Phosphatidylinositol-4,5-bisphosphate regulates NMDA receptor activity through alpha-actinin - PubMed (original) (raw)

Comparative Study

Phosphatidylinositol-4,5-bisphosphate regulates NMDA receptor activity through alpha-actinin

Ioannis E Michailidis et al. J Neurosci. 2007.

Abstract

Phosphatidylinositol-4,5-bisphosphate (PIP2) 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 PIP2 supports NMDAR activity. In Xenopus oocytes, overexpression of phospholipase Cgamma (PLCgamma) or preincubation with 10 microm wortmannin markedly reduced NMDA currents. Stimulation of the epidermal growth factor receptor (EGFR) promoted the formation of an immunocomplex between PLCgamma and NMDAR subunits. Stimulation of EGFR or the PLCbeta-coupled M1 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 alpha-actinin. Mutation of the PIP2-binding residues of alpha-actinin dramatically reduced NMDA currents and occluded the effect of EGF. Interestingly, EGF or wortmannin affected the interaction between NMDAR subunits and alpha-actinin, suggesting that this protein mediates the effect of PIP2 on NMDARs. In mature hippocampal neurons, expression of the mutant alpha-actinin reduced NMDA currents and accelerated inactivation. We propose a model in which alpha-actinin supports NMDAR activity via tethering their intracellular tails to plasma membrane PIP2. Thus, our results extend the influence of PIP2 to the NMDA ionotropic glutamate receptors and introduce a novel mechanism of "indirect" regulation of transmembrane protein activity by PIP2.

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Figures

Figure 1.

Figure 1.

PLCγ inhibits NMDA currents in Xenopus laevis oocytes. A, Left, Representative traces of whole-cell NR1/2A currents recorded with the two-electrode voltage-clamp technique at −80 mV (in this and subsequent figures). NR1/2A currents were evoked by NMDA (100 μ

m

) and glycine (Gly; 10 μ

m

) applied as indicated by arrows, in control cells (b), cells overexpressing PLCγ (a), or cells expressing the inactive mutant PLCγY783F (c), as described in Results. Right, Bars representing mean ± SE percentage steady-state current after agonist application, normalized to the average of the control group. The means for the PLCγ group were significantly different (*p < 0.01, t test). B, Bars obtained similarly as in A but for NR1/2A or NR1/2B currents from control oocytes or oocytes preincubated for 1 h with 10 μ

m

wortmannin (Wtmn). The means were significantly different in all sets of experiments (p < 0.001, t test).

Figure 2.

Figure 2.

Stimulation of hormonal receptors that activate PLC transiently inhibits NMDA currents. A, Representative traces of the effect to the NR1/2A current of 100 ng/ml EGF or 5 μ

m

ACh applied as shown. Bars represent mean ± SE percentage current of current before (control current, taken as always 100% before EGF or ACh application in this and subsequent figures when comparison with inhibited states is made) or of maximally inhibited current after EGF or ACh application. Both means representing inhibited current were significantly different from control (*p < 0.001, t test). B, Xenopus oocytes expressing NR1/2A subunits and EGFR were stimulated with EGF (100 ng/ml) as indicated, and membrane preparations were obtained as described in Materials and Methods. Top, Western immunoblot (WB) of a sample of the membrane preparations with an antibody against NR1. The rest of the same membrane preparations were subjected to immunoprecipitation (IP) using an antibody against PLCγ (see Materials and Methods). Bottom, NR1 Western blotting of oocyte membrane immunoprecipitates pulled down with PLCγ antibodies. Blots are representative of two independent experiments with similar results.

Figure 3.

Figure 3.

Wortmannin blocks the recovery of NMDA currents in response to PLC-coupled receptor stimulation. A, B, Left traces, Effect to the NR1/2C current of 100 ng/ml EGF or 5 μ

m

ACh applied as indicated. Right traces, In addition to EGF or ACh, wortmannin (Wtmn; 20–40 μ

m

) was perfused to the oocyte (different oocytes from the ones used in experiments without wortmannin) as indicated. Bars represent mean ± SE percentage current of current before (control current) or of maximally inhibited current after EGF or ACh application (I) or of recovered current (R) from cells with or without wortmannin (I, R + wortmannin). *p < 0.001, the mean maximally recovered currents in cells perfused with wortmannin were significantly different than control cells (for both EGF and Ach; t test). The arrows indicate where the I and R measurements were made. Gly, Glycine.

