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. 2018 Sep 18;115(38):E9006-E9014.
doi: 10.1073/pnas.1802567115. Epub 2018 Sep 4.

Glutamate-activated BK Channel Complexes Formed With NMDA Receptors

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Free PMC article

Glutamate-activated BK Channel Complexes Formed With NMDA Receptors

Jiyuan Zhang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The large-conductance calcium- and voltage-activated K+ (BK) channel has a requirement of high intracellular free Ca2+ concentrations for its activation in neurons under physiological conditions. The Ca2+ sources for BK channel activation are not well understood. In this study, we showed by coimmunopurification and colocalization analyses that BK channels form complexes with NMDA receptors (NMDARs) in both rodent brains and a heterologous expression system. The BK-NMDAR complexes are broadly present in different brain regions. The complex formation occurs between the obligatory BKα and GluN1 subunits likely via a direct physical interaction of the former's intracellular S0-S1 loop with the latter's cytosolic regions. By patch-clamp recording on mouse brain slices, we observed BK channel activation by NMDAR-mediated Ca2+ influx in dentate gyrus granule cells. BK channels modulate excitatory synaptic transmission via functional coupling with NMDARs at postsynaptic sites of medial perforant path-dentate gyrus granule cell synapses. A synthesized peptide of the BKα S0-S1 loop region, when loaded intracellularly via recording pipette, abolished the NMDAR-mediated BK channel activation and effect on synaptic transmission. These findings reveal the broad expression of the BK-NMDAR complexes in brain, the potential mechanism underlying the complex formation, and the NMDAR-mediated activation and function of postsynaptic BK channels in neurons.

