Phosphorylation factors control neurotransmitter and neuromodulator actions at the gamma-aminobutyric acid type A receptor
Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence of gamma-aminobutyric acid (GABA)A receptor function on intracellular phosphorylation factors. Without ATP in the intracellular solution, repeated application of 30 mic...
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Published in | Molecular pharmacology Vol. 46; no. 3; pp. 542 - 549 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Pharmacology and Experimental Therapeutics
01.09.1994
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Subjects | |
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Abstract | Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence
of gamma-aminobutyric acid (GABA)A receptor function on intracellular phosphorylation factors. Without ATP in the intracellular
solution, repeated application of 30 microM GABA results in a progressive decline (run-down) of the currents evoked by GABA
in standard whole-cell recordings but not when the nystatin-perforated patch method is used. Run-down is also observed in
outside-out excised patch recordings, indicating that any enzymatic factors required for run-down must be closely associated
with the plasma membrane. Run-down is associated with decreases in both the maximum GABA-induced current and the GABA EC50.
Inclusion of magnesium adenosine-5'-O-(3-thio)triphosphate in the intracellular buffer prevents the decline in the maximum
GABA response but the GABA EC50 still decreases, resulting in a "run-up" of the response at low (3 microM) GABA concentrations.
Run-down is use dependent, requiring repeated activation of the GABAA receptor by high (30 microM) GABA concentrations. However,
use-independent run-down can be induced by the inclusion of alkaline phosphatase in the intracellular buffer. The response
to 3 microM GABA does not normally run down, but run-down is observed when the response to 3 microM GABA is potentiated with
pentobarbital or allopregnanolone, suggesting that run-down is consequence of GABA receptor activation and/or desensitization.
Run-down of the potentiated GABA response can be prevented by addition of magnesium adenosine-5'-O-(3-thio)triphosphate to
the intracellular solution. Strikingly, run-down results in a significant decrease in the potentiating effects of positive
modulators, whereas the inhibitory effects of negative modulators such as pregnenolone sulfate and ZnCl2 are unchanged. The
results demonstrate that phosphorylation factors have the capacity to control GABAA receptor pharmacology, affecting the potency
and efficacy of GABA, the kinetics of GABAA receptor desensitization, and the sensitivity of the receptor to modulators such
as steroids, benzodiazepines, and barbiturates. |
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AbstractList | Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence
of gamma-aminobutyric acid (GABA)A receptor function on intracellular phosphorylation factors. Without ATP in the intracellular
solution, repeated application of 30 microM GABA results in a progressive decline (run-down) of the currents evoked by GABA
in standard whole-cell recordings but not when the nystatin-perforated patch method is used. Run-down is also observed in
outside-out excised patch recordings, indicating that any enzymatic factors required for run-down must be closely associated
with the plasma membrane. Run-down is associated with decreases in both the maximum GABA-induced current and the GABA EC50.
Inclusion of magnesium adenosine-5'-O-(3-thio)triphosphate in the intracellular buffer prevents the decline in the maximum
GABA response but the GABA EC50 still decreases, resulting in a "run-up" of the response at low (3 microM) GABA concentrations.
Run-down is use dependent, requiring repeated activation of the GABAA receptor by high (30 microM) GABA concentrations. However,
use-independent run-down can be induced by the inclusion of alkaline phosphatase in the intracellular buffer. The response
to 3 microM GABA does not normally run down, but run-down is observed when the response to 3 microM GABA is potentiated with
pentobarbital or allopregnanolone, suggesting that run-down is consequence of GABA receptor activation and/or desensitization.
