PKM zeta maintains late long-term potentiation by N-ethylmaleimide-sensitive factor/GluR2-dependent trafficking of postsynaptic AMPA receptors

Abstract

Although the maintenance mechanism of late long-term potentiation (LTP) is critical for the storage of long-term memory, the expression mechanism of synaptic enhancement during late-LTP is unknown. The autonomously active protein kinase C isoform, protein kinase Mzeta (PKMzeta), is a core molecule maintaining late-LTP. Here we show that PKMzeta maintains late-LTP through persistent N-ethylmaleimide-sensitive factor (NSF)/glutamate receptor subunit 2 (GluR2)-dependent trafficking of AMPA receptors (AMPARs) to the synapse. Intracellular perfusion of PKMzeta into CA1 pyramidal cells causes potentiation of postsynaptic AMPAR responses; this synaptic enhancement is mediated through NSF/GluR2 interactions but not vesicle-associated membrane protein-dependent exocytosis. PKMzeta may act through NSF to release GluR2-containing receptors from a reserve pool held at extrasynaptic sites by protein interacting with C-kinase 1 (PICK1), because disrupting GluR2/PICK1 interactions mimic and occlude PKMzeta-mediated AMPAR potentiation. During LTP maintenance, PKMzeta directs AMPAR trafficking, as measured by NSF/GluR2-dependent increases of GluR2/3-containing receptors in synaptosomal fractions from tetanized slices. Blocking this trafficking mechanism reverses established late-LTP and persistent potentiation at synapses that have undergone synaptic tagging and capture. Thus, PKMzeta maintains late-LTP by persistently modifying NSF/GluR2-dependent AMPAR trafficking to favor receptor insertion into postsynaptic sites.

Figure 1.

PKMζ enhances AMPAR-mediated synaptic transmission through NSF/GluR2 interactions. A, Postsynaptic perfusion of PKMζ through a whole-cell recording pipette enhances AMPAR responses at Schaffer collateral/commissural-CA1 pyramidal cell synapses (black open circles); pep2m (100 μm) together with PKMζ blocks AMPAR potentiation (red filled circles). pep2m alone has minimal effect on baseline synaptic transmission at 0.067 Hz (red filled squares) compared with baseline recordings without pep2m (black open squares). Left insets for all panels show representative traces recorded ∼1 min (left) and ∼13 min (right) after cell breakthrough. Right insets for all panels show the subtraction of baseline responses from responses with PKMζ alone (black open circles) and the subtraction of baseline responses in the presence of the agent from responses with PKMζ together with the agent (red filled circles). The number of experiments for each condition is five to six. B, Inactive scrambled version of pep2m (100 μm) has no effect on PKMζ-mediated potentiation of AMPAR responses (n = 4–5). C, pep-NSF3 (100 μm), which blocks the ATPase activity of NSF, prevents PKMζ-mediated potentiation of AMPAR responses (n = 4). D, Blockade of exocytosis by Botox B (0.5 μm) does not affect PKMζ-mediated potentiation of AMPAR responses (n = 4). Right inset, Responses with baselines subtracted show no significant difference.

Figure 2.

Disruption of GluR2/PICK1 interactions mimics and occludes PKMζ-mediated AMPAR potentiation. A, Intracellular perfusion of pep2-EVKI with 100 μm (red filled squares; n = 6) and 500 μm (red open squares; n = 3) in the recording pipette produce similar degrees of AMPAR potentiation. Inactive peptide pep2-EVKE did not cause potentiation (100 μm; blue filled squares; n = 5). B, PKMζ alone (black open circles; n = 5) and PKMζ with pep2-EVKI (100 μm; red filled circles; n = 5) show equivalent potentiation.

Figure 3.

PKMζ-mediated NSF/GluR2 trafficking is critical for AMPAR potentiation during LTP. A, Bath application of myr-pep2m (10 μm) blocks PKMζ-mediated potentiation of AMPAR responses (red filled circles are responses in the presence of the agent; black open circles in its absence; n = 5). Insets above show representative EPSC responses 1 min (left) and 13 min (right) after cell breakthrough. B, myr-pep2m blocks the persistence of LTP (red filled circles are responses in the presence of myr-pep2m and black open circles in its absence; tetanization is denoted by an arrow; n = 10). Top left insets, Representative field responses 1 min before and 60 min after tetanization. Top right inset, Application of myr-pep2m does not affect baseline synaptic transmission (n = 4; the SEM symbols are all smaller than the mean symbols). C, Synaptosomal GluR2 and GluR3 subunits increase and GluR1 subunits decrease 1 h after tetanization (n = 8–10). Applications of myr-pep2m (10 μm) and the PKMζ inhibitor ZIP (2 μm) block the increase of synaptosomal GluR2 and GluR3 (n = 4). Left, Representative immunoblots; GAPDH was used to normalize AMPAR immunostaining levels with respect to protein loading on immunoblots. Right, Mean data; significant differences denoted by asterisks. D, myr-pep2m does not prevent the increase of PKMζ 1 h after tetanization. Top, Representative immunoblots; bottom, mean data (no drug, n = 10; myr-pep2m, n = 4; asterisks denote significant differences relative to nontetanized slices; ns denotes no significant difference between PKMζ levels with and without myr-pep2m). E, Surface (measured by biotin labeling) and total GluR2 levels do not change 1 h after tetanization. Top, Representative immunoblots; bottom, mean data (surface, n = 4; total, n = 8).

Figure 4.

NSF/GluR2 interactions mediate the persistence of late-LTP. A, myr-pep2m (10 μm) reverses late-LTP when applied 3 h after tetanic stimulation (open circles). The inhibitor has no effect on an independent pathway simultaneously recorded within each slice (open squares) (n = 4). B, An inactive version of myr-pep2m (scr-myr-pep2m; 10 μm) has no effect on potentiation (n = 4).

Figure 5.

In late-LTP maintenance, PKMζ persistently upregulates an AMPAR trafficking mechanism that maintains a constant number of postsynaptic receptors under basal conditions. Left, Illustration of NSF/GluR2-mediated and exocytotic pathways maintaining the postsynaptic AMPAR pool under basal conditions. After afferent activity, NSF acts to traffic AMPARs back to the synapse (or prevent their release from the synapse), which can be blocked by pep2m. The inhibitory action of Botox B on trafficking of AMPARs is shown as blocking exocytosis from a putative internal pool to the synapse. Right, PKMζ maintains LTP through enhancing NSF/GluR2-mediated trafficking of AMPARs to the synapse by releasing receptors from an extrasynaptic pool.