GABAA receptor modulation by protein tyrosine kinase in the rat diagonal band of Broca

doi:10.1016/S0006-8993(97)00892-5

Brain Research

Volume 775, Issues 1–2, 14 November 1997, Pages 127-133

https://doi.org/10.1016/S0006-8993(97)00892-5 Get rights and content

Abstract

The diagonal band of Broca (DBB) is involved in a wide array of physiological functions which are, in part, mediated by activation of GABA_A receptors. DBB is enriched in GABA and protein tyrosine kinase (PTK) immunoreactivity. Whole-cell patch-clamp recording were performed from acutely dissociated DBB neurons to investigate the involvement of PTK in GABA_A receptor function. The activation of GABA_A receptor by the selective agonist, muscimol (5 μM) was dependent on the presence of intracellular ATP. Omission of ATP in the intracellular medium resulted in a fast decrement of the response whereas inclusion of sodium orthovanadate (100 μM), a non-specific phosphatase inhibitor, augmented the response and inhibited `run down' of the response. Genistein (100 μM) and tyrphostin B-44 (–), specific inhibitors of PTK, attenuated the response to muscimol. The muscimol response was not affected by daidzein (100 μM; an inactive analogue of genistein) nor by tetraethylammonium bromide (1 mM). These observations suggest that phosphorylation is important for the activation and long term maintenance of GABA_A receptor function. PTK phosphorylation, which has been previously identified as an important event in signal transduction, may modulate GABA mediated neurotransmission in the forebrain.

Introduction

The diagonal band of Broca (DBB), a basal forebrain nucleus, is an important site for a variety of physiological functions including those related to learning and memory, the generation of theta rhythm and central cardiovascular regulation [11]. Multiple chemical transmitter phenotypes are represented within DBB's extensive connectivity with other brain regions including the hippocampus, hypothalamus, brain-stem and cerebral cortex. Amongst these γ-aminobutyric acid (GABA), a monocarboxylic amino acid, is a major inhibitory neurotransmitter. The DBB is enriched in GABA immunoreactivity and binding sites 6, 23. Applications of GABA agonists and antagonists within DBB interfere with hippocampal theta rhythm [18]and impair cognitive function 2, 5. Thus modulation of GABA_A receptor function within the DBB may play an important role not only under physiological conditions but also within the context of pathology involving this region such as in Alzheimer's disease 4, 27.

The GABA_A receptors are pentameric structures composed of several distinct subunits (α, β, γ, and δ) and belong to the family of ligand-gated ion channels. Binding of GABA to the GABA_A receptor opens the chloride ionic conductance resulting in activation of chloride currents. Phosphorylation by various protein kinases is a common mechanism for modulating the activity of certain ligand- and voltage-gated ion channels 9, 10, 16. The GABA_A receptor-gated chloride currents have been shown to be subject to such modulation by serine and threonine kinases. Phosphorylation of these receptors by cAMP-dependent protein kinase (PKA) 7, 15, 22and protein kinase C [17]increases the rate of desensitization resulting in a decrease in the GABA_A-receptor gated chloride fluxes. These two kinases are thus less likely to be involved in maintenance of long term activation of GABA_A-receptor gated chloride channels. Phosphorylation of the receptor protein by protein tyrosine kinase (PTK) is a relatively recently described mechanism for amino acid transmitters 14, 24, 26. A potential tyrosine phosphorylation site has been proposed to exist in the large cytoplasmic loop of the γ2 subunit (tyrosine 367/369) of the GABA_A receptor 10, 21. Tyrosine phosphorylation has been shown to augment whole-cell currents in human embryonic kidney cells in which α1, β1 and γ2L subunits were co-expressed with the tyrosine kinase vSRC (the transforming gene product of the Rous sarcoma virus; [14]). Inhibitors of PTK phosphorylation block ³⁶Cl⁻ uptake in mouse brain membrane vesicles [24]. Thus from these preparations there is emerging evidence for PTK involvement in the GABA_A receptor function.

PTK activity is localized within widespread areas of the basal forebrain, which includes the DBB [8]. Since PTK activity and GABA_A receptor have been found to be co-localized in the DBB neurons, and GABA_A receptor function is crucial to the physiological functions of DBB, we tested the hypothesis if GABA_A receptor function in the DBB neurons is modulated by PTK activity.

