Constitutive Activation of Kappa Opioid Receptors at Ventral Tegmental Area Inhibitory Synapses Following Acute Stress.

Stressful experiences potently activate kappa opioid receptors (κORs). κORs in the ventral tegmental area regulate multiple aspects of dopaminergic and non-dopaminergic cell function. Here we show that at GABAergic synapses on rat VTA dopamine neurons, a single exposure to a brief cold-water swim stress induces prolonged activation of κORs. This is mediated by activation of the receptor during the stressor followed by a persistent, ligand-independent constitutive activation of the κOR itself. This lasting change in function is not seen at κORs at neighboring excitatory synapses, suggesting distinct time courses and mechanisms of regulation of different subsets of κORs. We also provide evidence that constitutive activity of κORs governs the prolonged reinstatement to cocaine-seeking observed after cold water swim stress. Together, our studies indicate that stress-induced constitutive activation is a novel mechanism of κOR regulation that plays a critical role in reinstatement of drug seeking

A Significant but Rather Mild Contribution of T286 Autophosphorylation to Ca2+/CaM-Stimulated CaMKII Activity

Autophosphorylation of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) at T286 generates partially Ca(2+)/CaM-independent "autonomous" activity, which is thought to be required for long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. A requirement for T286 autophosphorylation also for efficient Ca(2+)/CaM-stimulated CaMKII activity has been described, but remains controversial.In order to determine the contribution of T286 autophosphorylation to Ca(2+)/CaM-stimulated CaMKII activity, the activity of CaMKII wild type and its phosphorylation-incompetent T286A mutant was compared. As the absolute activity can vary between individual kinase preparations, the activity was measured in six different extracts for each kinase (expressed in HEK-293 cells). Consistent with measurements on purified kinase (from a baculovirus/Sf9 cell expression system), CaMKII T286A showed a mildly but significantly reduced rate of Ca(2+)/CaM-stimulated phosphorylation for two different peptide substrates (to ~75-84% of wild type). Additional slower CaMKII autophosphorylation at T305/306 inhibits stimulation by Ca(2+)/CaM, but occurs only minimally for CaMKII wild type during CaM-stimulated activity assays. Thus, we tested if the T286A mutant may show more extensive inhibitory autophosphorylation, which could explain its reduced stimulated activity. By contrast, inhibitory autophosphorylation was instead found to be even further reduced for the T286A mutant under our assay conditions. On a side note, the phospho-T305 antibody showed some basal background immuno-reactivity also with non-phosphorylated CaMKII, as indicated by T305/306A mutants.These results indicate that Ca(2+)/CaM-stimulated CaMKII activity is mildly (~1.2-1.3fold) further increased by additional T286 autophosphorylation, but that this autophosphorylation is not required for the major part of the stimulated activity. This indicates that the phenotype of CaMKII T286A mutant mice is indeed due to the lack of autonomous activity, as the T286A mutant showed no dramatic reduction in stimulated activity

CaMKII T305/306 “burst” auto-phosphorylation <i>in vitro</i>.

<p><i>A</i>, The sequence of the CaMKII regulatory domain with T286 in the autoinhibitory region and T305/306 in the CaM-binding region indicated. <i>B</i>, T305/306 phosphorylation was assessed by Western analysis. CaMKII was pre-phosphorylated at T286 on ice; the “burst” was induced by EGTA addition at 30°C or on ice, and stopped after different reaction times. Note the band-shift caused by phosphorylation of additional sites during the “burst” at 30°C, and the basal immuno-detection without phosphorylation reaction. <i>C</i>, Quantification of the relative T305/306 phosphorylation during the “burst” by arbitrary relative immuno-detection values (IDV). Basal immuno-detection prior to the phosphorylation reactions was subtracted in the quantification shown. No increase in phosphorylation was detected on ice. For 30°C, the results indicate an initial phosphorylation rate (at reaction times of 1 min or less) of ∼0.45 min⁻¹ (assuming that saturation represents near-complete phosphorylation; otherwise the rate is even lower). <i>D</i>, The basal immuno-detection with the phospho-T305 antibody prior to the phosphorylation reactions was likely due to background immuno-reactivity, as indicated by comparison to T305/306A mutant CaMKII.</p

Maximal stimulated activity of CaMKII wild type and its T286A mutant was induced by 1 µM Ca²⁺/CaM and measured by the phosphorylation rate of two different substrates (syntide 2 and AC2) in biochemical assays.

<p>Error bars indicate mean ± s.e.m; **: p<0.01, *: p<0.05 in two-tailed t-test. <i>A</i>, CaMKII activity in the different extracts for each kinase form (n = 5 individual assays). <i>B</i>, The average activity of CaMKII from the individual extracts shown separately in panel A (N = 6 different extracts). <i>C</i>, The relative activity of the T286A mutant (compared to wild type activity normalized as 1 for each substrate) based on the results shown in panel B.</p

CaMKII T305 phosphorylation is further reduced for the T286A mutant.

<p><i>A</i>, T305 phosphorylation was detected by Western analysis after reactions corresponding to the kinase activity assays shown in Fig. 1 (1 min at 30°C, but without substrate peptide) or after extended reaction times (10 min). <i>B</i>, Quantification of the relative T305 phosphorylation by the ratio of the phospho-T305 and total CaMKII immuno-detection values (IDV) shows significantly lower phosphorylation of the T286A mutant in our 1 min kinase assay conditions (N = 3 different extracts for each kinase form; *: p<0.05 in two-tailed t-test). The IDV ratio for T286A was ∼28% (±7.6%) of wild type after the 1 min reactions; in the 10 min reaction experiment shown in panel A, this ratio was ∼83%, consistent with the faster wild type reaction reaching saturation faster.</p

Submaximal stimulated activity of CaMKII wild type and its T286A mutant was induced by 0.1 µM Ca²⁺/CaM and measured by the phosphorylation rate of the substrate syntide 2 (in 1 min reactions at 30°C).

<p>Error bars indicate mean ± s.e.m; **: p<0.01, n.s.: p>0.05 in two-tailed t-test. <i>A</i>, T286 autophosphorylation of CaMKII wild type was assessed by Western analysis (left), and quantified by arbitrary relative immuno-detection values (IDV; right). T286 autophosphorylation stimulated by 0.1 µM Ca²⁺/CaM was slower compared to stimulation by 1 µM Ca²⁺/CaM, but the same level of maximal autophosphorylation was still achieved within 1 min reaction time at 30°C. <i>B</i>, Submaximal activation by 0.1 µM Ca²⁺/CaM was verified by comparing one individual preparation of each CaMKII wild type and T286A mutant to the activity induced by 1 µM Ca²⁺/CaM (n = 4 individual assays). <i>C</i>, The average activity of CaMKII from multiple preparations (N = 5) stimulated by 0.1 µM Ca²⁺/CaM did not differ significantly between CaMKII wild type and the T286A mutant. While the ratio of the mean activities of T286A over wild type was similar as observed at maximal stimulation (compare Fig. 1B,C), the variability at submaximal stimulation was greater (with standard deviations of 35–36% of the mean at submaximal stimulation compared to 13–17% at maximal stimulation for syntide 2 substrate).</p