Agonist-induced GABA(A) receptor down-regulation and uncoupling in neuronal culture: Multiple signal transduction pathways and a role for intracellular calcium

• Main Author: Lyons, Helen Rosemary
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-1998
• Subjects: Cellular biology; Neurology; Neurosciences; Pharmacology
• ISBN: 9780591780864; 0591780860

Fast inhibitory neurotransmission is mediated primarily by activation of the type A $\gamma$-aminobutyric acid (GABA$\sb{\rm A}$) receptor, a hetero-pentameric ligand-gated chloride channel. In addition to mediating fast synaptic transmission, activation of ligand-gated ion channels (ionotropic receptors) can cause lasting adaptive changes in neuronal gene expression. As first described in our laboratory, chronic treatment of chick cortical neurons in culture with GABA downregulates GABA$\sb{\rm A}$ receptor number and subunit mRNAs without increasing the rate of receptor degradation. The signal transduction mechanisms that allow GABA$\sb{\rm A}$ receptor activation to regulate GABA$\sb{\rm A}$ receptor number is an important but unexplored area of molecular neurobiology. The GABA$\sb{\rm A}$ receptor is assembled from multiple subunits, including $\alpha$1-6, $\beta$1-4 and $\gamma$1-4, some with alternatively spliced short (S) and (L) forms. The present results demonstrate coordinate downregulation of $\alpha1,\ \beta2$S and $\gamma$1 subunit mRNA levels. Downregulation of $\alpha$1 mRNA (t$\sb{1/2}$ = 8 h) occurs faster than receptor downregulation (t$\sb{1/2}$ 25 h), indicating that inhibition of mRNA synthesis or stimulation of mRNA degradation precedes receptor downregulation. GABA does not accelerate degradation of the $\alpha1,\ \beta2$S or $\gamma$1 mRNAs suggesting that autologous downregulation of GABA$\sb{\rm A}$ receptor subunit mRNAs is under transcriptional control. GABA$\sb{\rm A}$ receptor downregulation is blocked by inhibiting neuronal activity with the voltage-gated Na$\sp+$ channel inhibitor, tetrodotoxin, or the ionotropic glutamate receptor antagonists, DNQX + APV. Chronic depolarization with 25 mM K$\sp+$, but not 10 mM K$\sp+$, also blocks downregulation consistent with inhibition of neuronal activity through inactivation of voltage-gated ion channels. In addition to downregulation, GABA treatment uncouples the allosteric interaction between binding sites for GABA and allosteric modulators. Uncoupling is not blocked by inhibitors of downregulation, demonstrating that downregulation and uncoupling occur through different molecular mechanisms. GABA$\sb{\rm A}$ receptor activation causes a rapid, transient Ca$\sp{2+}$ influx which is blocked by nifedipine, a voltage-gated Ca$\sp{2+}$ channel inhibitor. Nifedipine also blocks GABA-induced downregulation demonstrating that voltage-gated Ca$\sp{2+}$ channel activation is required. These are the first results to suggest a role for Ca$\sp{2+}$ in the signaling pathways linking GABA$\sb{\rm A}$ receptor activation with GABA$\sb{\rm A}$ receptor downregulation.