Jennifer J. Carlisle Michel

Crystal structure of the ATP-gated P2X4 ion channel in the closed state

P2X receptors are cation-selective ion channels gated by extracellular ATP, and are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is at present no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X4 receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit–subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in β-strands, have large acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. In the transmembrane pore, the ‘gate’ is defined by an ∼8 Å slab of protein. We define the location of three non-canonical, intersubunit ATP-binding sites, and suggest that ATP binding promotes subunit rearrangement and ion channel opening.

The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways.

Cyclic adenosine 3′, 5′-monophosphate (cAMP) is a ubiquitous mediator of intracellular signalling events. It acts principally through stimulation of cAMP-dependent protein kinases (PKAs) but also activates certain ion channels and guanine nucleotide exchange factors (Epacs). Metabolism of cAMP is catalysed by phosphodiesterases (PDEs). Here we identify a cAMP-responsive signalling complex maintained by the muscle-specific A-kinase anchoring protein (mAKAP) that includes PKA, PDE4D3 and Epac1. These intermolecular interactions facilitate the dissemination of distinct cAMP signals through each effector protein. Anchored PKA stimulates PDE4D3 to reduce local cAMP concentrations, whereas an mAKAP-associated ERK5 kinase module suppresses PDE4D3. PDE4D3 also functions as an adaptor protein that recruits Epac1, an exchange factor for the small GTPase Rap1, to enable cAMP-dependent attenuation of ERK5. Pharmacological and molecular manipulations of the mAKAP complex show that anchored ERK5 can induce cardiomyocyte hypertrophy. Thus, two coupled cAMP-dependent feedback loops are coordinated within the context of the mAKAP complex, suggesting that local control of cAMP signalling by AKAP proteins is more intricate than previously appreciated.

NMDA receptor structures reveal subunit arrangement and pore architecture.

N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1–GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

Screening and large-scale expression of membrane proteins in mammalian cells for structural studies

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8–tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI− (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4–6 weeks.

The mAKAP complex participates in the induction of cardiac myocyte hypertrophy by adrenergic receptor signaling.

Maladaptive cardiac hypertrophy can progress to congestive heart failure, a leading cause of morbidity and mortality in the United States. A better understanding of the intracellular signal transduction network that controls myocyte cell growth may suggest new therapeutic directions. mAKAP is a scaffold protein that has recently been shown to coordinate signal transduction enzymes important for cytokine-induced cardiac hypertrophy. We now extend this observation and show mAKAP is important for adrenergic-mediated hypertrophy. One function of the mAKAP complex is to facilitate cAMP-dependent protein kinase A-catalyzed phosphorylation of the ryanodine receptor Ca2+-release channel. Experiments utilizing inhibition of the ryanodine receptor, RNA interference of mAKAP expression and replacement of endogenous mAKAP with a mutant form that does not bind to protein kinase A demonstrate that the mAKAP complex contributes to pro-hypertrophic signaling. Further, we show that calcineurin Aβ associates with mAKAP and that the formation of the mAKAP complex is required for the full activation of the pro-hypertrophic transcription factor NFATc. These data reveal a novel function of the mAKAP complex involving the integration of cAMP and Ca2+ signals that promote myocyte hypertrophy.

PKA-phosphorylation of PDE4D3 facilitates recruitment of the mAKAP signalling complex.

mAKAP (muscle-selective A-kinase-anchoring protein) co-ordinates a cAMP-sensitive negative-feedback loop comprising PKA (cAMP-dependent protein kinase) and the cAMP-selective PDE4D3 (phosphodiesterase 4D3). In vitro and cellular experiments demonstrate that PKA-phosphorylation of PDE4D3 on Ser-13 increases the affinity of PDE4D3 for mAKAP. Our data suggest that activation of mAKAP-anchored PKA enhances the recruitment of PDE4D3, allowing for quicker signal termination.

Anchored p90 Ribosomal S6 Kinase 3 is Required for Cardiac Myocyte Hypertrophy

Rationale: Cardiac myocyte hypertrophy is the main compensatory response to chronic stress on the heart. p90 ribosomal S6 kinase (RSK) family members are effectors for extracellular signal-regulated kinases that induce myocyte growth. Although increased RSK activity has been observed in stressed myocytes, the functions of individual RSK family members have remained poorly defined, despite being potential therapeutic targets for cardiac disease. Objective: To demonstrate that type 3 RSK (RSK3) is required for cardiac myocyte hypertrophy. Methods and Results: RSK3 contains a unique N-terminal domain that is not conserved in other RSK family members. We show that this domain mediates the regulated binding of RSK3 to the muscle A-kinase anchoring protein scaffold, defining a novel kinase anchoring event. Disruption of both RSK3 expression using RNA interference and RSK3 anchoring using a competing muscle A-kinase anchoring protein peptide inhibited the hypertrophy of cultured myocytes. In vivo, RSK3 gene deletion in the mouse attenuated the concentric myocyte hypertrophy induced by pressure overload and catecholamine infusion. Conclusions: Taken together, these data demonstrate that anchored RSK3 transduces signals that modulate pathologic myocyte growth. Targeting of signaling complexes that contain select kinase isoforms should provide an approach for the specific inhibition of cardiac myocyte hypertrophy and for the development of novel strategies for the prevention and treatment of heart failure.