root ganglion (DRG) neurons known to project primarily to the superficial dorsal horn. Expression of the heat-sensitive vanilloid receptor 1 (VR1) and sensitivity to capsaicin were used to characterize DRG neurons sensitive to noxious heat. Both P2X and GABA_A receptors were expressed on the majority of DRG neurons examined. Recording compound action potentials (CAPs) from dorsal roots in the presence of muscimol, α,β-methylene-ATP (α,β-meATP) or capsaicin resulted in depression of CAP in the slow and medium conducting fibres, indicating cognate receptor expression on the small diameter axons. Dorsal root-evoked dorsal root potentials (DR-DRPs), reflecting depolarization of primary afferent terminals by endogenously released substances, were depressed by the GABA_A receptor antagonist SR95531 and α,β-meATP. These results suggest that GABA_A and P2X receptors are expressed on DRG cell bodies and slow fibre axons, many of which are heat-nociceptive. These fibres project to the superficial lamina of the dorsal horn where the receptors may function to modulate transmitter release near their central terminals.     

Binding site stoichiometry and the effects of phosphorylation on human α1 homomeric glycine receptors

The kinetic properties of the human α1 homomeric glycine receptor were investigated. Receptors were expressed in HEK 293 cells, and glycine was applied to outside-out membrane patches with sub-millisecond solution exchange. The activation time course of the glycine response was used to investigate receptor stoichiometry. The unbinding of three strychnine molecules and the cooperative binding of two glycine molecules were required to activate the channel. The effects of phosphorylation on glycine receptor kinetics were investigated by pretreating cells with phosphorylators or with phosphatases. Phosphorylation accelerated desensitisation, but slowed deactivation and recovery from desensitisation. A chemical-kinetic model was developed that reproduced the experimental observations. The model suggests that only three binding sites on the glycine channel are functional, while the remaining two binding sites are 'silent', possibly due to strong negative cooperativity.     

ATP participates in three excitatory postsynaptic potentials in the submucous plexus of the guinea pig ileum

Synaptic transmission between neurones intrinsic to the wall of the intestine involves multiple neurotransmitters. This study aimed to identify neurotransmitters responsible for non-cholinergic excitatory synaptic transmission in the submucous plexus of the guinea pig ileum. Intracellular recordings were made from secretomotor and vasodilator neurones. A single electrical stimulus to a fibre tract evoked excitatory postsynaptic potentials (EPSPs) with three different time courses – fast, slow and an EPSP with an intermediate time course (latency 96 ms, duration 1.2 s). In all neurones, blocking nicotinic receptors reduced fast EPSPs, but they were abolished in only 57 of 78 neurones. Fast EPSPs were also reduced by P2 purinoceptor blockade (5 of 27 neurones) or 5-HT₃ receptor blockade (3 of 20 neurones). The intermediate EPSP was abolished by P2 receptor blockade (13 of 13 neurones) or by the specific P2Y₁ receptor antagonist MRS 2179 (5 of 5 neurones) and was always preceded by a nicotinic or mixed nicotinic/purinergic fast EPSP. Intermediate EPSPs were observed in over half of all neurones including most non-cholinergic secretomotor neurones identified by immunoreactivity for vasoactive intestinal peptide. The slow EPSP evoked by a single pulse stimulus was also abolished by P2 receptor blockade (5 of 5 neurones) or by MRS 2179 (3 of 3 neurones). We conclude that fast EPSPs in submucous neurones are mediated by acetylcholine acting at nicotinic receptors, ATP acting at P2X receptors and 5-HT acting at 5-HT₃ receptors. Both the intermediate EPSP and the single stimulus slow EPSP are mediated by ATP acting at P2Y₁ receptors.     

GABAA, GABAC and glycine receptor-mediated inhibition differentially affects light-evoked signalling from mouse retinal rod bipolar cells

Rod bipolar cells relay visual signals evoked by dim illumination from the outer to the inner retina. GABAergic and glycinergic amacrine cells contact rod bipolar cell terminals, where they modulate transmitter release and contribute to the receptive field properties of third order neurones. However, it is not known how these distinct inhibitory inputs affect rod bipolar cell output and subsequent retinal processing. To determine whether GABA_A, GABA_C and glycine receptors made different contributions to light-evoked inhibition, we recorded light-evoked inhibitory postsynaptic currents (L-IPSCs) from rod bipolar cells mediated by each pharmacologically isolated receptor. All three receptors contributed to L-IPSCs, but their relative roles differed; GABA_C receptors transferred significantly more charge than GABA_A and glycine receptors. We determined how these distinct inhibitory inputs affected rod bipolar cell output by recording light-evoked excitatory postsynaptic currents (L-EPSCs) from postsynaptic AII and A17 amacrine cells. Consistent with their relative contributions to L-IPSCs, GABA_C receptor activation most effectively reduced the L-EPSCs, while glycine and GABA_A receptor activation reduced the L-EPSCs to a lesser extent. We also found that GABAergic L-IPSCs in rod bipolar cells were limited by GABA_A receptor-mediated inhibition between amacrine cells. We show that GABA_A, GABA_C and glycine receptors mediate functionally distinct inhibition to rod bipolar cells, which differentially modulated light-evoked rod bipolar cell output. Our findings suggest that modulating the relative proportions of these inhibitory inputs could change the characteristics of rod bipolar cell output.     