Figure 4.

Figure 4.

Intracellular Ca2+ store depletion does not prevent NMDA current inhibition in response to PLC-coupled receptor stimulation. A, B, Traces represent NMDA currents recorded from oocytes expressing NR1/2A (A) or NR1/2B (B) subunits on which EGF or ACh were administered as indicated. Thapsigargin (1–5 μ

m

) was used to pretreat the oocytes for 30–60 min before experiments. C, Bars represent mean ± SE percentage current of current before (control current) or of maximally inhibited current after EGF (100 ng/ml) or ACh (5 μ

m

) application. All sets of means were significantly different (*p < 0.001, t test). Gly, Glycine.

Figure 5.

Figure 5.

The C0 domain of the NR1 subunit is required for the inhibition of NR1/2A currents. A, The C terminus of NR1 (NR1–1b). Shown in bold are the α-actinin-binding residues within C0. B, Traces represent recordings of NMDA currents from oocytes expressing the NR1 deletion mutant subunits indicated (see Results) with wild-type NR2A subunit, in which EGF (1 ng/ml) or ACh (5 μ

m

) were applied as indicated. In NR1838 and NR1ΔC0, the C terminus of NR1 or only its C0 domain have been deleted, respectively. Bars represent mean ± SE percentage current of current before (control current) or of current after EGF or ACh application. The means were not significantly different (t test). Gly, Glycine; TM, transmembrane domain.

Figure 6.

Figure 6.

α-Actinin-binding sites within the C0 domain of NR1 and the distal α-actinin-binding region of the C terminus of NR2B are required for inhibition of NR1/2B currents. A–C, Traces represent recordings of NMDA currents from thapsigargin-treated oocytes expressing wild-type NR1 or NR1 deletion mutants, together with wild-type NR2B or an NR2B mutant as indicated. EGF (100 ng/ml) or ACh (5 μ

m

) were applied as indicated. The constructs have been named after the last residue expressed in the mutant subunit (see Results). D, Bars represent mean ± SE percentage current of current before (control current) or of steady-state current after EGF or ACh application. The means were significantly different only for NR1 or NR2B subunit combinations that contained an α-actinin-binding site (p < 0.01, t test). D, Schematic of NR1 and NR2B subunits with sites of mutants used. α-Actinin binds residues 858–863 of NR1 (see also Fig. 5) and the distal C terminus of NR2B. Gly, Glycine; TM, transmembrane domain.

Figure 7.

Figure 7.

Mutation of the PIP2-binding residues of α-actinin affects NMDA currents in oocytes. Top left, Bars representing mean ± SE percentage steady-state normalized NMDA current recorded from oocytes expressing NR1/2A together with endogenous α-actinin (control), overexpressed wild-type α-actinin 2, single-point mutant α-actinin 2 K184Q, or double-point mutant α-actinin 2 R172Q–K184Q. The means were significantly different from control (p < 0.001 for all comparisons, t tests). Top right, Representative NMDA current trace of a control oocyte expressing NR1/2A in response to EGF (1 ng/ml) applied as indicated. Bottom left, Representative NMDA current trace of an oocyte expressing NR1/2A and single-point mutant α-actinin 2 K184Q in response to 1 ng/ml EGF. Bottom right, Bars represent mean ± SE percentage current of current before (control current) or of steady-state current after EGF application from oocytes expressing NR1/2A, and NR1/2A together with wild-type α-actinin 2 or single-point mutant α-actinin 2 K184Q. *p < 0.001, statistically significant difference between the NR1/2A group and the K184Q group (t test).

Figure 8.

Figure 8.

Disruption of the α-actinin–PIP2 interaction affects NMDA currents in hippocampal neurons. A, Overlaid normalized representative NMDA–glycine-evoked current traces from neurons clamped at −60 mV expressing α-actinin–EGFP (red) or the α-actinin–EGFP R172Q–K184Q mutant (blue). For α-actinin–EGFP and α-actinin–EGFP R172Q–K184Q expressing neurons compared are mean ± SE. B, Percentage normalized peak NMDAR/K+ channel current ratios obtained as described in Materials and Methods. C, Percentage of the peak NMDA current remaining 1 s after peak. D, Time constants (tau) of inactivation of agonist-evoked NMDAR currents in neurons. All pairs of means were significantly different (*p < 0.001, t test). Gly, Glycine; α-act, α-actinin.

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