Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type CaV1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in CaV1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the CaV1.2 channel pore-forming subunit (α1C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in CaV1.2 channel activity were associated with PKA activity, leading to α1C phosphorylation at Ser1928. Compared to arteries from low-fat diet (LFD)–fed mice and nondiabetic patients, arteries from high-fat diet (HFD)–fed mice and from diabetic patients had increased Ser1928 phosphorylation and CaV1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser1928 phosphorylation and CaV1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a CaV1.2 with Ser1928 mutated to alanine (S1928A) lacked glucose-mediated increases in CaV1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in CaV1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1C phosphorylation at Ser1928 in stimulating CaV1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes.

Agonist-triggered downregulation of b-adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of b 2 AR signaling highly specific for Ca v 1.2. We find that b 2 -AR binding to Ca v 1.2 residues 1923-1942 is required for b-adrenergic regulation of Ca v 1.2. Despite the prominence of PKA-mediated phosphorylation of Ca v 1.2 S1928 within the newly identified b 2 AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the b 2 AR from Ca v 1.2 upon b-adrenergic stimulation rendering Ca v 1.2 refractory for several minutes from further b-adrenergic stimulation. This effect is lost in S1928A knock-in mice. Although AMPARs are clustered at postsynaptic sites like Ca v 1.2, b 2 AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the b 2 AR from Ca v 1.2 is a uniquely specific desensitization mechanism of Ca v 1.2 regulation by highly localized b 2 AR/cAMP/PKA/ S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT-LTP) depends on Ca v 1.2 and its regulation by channel-associated b 2 AR.

The L-type Ca2+ channel Cav1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser1928 in Cav1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser1928 is not required for regulation of cardiac Cav1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Cav1.2 and enhancement of its activity by the β2-adrenergic receptor (β2AR)–cAMP–PKA cascade. Thus, there is an unexpected dichotomy in the control of Cav1.2 by PKA in cardiomyocytes and hippocampal neurons.

Highlights d Disrupting CaMKII/GluN2B binding protects from neuronal injury after cardiac arrest d Neuroprotective CaMKII and DAPK1 inhibitors are selective for their respective kinase d CaMKII, but not DAPK1, accumulates at synaptic GluN2B during excitotoxic insults d Extra-synaptic GluN2B decreases during excitotoxic and ischemic insults

Higher brain functions are thought to require synaptic frequency decoding that can lead to long-term potentiation (LTP) or depression (LTD). We show that the LTP versus LTD decision is determined by complex cross-regulation of T286 and T305/306 autophosphorylation within the 12meric CaMKII holoenzyme, which enabled molecular computation of stimulus frequency, amplitude, and duration. Both LTP and LTD require T286 phosphorylation, but T305/306 phosphorylation selectively promoted LTD. In response to excitatory LTP versus LTD stimuli, the differential T305/306 phosphorylation directed CaMKII movement to either excitatory or inhibitory synapses, thereby coordinating plasticity at both synapse types. Fast T305/306 phosphorylation required prior T286 phosphorylation and then curbed CaMKII activity by two mechanisms: (i) a cis-subunit reaction reduced both Ca2+ stimulation and autonomous activity and (ii) a trans-subunit reaction enabled complete activity shutdown and feed-forward inhibition of further T286 phosphorylation. These are fundamental additions to the long-studied CaMKII regulation and function in neuronal plasticity.