Keywords: BK channel; NMDA receptor; dentate gyrus granule cells; protein interactions; synaptic transmission.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
BK channels and NMDARs form complexes in rat brains. (A) Schematic of the sample-preparation protocols for the immunopurified NMDAR (IP-NMDAR) and BK channel (IP-BK), tandem immunopurified BK–NMDAR complex (TIP-BK/NMDAR), and negative control samples. (B) SDS/PAGE and silver stain analysis of the IP-NMDAR and control samples. (C) Tandem mass spectrometric spectra of a unique peptide of BKα identified in the IP-NMDAR sample. (D) Immunoblot (IB) analysis of BKα and GluN1 in the IP-NMDAR sample. (E) Tandem mass spectrometric spectra for a unique peptide of GluN1 identified in the IP-BK sample. (F) Immunoblot analysis of GluN1 and BKα in the IP-BK sample. (G) Immunoblot analysis of GluN2A and GluN2B in the TIP-BK/NMDAR sample. (H) Immunoblot analysis of BKα and GluN1 in IP-NMDAR (Left) and IP-BK (Right) samples prepared from different brain regions.
Fig. 2.
Fig. 2.
BK channels and NMDARs interact via BKα and GluN1 in HEK-293 cells. (A) Reciprocal pull-down of GluN1 and BKα by anti-BKα and -GluN1 antibodies in the absence of expression of any other NMDAR subunit. IB, immunoblot; IP, immunopurification. (B) Pull-down of GluN2A and GluN2B by an anti-BKα antibody only in the presence of GluN1 expression. (C) Pull-down of BKα by an anti-GluN2A or -GluN2B antibody only in the presence of GluN1 expression. (D) Reciprocal pull-down assay showed that the BKα was largely defective in its association with GluN1 upon deletion of its S0–S1 loop region (residues 46–93). (E) Pull-down of BKα by an anti-GluN1 was interfered by a synthesized peptide of the BKα’s S0–S1 loop region (residues 46–93), but not by a scrambled peptide. The peptides were added at a concentration of 1 mg/mL to cell lysates that were equally divided from the same lysate of cells coexpressing BKα and GluN1. (F) Pull-down of GluN1 by an anti-BKα antibody showed that the BKα–GluN1 association was markedly decreased with the GluN1 N1/K2C mutant in which the C-terminal part, including TM domain (residues 527–647 and 781–826) and C-terminal domain (residues 827–920), was replaced by those of GluK2. (G) Pull-down of the GluN1’s cytosolic regions by the BKα46–93 peptide, but not by the scrambled peptide. The peptides were biotinylated on their N termini and immobilized on streptavidin agarose. The fusion construct of the GluN1’s cytosolic regions (residues 563–587 and 813–920) was 6× His-tagged in its C terminus, expressed in E. coli, and purified with IMAC chromatography.
Fig. 3.
Fig. 3.
In situ PLA of BKα and GluN1 colocalization in HEK-293 cells and mouse brains. (A) PLA signals (red dots) probed with anti-V5 and -FLAG antibodies under a nonpermeabilized condition were detected in HEK-293 cells coexpressing V5-BKα and FLAG-GluN1. (B and C) In situ PLA of BKα–GluN1 complexes in the hippocampal dentate gyrus of control (Nestin-Cre/KCNMA1fl/fl) mice (B) and neuron-specific BKα KO (Nestin-Cre+/KCNMA1fl/fl) mice (C). Nuclei are shown in blue (DAPI). (Scale bars: 40 or 10 μm.)
Fig. 4.
Fig. 4.
Glutamate-induced BK channel activation via NMDARs in mature dentate gyrus granule cells. (A) Whole-cell recording of glutamate-induced currents at different holding membrane voltages upon somatic puff application of 100 μM glutamate and 10 μM glycine for 100 ms. (B) Representative glutamate-induced currents at −10 mV in the absence and presence of the BK channel blocker paxilline (Pax; 10 μM) and NMDAR antagonist AP5 (200 μM) and in the presence of the intracellular chelators EGTA and BAPTA. (C) Averaged effects of paxilline (n = 13), AP5 (n = 13), and intracellular chelators (0.2 mM EGTA, n = 26; 2 mM EGTA, n = 8; 2 mM BAPTA, n = 8) on the glutamate-induced outward currents. (D) Representative glutamate-induced currents at −10 mV with recording pipette solution containing 0.5 mg/mL BKα46–93 peptide or scrambled peptide and the bath solution perfused with/without AP5 (200 μM). Pep., peptide. (E) Averaged amplitudes of glutamate-induced outward currents in the presence of the intracellularly loaded BKα46–93 (n = 9) or scrambled (n = 8) peptide. The current amplitudes were calculated from the peak amplitudes of outward currents relative to the current levels before drug application. Data are shown as means ± SEM. Statistical differences were evaluated using a t test. N.S., not significant. *P < 0.05; ***P < 0.001; ****P < 0.0001.
Fig. 5.
Fig. 5.
Postsynaptic BK channels regulate synaptic transmission in mature dentate granule cells via NMDAR-mediated channel activation. (A) Effects of paxilline (Pax) alone and combined with AP5 on the amplitudes of evoked EPSPs (n = 13). (B) Effects of AP5 alone and combined with paxilline on the amplitudes of evoked EPSPs (n = 13). For comparison, the effects of AP5 alone on evoked EPSPs over a similar extended time course (n = 6) were included in the averaged plot. (C) Effects of paxilline alone and combined with AP5 on the amplitudes of evoked EPSPs in mature granule cells in BKα-KO (Nestin-Cre+/KCNMA1fl/fl) mice (n = 9). (D) The effect of paxilline alone (bath) and combined with AP5 (bath) on evoked EPSPs in the presence of intracellularly applied MK-801 (n = 9). (E) Effects of paxilline on evoked EPSPs in the presence of intracellularly applied paxilline (n = 8). (F) Effects of paxilline on evoked EPSPs in the presence of intracellularly applied 0.5 mg/mL BKα46–93 (n = 8) or scrambled (n = 7) peptide. Pep., peptide. (G) Effects of extracellularly and intracellularly applied paxilline (n = 9 and 7, respectively) and KO of BK channels (Nestin-Cre+/KCNMA1fl/fl) (n = 5) on paired-pulse ratios. All experiments were done with regular C57BL/C6 mice except as specified. Control, data obtained without or before drug application. Paxilline and AP5 were perfused in the bath solution at a concentration of 10 and 200 µM, respectively, and they were applied either individually or combined together at a later stage of the experiment. Statistical differences were evaluated by using a t test. N.S., not significant. *P < 0.05; **P < 0.01; ***P < 0.001.

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