Section snippets

Materials and methods

Whole-cell patch-clamp recordings were performed on acutely dissociated DBB neurons from 15–21 day old male Sprague-Dawley rats using Axopatch-1D patch-clamp amplifier. The cells were prepared by enzymatic treatment of brain slices from DBB with trypsin (0.65 mg/ml) followed by mechanical trituration followed by plating on poly-l-lysine (0.005% w/v) coated cover slips. The external perfusing solution, an artificial cerebrospinal fluid (ACSF), contained (in mM): NaCl 140, KCl 2.5, CaCl₂ 1.5, MgCl

GABA_A receptor activation is dependent on presence of intracellular ATP

Application of muscimol, a selective GABA_A receptor agonist, resulted in readily reversible activation of chloride conductances (Fig. 1A,B,C). The drug application was repeated at 5 min intervals and voltage ramp applied at the peak response to estimate the activatable amount of voltage-activated conductances. When the internal patch pipette solution contained 2.2 mM ATP the responses could be reproducibly evoked on repeated applications (1 min) of the agonist (5 μM), at 5 min intervals, for

Discussion

In the rat DBB neurons, muscimol generated an inward current at potentials negative to E_Cl and an outward current positive to E_Cl indicating selective activation of GABA _A receptor-gated chloride conductances. The long term maintenance of the muscimol-evoked current was dependent on inclusion of ATP in the intracellular medium suggesting the involvement of phosphorylation mechanisms in the maintenance of GABA_A receptor function. Previously, it has been shown that the maintenance of

Acknowledgements

This work was supported by the Medical Research Council of Canada. The authors thank Drs C. Bourque and J. Greer for comments and suggestions towards this manuscript. We also thank Mr Kim Harris, Dr Jacob Easaw and Ms Caroline Cho for assistance with experiments and illustrations, and Ms C. Krys with typing the manuscript. Dr B.S. Jassar was a recipient of fellowships from the H.M. Toupin Foundation and Alzheimer Society of Canada. Ms P.M. Ostashewski held an AHFMR Summer Studentship award.

References (27)

T. Akiyama et al.
Genistein, a specific inhibitor of tyrosine-specific protein kinases
J. Biol. Chem.
(1987)
J.J. Chrobak et al.
Intraseptal administration of muscimol produces dose-dependent memory impairment in the rat
Behav. Neural Biol.
(1989)
J.E. Cheun et al.
Noradrenergic potentiation of cerebellar purkinje cell response to GABA: cyclic AMP as intracellular intermediary
Neuroscience
(1996)
R.L. Huganir et al.
Regulation of receptor function by protein phosphorylation
Trends Pharmacol. Sci.
(1987)
R.L. Huganir et al.
Regulation of neurotransmitter receptor desensitization by protein phosphorylation
Neuron
(1990)
J.H. Jhamandas et al.
Diagonal band neurons may mediate arterial baroreceptor input to hypothalamic vasopressin secreting neurons
Neurosci. Lett.
(1986)
J. Kang et al.
Modulation of net outward current in cultured neurons by angiotensin. II. Involvement of AT1 and AT2 receptors
Brain Res.
(1992)
N.M. Porter et al.
cAMP-dependent protein kinase decreases GABA_A receptor current in mouse spinal neurons
Neuron
(1990)
L.A. Raymond et al.
Phosphorylation of amino acid neurotransmitter receptors in synaptic plasticity
Trends Neurosci.
(1993)
J.W. Smythe et al.
The extrinsic modulation of hippocampal theta depends on the co-activation of cholinergic and GABA-ergic medial septal inputs
Neurosci. Biobehav. Rev.
(1992)

G. Swarup et al.

Activation of cellular tyrosine-specific protein kinase by phosphorylation

FEBS Lett.

(1985)

C.F. Valenzuela et al.

Tyrosine kinase phosphorylation of GABA_A receptors

Mol. Brain Res.

(1995)

J.T. Coyle et al.

Alzheimer's disease. A disorder of cortical cholinergic innervation

Science (Washington)

(1983)

Cited by (16)

