EPAC inhibition of SUR1 receptor increases glutamate release and seizure vulnerability

Abstract

EPAC (Exchange Proteins Activated by cAMP) regulates glutamate transmitter release in the central neurons, but a role underlying this regulation has yet to be identified. Here we show that EPAC binds directly to the intracellular loop of an ATP-sensitive potassium (KATP) channel type-1 sulfonylurea receptor (SUR1) receptor consisting of amino acids 859-881 (SUR1(859-881)). Ablation of EPAC or expression of SUR1(859-881), which intercepts EPAC-SUR1 binding, increases the open probability of KATP channels consisting of the Kir6.1 subunit and SUR1. Opening of KATP channels inhibits glutamate release and reduces seizure vulnerability in adult mice. Therefore, EPAC interaction with SUR1 controls seizure susceptibility and possibly acts via regulation of glutamate release.

Figure 1.

EPAC binds to SUR1 directly. A–C, Synaptosomes were prepared from SUR1^+/+ mice (A,B) and SUR1^−/− mice (C) and precipitated using antibodies against EPAC1 and EPAC2 or Kir6.1 and SUR1, as indicated. The precipitates were blotted with antibodies against EPAC1, EPAC2, SUR1, Kir6.1, and syntaxin-1A. Input: 20 μg of proteins without precipitation were loaded. Similar results were seen in each of the four experiments. D, Synaptosomes were precipitated using GST-EPAC2^1–158, GST-EPAC2^206–334, or GST-EPAC2^351–458 and blotted with anti-SUR1. Input: 20 μg of protein without precipitation was blotted with antibodies against GST and SUR1, as indicated. Similar results were seen in each of the four experiments. E, GST-EPAC2^351–458 or GST alone was coexpressed with Flag-tagged SUR1^859–881 in HEK293 cells. Then, 72 h after expression, cell lysates were precipitated using anti-GST and blotted with anti-Flag antibody. Input: 10 μg of proteins without precipitation were loaded. Similar results were seen in each of the four experiments. F, GST-EPAC2^351–458 was coexpressed with Flag-tagged SUR1^859–881 in HEK293 cells. Then, 72 h after expression, cell lysates were precipitated using anti-GST in the presence of 5 μg/ml SUR1^859–881 peptide or its scrambled control (S-SUR1^859–881) and blotted with anti-Flag antibody. Similar results were seen in each of the five experiments. G, H, The rAAV1/2 vector (G) for expression of SUR1^859–881 and a representative image (H) of the dentate gyrus area taken 15 d after injection of the rAAV1/2-CAP/SUR1^859–881-IRES-eGFP virus particles. Synaptosomes were prepared from the dentate gyrus 15 d after expression of SUR1^859–881 (lane 1) or its scrambled control (lane 0) and precipitated with anti-EPAC1 and anti-EPAC2, respectively. The precipitates were blotted with antibodies against EPAC1, EPAC2, or SUR1, as indicated. Similar results were seen in each of the five experiments.

Figure 2.

Deletion EPAC increases K_ATP channel open probability. A, Traces are representative single-channel recordings from the dentate granule cells. Downward deflections of the current traces indicate single-channel openings. B, C, Graphs of open (B) and closed (C) time distributions of single K_ATP channel currents. D, Bar graph summarizes single-channel currents in cell-attached patches from the dentate granule neurons of EPAC^+/+/SUR1^+/+, EPAC^−/−/SUR1^+/+, and EPAC^−/−/SUR1^−/− mice. Data are mean ± SEM (n = 8 recordings/4 mice/genotypes, *p < 0.01). E, Interception of EPAC–SUR1 binding increases K_ATP channel open probability. The K_ATP single-channel currents were recorded from the dentate granule cells 15 d after expression of SUR1^859–881 or its scrambled control. Data are mean ± SEM (n = 8 recordings/4 mice/genotypes, *p < 0.01).

Figure 3.

EPAC inhibition of SUR1 reduces glutamate release. A–D, Representative spontaneous EPSCs (A), averaged responses (B), cumulative probability (C), and average frequency and amplitude (D) in CA3 pyramidal neurons of EPAC^+/+, EPAC^−/−, and EPAC^+/+ mice expressing SUR1^859–881 or scrambled SUR1^859–881. Data are mean ± SEM (n = 12 recording/6 mice per genotype, *p < 0.01). All recordings are at a hold potential of −70 mV. E, Representative NMDA-receptor-mediated EPSCs from CA3 pyramidal neurons at a holding potential of +60 mV in the presence of 20 μm NBQX and 10 μm bicuculline are evoked by paired pulse stimulation of the mossy-fiber tracks with the interstimulus intervals of 50 ms (top) and 500 ms (bottom), respectively. F, The paired-pulse ratios versus interstimulus intervals from EPAC^+/+ (brown triangles), EPAC^−/−(red triangles), and EPAC^+/+ mice expressing SUR1^859–881 (blue circles) or scrambled SUR1^859–881 (green circles). Data are mean ± SEM (n = 12 recordings/6 mice/genotype, *p < 0.01). G, The paired-pulse ratios increase with elevation of extracellular Ca²⁺ in EPAC^+/+ (blue triangles) and EPAC^−/−(green circles). Data are mean ± SEM (n = 9 recordings/3 mice/genotype).

Figure 4.

EPAC^−/− mice have decreased vulnerability to epileptic seizures. A, Bar graph showing the mortality rate due to convulsive responses within 3 h after intraperitoneal injection of KA at the doses from 25 to 40 mg/kg. Data are mean ± SEM (n = 13 mice/genotype). B, Bar graph showing seizure index after a single intraperitoneal dose of 30 mg/kg. Data are mean ± SEM (n = 13 mice/genotype). C, D, Working model: EPAC controls glutamate release via tonic inhibition of SUR1 receptor at the presynaptic terminals. Opening of Kir6.1/SUR1 channels hyperpolarizes cells, leading to a reduction of Ca²⁺-dependent transmitter release (C). Deletion of EPAC genes increases the open probability of K_ATP channels in the dentate granule neurons and reduces Ca²⁺-dependent glutamate release (D), thereby antagonizing epileptic seizures.