Phosphatidylinositol-4,5-Bisphosphate Regulates NMDA Receptor Activity through α-Actinin

Abstract

Phosphatidylinositol-4,5-bisphosphate (PIP 2 ) has been shown to regulate many ion channels, transporters, and other signaling proteins, but it is not known whether it also regulates neurotransmitter-gated channels. The NMDA receptors (NMDARs) are gated by glutamate and serve as a critical control point in synaptic function. Here we demonstrate that PIP 2 supports NMDAR activity. In Xenopus oocytes, overexpression of phospholipase C␥ (PLC␥) or preincubation with 10 M wortmannin markedly reduced NMDA currents. Stimulation of the epidermal growth factor receptor (EGFR) promoted the formation of an immunocomplex between PLC␥ and NMDAR subunits. Stimulation of EGFR or the PLC␤-coupled M 1 acetylcholine receptor produced a robust transient inhibition of NMDA currents. Wortmannin application blocked the recovery of NMDA currents from the inhibition. Using mutagenesis, we identified the structural elements on NMDAR intracellular tails that transduce the receptor-mediated inhibition, which pinpoint to the binding site for the cytoskeletal protein ␣-actinin. Mutation of the PIP 2 -binding residues of ␣-actinin dramatically reduced NMDA currents and occluded the effect of EGF. Interestingly, EGF or wortmannin affected the interaction between NMDAR subunits and ␣-actinin, suggesting that this protein mediates the effect of PIP 2 on NMDARs. In mature hippocampal neurons, expression of the mutant ␣-actinin reduced NMDA currents and accelerated inactivation. We propose a model in which ␣-actinin supports NMDAR activity via tethering their intracellular tails to plasma membrane PIP 2 . Thus, our results extend the influence of PIP 2 to the NMDA ionotropic glutamate receptors and introduce a novel mechanism of "indirect" regulation of transmembrane protein activity by PIP 2 .

Phosphatidylinositol-4,5-Bisphosphate and α-Actinin: Two-Component Hinge for the NMDA Receptor: Figure 1.

“…In the first set of experiments, the authors showed that PLC-catalyzed PIP2 hydrolysis suppressed NMDA currents in oocytes [Michailidis et al (2007) …”

“…One complication of this experiment is that hydrolysis of PIP2 leads to production of inositol trisphosphate and diacylglycerol, which mobilize intracellular Ca 2ϩ release. However, even after "dumping" most of the Ca 2ϩ from internal stores, NMDA receptor currents were still inhibited by PLC-coupled receptor stimulation [Michailidis et al (2007) The authors then identified sites on NMDA receptors responsible for "sensing" PIP2, consistent with ␣-actinin binding sites on NMDA receptors [Michailidis et al (2007), their Figs. 5 (http://www.…”

“…A recent paper published in The Journal of Neuroscience (Michailidis et al, 2007) provides evidence that PIP2 regulates NMDA receptor activity through a cytoplasmic interaction with ␣-actinin. The authors could not detect any effect on NMDA receptor activity with direct application of PIP2 or PIP2 antibody to excised inside-out patches, presumably because cytoplasmic components such as ␣-actinin are easily lost in excised insideout patches.…”

“…In the first set of experiments, the authors showed that PLC-catalyzed PIP2 hydrolysis suppressed NMDA currents in oocytes [Michailidis et al (2007), their Figs. 1 A (http://www.jneurosci.org/cgi/ content/full/27/20/5523/F1), 2 A (http:// www.jneurosci.org/cgi/content/full/27/ 20/5523/F2)].…”

“…The interaction of ␣-actinin with NMDA receptors prevents the intracellular C termini from tethering to the actin cytoskeleton and keeps the channel in an open state (Krupp et al, 1999). Because PIP2 also binds to ␣-actinin (Fukami et al, 1992), Michailidis et al (2007) investigated whether ␣-actinin mediated the PIP2 effect on NMDA receptors…”