Tyrosine Phosphorylation Determines Afterdischarge Initiation by Regulating an Ionotropic Cholinergic Receptor
2018, Neuroscience
Citation Excerpt :
Genistein blocks both EGFR and src, but fails to act on PKA, PKC, or phosphorylase kinase (Akiyama et al., 1987). Genistein was previously shown to inhibit the related pentameric cys-loop γ-aminobutyric acid receptor in both cerebellar granule cells and forebrain neurons (Jassar et al., 1997; Balduzzi et al., 2002). For Aplysia sensory neurons, genistein prevents certain serotonin-induced changes, including enhanced excitability, increased glutamate uptake, and PKC translocation, (Purcell et al., 2003; Khabour et al., 2004; Nagakura et al., 2010).
Changes to neuronal activity often involve a rapid and precise transition from low to high excitability. In the marine snail, Aplysia, the bag cell neurons control reproduction by undergoing an afterdischarge, which begins with synaptic input releasing acetylcholine to open an ionotropic cholinergic receptor. Gating of this receptor causes depolarization and a shift from silence to continuous action potential firing, leading to the neuroendocrine secretion of egg-laying hormone and ovulation. At the onset of the afterdischarge, there is a rise in intracellular Ca²⁺, followed by both protein kinase C (PKC) activation and tyrosine dephosphorylation. To determine whether these signals influence the acetylcholine ionotropic receptor, we examined the bag cell neuron cholinergic response both in culture and isolated clusters using whole-cell and/or sharp-electrode electrophysiology. The acetylcholine-induced current was not altered by increasing intracellular Ca²⁺ via voltage-gated Ca²⁺ channels, clamping intracellular Ca²⁺ with exogenous Ca²⁺ buffers, or activating PKC with phorbol esters. However, lowering phosphotyrosine levels by inhibiting tyrosine kinases both reduced the cholinergic current and prevented acetylcholine from triggering action potentials or afterdischarge-like bursts. In other systems, acetylcholine receptors are often modulated by multiple signals, but bag cell neurons appear to be more restrictive in this regard. Prior work finds that, as the afterdischarge proceeds, tyrosine dephosphorylation leads to biophysical alterations that promote persistent firing. Because this firing is subsequent to the cholinergic input, inhibiting the acetylcholine receptor may represent a means of properly orchestrating synaptically induced changes in excitability.
Interaction of pentobarbital with gabaergic drugs acting on the Cl<sup>-</sup>-ATPase activity of the plasma membranes from bream brain (Abramis brama L.)
2002, Neuroscience Letters
The present study was designed to investigate the role of the interaction of pentobarbital with γ-aminobutyric acid (GABA)ergic drugs acting on the Cl⁻-adenosine triphosphate (ATP)ase activity of the plasma membranes fraction of bream brain. The preincubation and then incubation of the membranes with pentobarbital as well as with other GABAergic ligands was conducted at physiologic pH (7.4), i.e. at the condition where the Cl⁻-ATPase activity is not detected. Pentobarbital (1–100 μM) induces Cl⁻-ATPase activity, however at high concentration (1000 μM) no effect of the ligand was found. In addition pentobarbital (50 μM) enhances the effect of low concentration of GABA (1 μM) on the Cl⁻-ATPase activity, but inhibits the action of high concentration of GABA (100 μM) on the enzyme. Whereas no activating effect of pentobarbital in the presence of baclofen (1 μM) was found. The blocker of GABA_A-receptors, picrotoxin (50 μM) and bicuculline (5 μM) eliminated the action of pentobarbital on the enzyme. The present results provide evidence for the first time that at physiologic pH in incubation medium the interaction of pentobarbital with GABAergic drugs on the Cl⁻-ATPase activity is similar to the effects of these ligands on the GABA_A-receptor.
Zinc modulation of ionic currents in the horizontal limb of the diagonal band of Broca
1999, Neuroscience
We examined modulation of ionic currents by Zn²⁺ in acutely dissociated neurons from the rat's horizontal limb of the diagonal band of Broca using the whole-cell patch-clamp technique. Application of 50 μM Zn²⁺ increased the peak amplitude of the transiently activated potassium current, I_A (at +30 mV), from 2.20±0.08 to 2.57±0.11 nA (n=27). This response was reversible and could be repeated in 0 Ca²⁺/1 μM tetrodotoxin (n=15). Zn²⁺ shifted the inactivation curve to the right, resulting in a shift in the half-inactivation voltage from −76.4±2.2 to −53.4±2.0 mV (n=11), with no effect on the voltage dependence of activation gating (n=15). There was no significant difference in the time to peak under control conditions (7.43±0.35 ms, n=14) and in the presence of Zn²⁺ (8.20±0.57 ms, n=14). Similarly, the time constant of decay of I_A (τ_d) at +30 mV showed no difference (control: 38.68±3.68 ms, n=15; Zn²⁺: 38.48±2.85 ms, n=15). I_A was blocked by 0.5–1 mM 4-aminopyridine. In contrast to its effects on I_A, Zn²⁺ reduced the amplitude of the delayed rectifier potassium current (I_K). The reduction of outward K⁺ currents was reproducible when cells were perfused with 1 μM tetrodotoxin in a 0 Ca²⁺ external solution. The amplitude of the steady-state outward currents at +30 mV under these conditions was reduced from 6.40±0.23 (control) to 5.76±0.18 nA in the presence of Zn²⁺ (n=16). The amplitudes of peak sodium currents (I_Na) were not significantly influenced (n=10), whereas barium currents (I_Ba) passing through calcium channels were potently modulated. Zn²⁺ reversibly reduced I_Ba at −10 mV by ∼85% from −2.06±0.14 nA under control conditions to −0.30±0.10 nA in the presence of Zn²⁺ (n=14). Further analyses of Zn²⁺ effects on specific calcium channels reveals that it suppresses all types of high-voltage-activated Ca²⁺ currents.
Under current-clamp conditions, application of Zn²⁺ resulted in an increase in excitability and loss of accommodation (n=13), which appears to be mediated through its effects on Ca²⁺-dependent conductances.
The tyrosine kinase inhibitor genistein directly inhibits GABA(A) receptors
1999, Molecular Brain Research
The protein tyrosine kinase (PTK) inhibitor genistein has been widely used to examine potential effects of tyrosine phosphorylation on neurotransmitter function. We report here that genistein inhibits GABA_A receptors through a direct effect. Extracellular application of genistein and GABA reversibly inhibited GABA-activated currents recorded from HEK293 cells expressing rat α1β2γ2S or α1β2 receptors, even when genistein was preequilibrated in the intracellular solution. Daidzein, an analog of genistein that does not block PTK, also inhibited GABA-activated current. Coapplication of lavendustin A, a specific inhibitor of PTK, had no effect on the GABA response. Our results demonstrate that genistein has a direct inhibitory effect on GABA_A receptors that is not mediated via inhibition of tyrosine kinase.
Inhibition of GABA(A) receptor function by tyrosine kinase inhibitors and their inactive analogues
1998, Molecular and Cellular Neurosciences
The effects of tyrosine kinase inhibitors which target the ATP binding site or the substrate binding site of tyrosine kinases were assessed on murine recombinant type A γ-aminobutyric acid (GABA_A) receptors expressed inXenopusoocytes or HEK cells using two-electrode voltage clamp or patch clamp recording. Genistein inhibited in a noncompetitive manner GABA-activated currents recorded from α1β1γ2S receptor constructs by reducing the maximum normalized response from 1.83 ± 0.04 to 0.71 ± 0.04 and reducing the EC₅₀from 35.7 ± 2.1 μM to 15.1 ± 3.9 μM. After mutating the two “functionally active” substrate tyrosine (Y) residues in γ2S and expressing the mutant receptor α1β1γ2S(Y365F, Y367F), genistein still noncompetitively inhibited the responses to GABA reducing the maximum current from 1.81 ± 0.03 to 0.26 ± 0.01 and the EC₅₀from 33.1 ± 2.3 μM to 5.8 ± 2.2 μM. The inactive compound, daidzein, also similarly inhibited responses to GABA on these two receptor constructs. Inhibitors targeting the substrate binding site of tyrosine kinases, the tyrphostins, also inhibited both the wild-type and the tyrosine mutant GABA_Areceptors. Tyrphostin A25 and the inactive tyrphostin A1 reduced the maximum normalized responses for α1β1γ2S and α1β1γ2S(Y365F, Y367F) receptors by 73 and 64%, respectively. The tyrosine kinase inhibitors and their inactive controls did not display any significant voltage sensitivity to the antagonism of GABA-activated responses. Moreover, genistein or tyrphostin A25 did not affect the potentiation of responses to GABA by pentobarbitone or diazepam. Mutating the two “functionally silent” tyrosine residues, Y370 and Y372, known to be substrates for tyrosine kinases in the β1 subunit and coexpression in the α1β1(Y370F, Y372F)γ2S(Y365F, Y367F) construct failed to affect the inhibitory action of genistein. The study concludes that tyrosine kinase inhibitors and their inactive controls can directly interact with GABA_Areceptors completely independent of any effects on tyrosine kinases.
Phenols and GABA<inf>A</inf> receptors: from structure and molecular mechanisms action to neuropsychiatric sequelae
2024, Frontiers in Pharmacology

View all citing articles on Scopus

View full text

Research reportGABAA receptor modulation by protein tyrosine kinase in the rat diagonal band of Broca

Abstract

Introduction

Section snippets

Materials and methods

GABAA receptor activation is dependent on presence of intracellular ATP

Discussion

Acknowledgements

J. Biol. Chem.

Behav. Neural Biol.

Neuroscience

Trends Pharmacol. Sci.

Neuron

Neurosci. Lett.

Brain Res.

Neuron

Trends Neurosci.

Neurosci. Biobehav. Rev.

FEBS Lett.

Mol. Brain Res.

Alzheimer's disease. A disorder of cortical cholinergic innervation

Science (Washington)

Research report
GABA_A receptor modulation by protein tyrosine kinase in the rat diagonal band of Broca

GABA_A receptor activation is dependent on presence of intracellular ATP