κ-Opioid receptor-mediated enhancement of the hyperpolarization-activated current (Ih) through mobilization of intracellular calcium in rat nucleus raphe magnus

The hyperpolarization-activated current ( I _h) is important in the control of resting membrane potential, in the regulation of network firing pattern and in the modulation of presynaptic transmitter release in central neurons. Recent studies on native and cloned I _h channels have demonstrated that the I _h channel is commonly modulated by cAMP through a positive shift in its voltage dependence without a change in its maximum current. The present study demonstrates that activation of κ-opioid receptors enhances I _h by increasing its maximum current in brainstem neurons in the nucleus raphe magnus. Agents that interfere with the release of intracellular calcium from calcium stores altered the maximum I _h and significantly attenuated the κ-receptor-mediated enhancement of I _h. These results suggest that κ-opioid receptors enhance the maximum I _h by mobilizing intracellular calcium from calcium stores. This provides a physiological function for κ-receptor-stimulated calcium release and may suggest another I _h-regulating mechanism by intracellular calcium in central neurons.     

Secondary IgE Responses In Vivo are Predominantly Generated Via γ1ɛ-Double Positive B Cells

We have recently developed a model in which mice were treated with 1L-4 after primary immunization, resulting in elevated total serum IgG₁ and IgE levels, but decreased antigen-specific levels and memory formation for these isotypes. In this report, we describe that these effects of IL-4 are mediated at the B cell and not the T-cell level. Major changes occurred in the γ1ɛ-double positive B-cell population which is increased as a result of IL-4 treatment. Moreover, it is shown that γ1ɛ-double positive B cells can develop in vitro out of γ1-positive primed B cells and that these double positive cells can differentiate into IgG₁- and IgE-secreting cells. The existence of γ1ɛ-double positive memory B cells can explain the differences in cytokine dependence of TNP-specific memory IgG₁ and IgE responses found after adoptively transferring primed spleen cells into irradiated naive recipients. Whereas the IL-4 independent TNP-specific memory IgG₁ responses could be blocked efficiently by neutralizing IL-5 and IL-6, TNP-specific memory IgE responses were virtually not susceptible to such treatment. These IgE responses were also not susceptible to IFN-γ, used in doses that could inhibit the primary IgE response. Inhibition of the TNP-specific memory IgG₁ response by neutralizing IL-5 and IL-6 is accompanied by a 10-fold increase of the IL-4 independent TNP-specific IgE memory response. These data indicate that secondary IgE responses primarily result from B cells that are either switched to IgG₁, or are double positive for IgG₁ and IgE, thereby suggesting a minor role for ɛ-single positive B cells in secondary IgE responses.     

Physical activity changes the regulation of mitochondrial respiration in human skeletal muscle

Electrophysiological and pharmacological properties of glycine receptors were characterized in hippocampal organotypic slice cultures. In the presence of ionotropic glutamate and GABA_B receptor antagonists, pressure-application of glycine onto CA3 pyramidal cells induced a current associated with increased chloride conductance, which was inhibited by strychnine. Similar chloride currents could also be induced with β-alanine or taurine. Whole-cell glycine responses were significantly greater in CA3 pyramidal cells than in CA1 pyramidal cells and dentate granule cells, while responses to GABA were similar among these three cell types. Although these results demonstrate the presence of functional glycine receptors in the hippocampus, no evidence for their activation during synaptic stimulation was found. Gabazine, a selective GABA_A receptor antagonist, totally blocked evoked IPSCs in CA3 pyramidal cells. Glycine receptor activation is not dependent on transporter-controlled levels of extracellular glycine, as no chloride current was observed in response to sarcosine, an inhibitor of glycine transporters. In contrast, application of guanidinoethanesulfonic acid, an uptake inhibitor of β-alanine and taurine, induced strychnine-sensitive chloride current in the presence of gabazine. These data indicate that modulation of transporters for the endogenous amino acids, β-alanine and taurine, can regulate tonic activation of glycine receptors, which may function in maintenance of inhibitory tone in the hippocampus.     

Functional glycine receptor maturation in the absence of glycinergic input in dopaminergic neurones of the rat substantia nigra

The postnatal maturation pattern of glycine receptor channels (GlyRs) expressed by dopaminergic (DA) neurones of the rat substantia nigra pars compacta (SNc) was investigated using single-channel and whole-cell patch-clamp recordings in brain slices from rats aged 7–21 postnatal days (P). In neonatal rats (P7-P10), GlyRs exhibited a main conductance state of 100–110 pS with a mean open time of 16 ms. In juvenile rats (P19-P22), both the GlyR main conductance state (46-55 pS) and the mean open time (6.8 ms) were decreased. In neonatal rats, application of 30 μ m picrotoxin, which is known to block homomeric GlyRs, strongly reduced glycine-evoked responses, while it was much less effective in juvenile rats. These results suggest that these GlyRs correspond functionally to α₂ homomeric GlyRs in neonatal rats and α₁/β heteromeric GlyRs in juvenile rats. A drastic but transient decrease in the glycine responsiveness of DA neurones occurred around P17 concomitant to the functional switch from the homomeric state to the heteromeric state. This age corresponds to a maturation phase for DA neurones. The application of 1 μ m gabazine blocked spontaneous or evoked inhibitory synaptic current, while the addition of 1 μ m strychnine had no effect, suggesting a lack of functional glycinergic synapses on DA neurones. Although it has been proposed that taurine is co-released with GABA at GABAergic synapses on DA neurones, in the present study the stimulation of GABAergic fibres failed to activate GlyRs. Blockade of taurine transporters and applications of high K⁺ and hyposmotic solutions were also unable to induce any strychnine-sensitive current. We conclude that functional maturation of GlyRs can occur in the absence of any detectable GlyR activation in DA neurones of the SNc.