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GABA(A) receptors expressed in undifferentiated human teratocarcinoma NT2 cells differ from those expressed by
T R Neelands1, J Zhang, R L Macdonald
1Graduate Program in the Neurosciences, University of Michigan Health Sciences Center, Ann Arbor, Michigan 48104-1687, USA.
Abstract:
During CNS development, changes occur in expression of GABA(A) receptor subunit subtypes and GABA(A) receptor pharmacological and biophysical properties. We used reverse transcription PCR and whole-cell-recording techniques to determine whether GABA(A) receptor expression and function also changed during retinoic acid-induced differentiation of human Ntera 2 (NT2) teratocarcinoma cells into neuron-like cells (NT2-N cells). In undifferentiated NT2 cells only alpha5, beta3, gamma3, and pi subtype mRNAs were detected. NT2 GABA(A) receptor currents had a maximal amplitude of 52 pA and an EC(50) of 4.0 microM, were relatively insensitive to enhancement by zolpidem and diazepam, and were enhanced by loreclezole and inhibited by lanthanum, zinc, and furosemide. In contrast, in NT2-N cells after 13 weeks of retinoic acid treatment, all GABA(A) receptor subtype mRNAs were detected. Maximal peak whole-cell currents were approximately 50-fold larger than NT2 cell currents, and the GABA EC(50) was higher (39.7 microM). In 13 week NT2-N cells, diazepam, zolpidem, loreclezole, and lanthanum had only small effects on GABA(A) receptor currents, and the zinc IC(50) for current inhibition was significantly higher than that for NT2 cells. In a previous study, we showed that NT2-N cells after 5 weeks of retinoic acid treatment had moderate peak currents, GABA EC(50,) and zinc IC(50) but that currents were robustly enhanced by diazepam, zolpidem, and loreclezole. During differentiation of NT2 cells to NT2-N cells, GABA(A) receptors underwent changes in subunit expression and pharmacology that were similar to many of the developmental changes in GABA(A) receptors that occur in CNS neurons.
Insights
GABA(A) receptor subunit expression and function change during neuronal differentiation. Retinoic acid treatment of NT2 cells induces changes in GABA(A) receptors, mimicking CNS development.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- GABA(A) receptors are crucial for CNS development.
- Receptor properties change during neuronal maturation.
- Human NT2 teratocarcinoma cells differentiate into neuron-like cells (NT2-N) upon retinoic acid treatment.
Purpose of the Study:
- To investigate changes in GABA(A) receptor subunit expression and function during retinoic acid-induced differentiation of NT2 cells.
- To compare these changes to those observed during CNS development.
Main Methods:
- Reverse transcription PCR to detect GABA(A) receptor subunit mRNAs.
- Whole-cell recording techniques to measure GABA(A) receptor currents and pharmacology.
- Retinoic acid treatment of NT2 cells for 5 and 13 weeks.
Main Results:
- Undifferentiated NT2 cells expressed limited GABA(A) receptor subunit mRNAs (alpha5, beta3, gamma3, pi) with specific current properties.
- NT2-N cells (13 weeks) expressed all GABA(A) receptor subunit mRNAs, exhibiting significantly larger currents and altered pharmacology (higher EC50, reduced sensitivity to modulators).
- Changes in subunit expression and pharmacology during NT2 cell differentiation resemble those during CNS neuronal development.
Conclusions:
- Retinoic acid-induced differentiation of NT2 cells provides a model for studying developmental changes in GABA(A) receptors.
- GABA(A) receptor subunit composition and function undergo significant alterations during neuronal differentiation.
- These observed changes mirror critical developmental processes in the central nervous system.