ABSTRACT Ca 2+ /calmodulin-dependent protein kinase II alpha (CaMKIIα) is an abundant neuronal signaling protein involved in synaptic plasticity and memory formation 1,2 . The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers 3-6 . Recent findings have suggested that the hub is also an allosteric determinant of kinase activity 7 , and is thus an emerging target for therapies to correct CaMKIIα dysregulation 8,9 . However, pharmacological modulation of the hub domain has never been demonstrated. Here we show that stabilization of the CaMKIIα hub domain confers neuroprotection. By combining photoaffinity labeling and chemical proteomics using small molecule analogs of the natural metabolite γ-hydroxybutyrate (GHB) 10 we reveal that CaMKIIα is the selective target for GHB. We further find that these GHB analogs bind to the hub interior by solving a 2.2 Å crystal structure of CaMKIIα with bound ligand. Using differential scanning fluorimetry, we show that binding of ligands to the hub interior increases the thermal stability of hub oligomers in a concentration-dependent manner. Moreover, we demonstrate the functional significance of this hub stabilization by showing substantial neuroprotective effects in cellular excitotoxicity assays and in a mouse model of cerebral ischemia. Together, our results reveal that CaMKIIα hub stabilization is the mechanism by which GHB provides endogenous neuroprotection and that small-molecule CaMKIIα-selective ligands have therapeutic potential.
γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA A receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA A receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC 50 = 140 nM) over α4β(2/3)δ (EC 50 = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [ 3 H]( E , RS )-(6,7,8,9-tetrahydro-5-hydroxy-5 H -benzocyclohept-6-ylidene)acetic acid showed a 39% reduction ( P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA A receptors and postulate a role for extrasynaptic α4δ-containing GABA A receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.
GABAA receptor subunit composition has a profound effect on the receptor’s physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence on extrasynaptic GABAA receptor function is less well understood. Here, we examine the pharmacological properties of a potentially native extrasynaptic GABAA receptor that incorporates the β1 subunit, specifically composed of α4β1δ and α4β1 subunits. GABA activated concentration-dependent responses at α4β1δ and α4β1 receptors with EC50 values in the nanomolar to micromolar range, respectively. The divalent cations Zn2+ and Cu2+, and the β1-selective inhibitor salicylidine salicylhydrazide (SCS), inhibited GABA-activated currents at α4β1δ receptors. Surprisingly the α4β1 receptor demonstrated biphasic sensitivity to Zn2+ inhibition that may reflect variable subunit stoichiometries with differing sensitivity to Zn2+. The neurosteroid tetrahydro-deoxycorticosterone (THDOC) significantly increased GABA-initiated responses in concentrations above 30 nM for α4β1δ receptors. With this study we report the first pharmacological characterisation of various GABAA receptor ligands acting at murine α4β1δ GABAA receptors, thereby improving our understanding of the molecular pharmacology of this receptor isoform. This study highlights some notable differences in the pharmacology of murine and human α4β1δ receptors. We consider the likelihood that the α4β1δ receptor may play a role as an extrasynaptic GABAA receptor in the nervous system.
Significance GHB is a natural brain metabolite of GABA, previously reported to be neuroprotective. However, the high-affinity binding site for GHB has remained elusive for almost 40 y. We here unveil CaMKIIα, a highly important neuronal kinase, as the long-sought-after GHB high-affinity target. Via a specific interaction within the central hub domain of CaMKIIα, GHB analogs act to stabilize the hub oligomer complex. This interaction potentially explains pronounced neuroprotective effects of GHB analogs in cultured neurons exposed to a chemical insult and in mice exposed to ischemia. The postischemic treatment effects of GHB analogs underline these compounds as selective and high-affinity potential drug candidates and CaMKIIα as a relevant pharmacological target for stroke therapy.
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