Engaging homeostatic plasticity to treat depression.

Major depressive disorder (MDD) is a complex and heterogeneous mood disorder, making it difficult to develop a generalized, pharmacological therapy that is effective for all who suffer from MDD. Through the fortuitous discovery of N-methyl-D-aspartate receptor (NMDAR) antagonists as effective antidepressants, we have gained key insights into how antidepressant effects can be produced at the circuit and molecular levels. NMDAR antagonists act as rapid-acting antidepressants such that relief from depressive symptoms occurs within hours of a single injection. The mode of action of NMDAR antagonists seemingly relies on their ability to activate protein-synthesis-dependent homeostatic mechanisms that restore top-down excitatory connections. Recent evidence suggests that NMDAR antagonists relieve depressive symptoms by forming new synapses resulting in increased excitatory drive. This event requires the mammalian target of rapamycin complex 1 (mTORC1), a signaling pathway that regulates synaptic protein synthesis. Herein, we review critical studies that shed light on the action of NMDAR antagonists as rapid-acting antidepressants and how they engage a neuron's or neural network's homeostatic mechanisms to self-correct. Recent studies notably demonstrate that a shift in γ-amino-butyric acid receptor B (GABABR) function, from inhibitory to excitatory, is required for mTORC1-dependent translation with NMDAR antagonists. Finally, we discuss how GABABR activation of mTORC1 helps resolve key discrepancies between rapid-acting antidepressants and local homeostatic mechanisms.

Rapid antidepressants stimulate the decoupling of GABA(B) receptors from GIRK/Kir3 channels through increased protein stability of 14-3-3η.

A single injection of N-methyl-D-aspartate receptor (NMDAR) antagonists produces a rapid antidepressant response. Lasting changes in the synapse structure and composition underlie the effectiveness of these drugs. We recently discovered that rapid antidepressants cause a shift in the γ-aminobutyric acid receptor (GABABR) signaling pathway, such that GABABR activation shifts from opening inwardly rectifiying potassium channels (Kir/GIRK) to increasing resting dendritic calcium signal and mammalian Target of Rapamycin activity. However, little is known about the molecular and biochemical mechanisms that initiate this shift. Herein, we show that GABABR signaling to Kir3 (GIRK) channels decreases with NMDAR blockade. Blocking NMDAR signaling stabilizes the adaptor protein 14-3-3η, which decouples GABABR signaling from Kir3 and is required for the rapid antidepressant efficacy. Consistent with these results, we find that key proteins involved in GABABR signaling bidirectionally change in a depression model and with rapid antidepressants. In socially defeated rodents, a model for depression, GABABR and 14-3-3η levels decrease in the hippocampus. The NMDAR antagonists AP5 and Ro-25-6981, acting as rapid antidepressants, increase GABABR and 14-3-3η expression and decrease Kir3.2. Taken together, these data suggest that the shift in GABABR function requires a loss of GABABR-Kir3 channel activity mediated by 14-3-3η. Our findings support a central role for 14-3-3η in the efficacy of rapid antidepressants and define a critical molecular mechanism for activity-dependent alterations in GABABR signaling.

Membrane topology of the amino-terminal region of the sulfonylurea receptor.

The sulfonylurea receptor (SUR) is a member of the ATP-binding cassette family that is associated with Kir 6.x to form ATP-sensitive potassium channels. SUR is involved in nucleotide regulation of the channel and is the site of pharmacological interaction with sulfonylurea drugs and potassium channel openers. SUR contains three hydrophobic domains, TM(0), TM(1), and TM(2), with nucleotide binding folds following TM(1) and TM(2). Two topological models of SUR have been proposed containing either 13 transmembrane segments (in a 4+5+4 arrangement) or 17 transmembrane segments (in a 5+6+6 arrangement) (Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P. t., Boyd, A. E., III, González, G., Herrera-Sosa, H., Nguy, K., Bryan, J., and Nelson, D. A. (1995) Science 268, 423-426; Tusnády, G. E., Bakos, E., Váradi, A., and Sarkadi, B. (1997) FEBS Lett. 402, 1-3; Aguilar-Bryan, L., Clement, J. P., IV, González, G., Kunjilwar, K., Babenko, A., and Bryan, J. (1998) Physiol. Rev. 78, 227-245). We analyzed the topology of the amino-terminal TM(0) region of SUR1 using glycosylation and protease protection studies. Deglycosylation using peptide-N-glycosidase F and site-directed mutagenesis established that Asn(10), near the amino terminus, and Asn(1050) are the only sites of N-linked glycosylation, thus placing these sites on the extracellular side of the membrane. To study in detail the topology of SUR1, we constructed and expressed in vitro fusion proteins containing 1-5 hydrophobic segments of the TM(0) region fused to the reporter prolactin. The fusion proteins were subjected to a protease protection assay that reported the accessibility of the prolactin epitope. Our results indicate that the TM(0) region is comprised of 5 transmembrane segments. These data support the 5+6+6 model of SUR1 topology.

