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Regulation of Neuronal L-type Voltage-Gated Calcium Channels by Flurazepam and Other Positive Allosteric GABAA Receptor Modulators

Earl, Damien E.
http://rave.ohiolink.edu/etdc/view?acc_num=mco1307379688

Abstract Details

2011, Doctor of Philosophy (PhD), University of Toledo, College of Medicine.
Benzodiazpines (BZs) are clinically useful anxiolytics, sedatives, and anticonvulsants. Their mechanism of action is positive allosteric modulation of γ-aminobutyric acid type A (GABAA) receptors, the main inhibitory neurotransmitter receptors in the mammalian central nervous system. Long-term administration of BZs and other positive allosteric GABAA receptor modulators, neurosteroids, barbiturates, and ethanol can lead to physical dependence manifested by a characteristic withdrawal syndrome. A common mechanism proposed to contribute to this withdrawal syndrome is functional up-regulation of L-type voltage-gated calcium channels (L-VGCCs). Our lab models BZ dependence using a 1-week oral treatment of rats with flurazepam (FZP) followed by 1 or 2 days of withdrawal. This treatment paradigm resulted in a near doubling of voltage-gated Ca2+ currents in hippocampal CA1 neurons. Enhanced L-VGCC-mediated Ca2+ influx may activate Ca2+/calmodulin-dependent protein kinase II (CaMKII), which potentiated excitatory synaptic function in CA1 neurons correlating with expression of FZP withdrawal anxiety. The current studies tested three hypotheses: 1) GABAA receptor modulators directly inhibit recombinantly expressed L-VGCCs containing neuronal α1 subunits, Cav1.2 or Cav1.3; 2) L-VGCC subunit expression is increased in the rat hippocampal CA1 region; and 3) CaMKII enhances CA1 excitatory synaptic function via activation and autophosphorylation at Thr286 and/or enhanced localization to the postsynaptic density (PSD). The findings suggested that while the barbiturate pentobarbital and ethanol directly inhibit L-VGCCs at clinically relevant concentrations, the concentrations of BZs and neurosteroids required to inhibit recombinant L-VGCCs were likely too high to be clinically relevant. Interestingly, Cav1.2 channels were more sensitive to inhibition by pentobarbital and FZP and were less sensitive to inhibition by the L-VGCC benzothiazepine (BTZ) antagonist, diltiazem, than Cav1.3 channels. Selective inhibition could independently block Ca2+ signaling cascades mediated by Cav1.2 and Cav1.3 L-VGCCs. Mutation studies revealed that the pentobarbital L-VGCC binding site may overlap that of dihydropyridines, and despite structural similarities amongst BTZs and BZs, the BZ L-VGCC binding site is distinct from that of BTZs. No alteration in L-VGCC subunit expression was observed in PSD-enriched CA1 homogenates or immunostained hippocampal slices as a function of FZP withdrawal. Taken together, the data suggested that mechanisms other than direct inhibition of L-VGCCs and increased L-VGCC subunit expression mediate the enhanced Ca2+ influx observed following long-term FZP treatment. Post-translational modifications and/or enhanced trafficking of L-VGCCs to the membrane due to persistent BZ enhancement of GABAA receptors are alternate possibilities. Additionally, after 2 days of FZP withdrawal, total CaMKIIα expression was decreased in CA1 PSDs with no alteration in the absolute amount of the autonomously active Thr286 autophosphorylated form of CaMKII. Alternate mechanisms of CaMKII activation by L-VGCC-mediated Ca2+ influx and for the loss of CaMKII from PSDs during FZP withdrawal are proposed.
Elizabeth Tietz, Ph.D. (Committee Chair)
Zi-Jian Xie, Ph.D. (Committee Member)
David Giovannucci, Ph.D. (Committee Member)
Scott Molitor, Ph.D. (Committee Member)
Bryan Yamamoto, Ph.D. (Committee Member)
205 p.
Neurosciences
CNS depressants; benzodiazepines; GABA receptor; recombinant; calcium channels; CaMKII; electrophysiology; electron microscopy; neuronal plasticity; Western blot; Cav1.2; Cav1.3; hippocampus

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Citations

  • Earl, D. E. (2011). Regulation of Neuronal L-type Voltage-Gated Calcium Channels by Flurazepam and Other Positive Allosteric GABAA Receptor Modulators [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1307379688

    APA Style (7th edition)

  • Earl, Damien. Regulation of Neuronal L-type Voltage-Gated Calcium Channels by Flurazepam and Other Positive Allosteric GABAA Receptor Modulators. 2011. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1307379688.

    MLA Style (8th edition)

  • Earl, Damien. "Regulation of Neuronal L-type Voltage-Gated Calcium Channels by Flurazepam and Other Positive Allosteric GABAA Receptor Modulators." Doctoral dissertation, University of Toledo, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=mco1307379688

    Chicago Manual of Style (17th edition)

mco1307379688
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© 2011, all rights reserved.
This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.