Abstract
Rationale
Neurosteroids and likely other lipid modulators access transmembrane sites on the GABAA receptor (GABAAR) by partitioning into and diffusing through the plasma membrane. Therefore, specific components of the plasma membrane may affect the potency or efficacy of neurosteroid-like modulators. Here, we tested a possible role for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that governs activity of many channels and transporters, in modulation or function of GABAARs.
Objectives
In these studies, we sought to deplete plasma-membrane PIP2 and probe for a change in the strength of potentiation by submaximal concentrations of the neurosteroid allopregnanolone (3α5αP) and other anesthetics, including propofol, pentobarbital, and ethanol. We also tested for a change in the behavior of negative allosteric modulators pregnenolone sulfate and dipicrylamine.
Methods
We used Xenopus oocytes expressing the ascidian voltage-sensitive phosphatase (Ci-VSP) to deplete PIP2. Voltage pulses to positive membrane potentials were used to deplete PIP2 in Ci-VSP-expressing cells. GABAARs composed of α1β2γ2L and α4β2δ subunits were challenged with GABA and 3α5αP or other modulators before and after PIP2 depletion. KV7.1 channels and NMDA receptors (NMDARs) were used as positive controls to verify PIP2 depletion.
Results
We found no evidence that PIP2 depletion affected modulation of GABAARs by positive or negative allosteric modulators. By contrast, Ci-VSP-induced PIP2 depletion depressed KV7.1 activation and NMDAR activity.
Conclusions
We conclude that despite a role for PIP2 in modulation of a wide variety of ion channels, PIP2 does not affect modulation of GABAARs by neurosteroids or related compounds.
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Acknowledgments
We thank Jianmin Cui for the gift of Ci-VSP, Kv7.1, and KCNE1 constructs and Joe Henry Steinbach for the discussion related to the conception of these experiments. Thanks to Hong-Jin Shu for the help in the RNA preparation. This work was funded by a gift from the Bantly Foundation and NIH grants GM47969, MH078823, MH099658, MH077791, and AA017413.
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Mennerick, S., Taylor, A.A. & Zorumski, C.F. Phosphatidylinositol 4,5-bisphosphate depletion fails to affect neurosteroid modulation of GABAA receptor function. Psychopharmacology 231, 3493–3501 (2014). https://doi.org/10.1007/s00213-014-3486-5
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DOI: https://doi.org/10.1007/s00213-014-3486-5