Tetrameric subunit structure of the native brain inwardly rectifying potassium channel Kir 2.2.

Strongly inwardly rectifying potassium channels of the Kir 2 subfamily (IRK1, IRK2, and IRK3) are involved in maintenance and modulation of cell excitability in brain and heart. Electrophysiological studies of channels expressed in heterologous systems have suggested that the pore-conducting pathway contains four subunits. However, inferences from electrophysiological studies have not been tested on native channels and do not address the possibility of nonconducting auxiliary subunits. Here, we investigate the subunit stoichiometry of endogenous inwardly rectifying potassium channel Kir 2.2 (IRK2) from rat brain. Using chemical cross-linking, immunoprecipitiation, and velocity sedimentation, we report physical evidence demonstrating the tetrameric organization of the native channel. Kir 2.2 was sequentially cross-linked to produce bands on SDS-polyacrylamide gel electrophoresis corresponding in size to monomer, dimer, trimer, and three forms of tetramer. Fully cross-linked channel was present as a single band of tetrameric size. Immunoprecipitation of biotinylated membranes revealed a single band corresponding to Kir 2.2, suggesting that the channel is composed of a single type of subunit. Hydrodynamic properties of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid-solubilized channel were used to calculate the molecular mass of the channel. Velocity sedimentation in H2O or D2O gave a sharp peak with a sedimentation coefficient of 17.3 S. Gel filtration yielded a Stokes radius of 5.92 nm. These data indicate a multisubunit protein with a molecular mass of 193 kDa, calculated to contain 3.98 subunits. Together, these results demonstrate that Kir 2.2 channels are formed by the homotetrameric association of Kir 2.2 subunits and do not contain tightly associated auxiliary subunits. These studies suggest that Kir 2.2 channels differ in structure from related heterooctomeric ATP-sensitive K channels and heterotetrameric G-protein-regulated inward rectifier K channels.

Expression of a putative ATPase suppresses the growth defect of a yeast potassium transport mutant: identification of a mammalian member of the Clp/HSP104 family.

A cDNA encoding a novel mammalian member of the Clp/HSP104 family was isolated from a mouse macrophage-like cell line (J774.1) cDNA library by suppression of the growth defect of a Saccharomyces cerevisiae trk1 trk2 double mutant. The full-length version of this cDNA, termed SKD3, encodes a putative 76-kDa protein of 677 amino acids (aa). The deduced aa sequence of the SKD3 polypeptide contains four ankyrin-like repeats in the N-terminal domain and a single ATP-binding consensus site in the C-terminal domain. The 378-aa C-terminal domain of SKD3 has 57-64% similarity (30-40% identity) with members of the Clp/HSP104 family, including the ClpA regulatory subunit of the Clp protease and S. cerevisiae heat-shock protein 104. Northern analysis showed that the 2.3-kb SKD3 transcript is present in a wide variety of tissues, is abundant in mouse heart, skeletal muscle and kidney, and is most abundant in testis. Members of the Clp/HSP104 family have been identified previously from bacteria, yeast and chloroplasts, and are ATPases regulating Clp protease activity and specificity, or mediating cellular responses involved in thermotolerance. SKD3 is the first member of this protein family identified in a higher eukaryote.

Molecular cloning and expression of a human heart inward rectifier potassium channel.

We have isolated a cDNA encoding an inwardly rectifying K+ channel (HH-IRK1) from human heart. The cDNA codes for a 427-amino acid protein, with two putative transmembrane domains and an H5 region. The primary structure of HH-IRK1 is homologous to that of the IRK1 channel cloned from a mouse macrophage-like cell line. Functional expression in Xenopus oocytes showed that HH-IRK1 is a K+ channel with strong inward rectification, blocked by extracellular Ba2+ and Cs+, and with a single-channel conductance of 30 pS. Northern analysis showed HH-IRK1 transcripts in human heart, brain, skeletal muscle, placenta, lung and kidney. HH-IRK1 was mapped to human chromosome 17.