Run-down of the potentiated GABA response can be prevented by addition of magnesium adenosine-5'-O-(3-thio)triphosphate to
the intracellular solution. Strikingly, run-down results in a significant decrease in the potentiating effects of positive
modulators, whereas the inhibitory effects of negative modulators such as pregnenolone sulfate and ZnCl2 are unchanged. The
results demonstrate that phosphorylation factors have the capacity to control GABAA receptor pharmacology, affecting the potency
and efficacy of GABA, the kinetics of GABAA receptor desensitization, and the sensitivity of the receptor to modulators such
as steroids, benzodiazepines, and barbiturates. Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence of gamma-aminobutyric acid (GABA)A receptor function on intracellular phosphorylation factors. Without ATP in the intracellular solution, repeated application of 30 microM GABA results in a progressive decline (run-down) of the currents evoked by GABA in standard whole-cell recordings but not when the nystatin-perforated patch method is used. Run-down is also observed in outside-out excised patch recordings, indicating that any enzymatic factors required for run-down must be closely associated with the plasma membrane. Run-down is associated with decreases in both the maximum GABA-induced current and the GABA EC50. Inclusion of magnesium adenosine-5'-O-(3-thio)triphosphate in the intracellular buffer prevents the decline in the maximum GABA response but the GABA EC50 still decreases, resulting in a "run-up" of the response at low (3 microM) GABA concentrations. Run-down is use dependent, requiring repeated activation of the GABAA receptor by high (30 microM) GABA concentrations. However, use-independent run-down can be induced by the inclusion of alkaline phosphatase in the intracellular buffer. The response to 3 microM GABA does not normally run down, but run-down is observed when the response to 3 microM GABA is potentiated with pentobarbital or allopregnanolone, suggesting that run-down is consequence of GABA receptor activation and/or desensitization. Run-down of the potentiated GABA response can be prevented by addition of magnesium adenosine-5'-O-(3-thio)triphosphate to the intracellular solution. Strikingly, run-down results in a significant decrease in the potentiating effects of positive modulators, whereas the inhibitory effects of negative modulators such as pregnenolone sulfate and ZnCl2 are unchanged. The results demonstrate that phosphorylation factors have the capacity to control GABAA receptor pharmacology, affecting the potency and efficacy of GABA, the kinetics of GABAA receptor desensitization, and the sensitivity of the receptor to modulators such as steroids, benzodiazepines, and barbiturates. Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence of gamma -aminobutyric acid (GABA) sub(A) receptor function on intracellular phosphorylation factors. Without ATP in the intracellular solution, repeated application of 30 mu M GABA results in a progressive decline (run-down) of the currents evoked by GABA in standard whole-cell recordings but not when the nystatin-perforated patch method is used. Run-down is also observed in outside-out excised patch recordings, indicating that any enzymatic factors required for run-down must be closely associated with the plasma membrane. Run-down is associated with decreases in both the maximum GABA-induced current and the GABA EC sub(50). Inclusion of magnesium adenosine-5'-O-(3-thio)triphosphate in the intracellular buffer prevents the decline in the maximum GABA response but the GABA EC sub(50) still decreases, resulting in a "run-up" of the response at low GABA concentrations. Run-down is use dependent, requiring repeated activation of the GABA sub(A) receptor by high GABA concentrations. However, use-independent run-down can be induced by the inclusion of alkaline phosphatase in the intracellular buffer. The response to 3 mu M GABA does not normally run down, but run-down is observed when the response to 3 mu M GABA is potentiated with pentobarbital or allopregnanolone, suggesting that run-down is a consequence of GABA receptor activation and/or desensitization. Run-down of the potentiated GABA response can be prevented by addition of magnesium adenosine-5'-O-(3-thio)triphosphate to the intracellular solution. Strikingly, run-down results in a significant decrease in the potentiating effects of positive modulators, whereas the inhibitory effects of negative modulators such as pregnenolone sulfate and ZnCl sub(2) are unchanged. The results demonstrate that phosphorylation factors have the capacity to control GABA sub(A) receptor pharmacology, affecting the potency and efficacy of GABA, the kinetics of GABA sub(A) receptor desensitization, and the sensitivity of the receptor to modulators such as steroids, benzodiazepines, and barbiturates. |
Author | T T Gibbs Q Wang M Gyenes D H Farb |
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Snippet | Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence
of gamma-aminobutyric acid... Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence of gamma-aminobutyric acid... Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence of gamma -aminobutyric acid... |
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SubjectTerms | Adenosine Triphosphate - pharmacology Animals Barbiturates - pharmacology Benzodiazepines - pharmacology Cells, Cultured Chick Embryo Chlorides - pharmacology Dose-Response Relationship, Drug Drug Interactions Electrophysiology gamma-Aminobutyric Acid - pharmacology Neurons - drug effects Neurons - metabolism Patch-Clamp Techniques Phosphorylation Receptors, GABA-A - drug effects Receptors, GABA-A - metabolism Spinal Cord - cytology Spinal Cord - embryology Spinal Cord - metabolism Zinc Compounds - pharmacology |
Title | Phosphorylation factors control neurotransmitter and neuromodulator actions at the gamma-aminobutyric acid type A receptor |
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