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Ca2+ influx through NMDA‐type glutamate receptor at excitatory synapses causes activation of post‐synaptic Ca2+/calmodulin‐dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo. Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B‐Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca2+/calmodulin activated form and the autonomously active Thr286‐autophosphorylated form of CaMKII. Green fluorescent protein–α‐CaMKII co‐expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione‐S‐transferase pull‐down assay. Thr286‐autophosphorylated α‐CaMKII or the autophosphorylation mimicking mutant, T286D‐α‐CaMKII, binds NR2B sequence independent of Ca2+/calmodulin unlike native wild‐type α‐CaMKII. We show enhancement of this binding by Ca2+/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B‐S1303D) abolishes the Ca2+/calmodulin‐independent binding whereas it allows the Ca2+/calmodulin‐dependent binding of α‐CaMKII in vitro. Similarly, the autonomously active mutants, T286D‐α‐CaMKII and F293E/N294D‐α‐CaMKII, exhibited Ca2+‐independent binding to non‐phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho‐Ser1303 on NR2B.

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Hereditary long‐QT syndrome (LQTS) is caused by mutations in ion channel genes resulting in a prolonged action potential duration (APD) in cardiomyocytes. Complications include syncope and sudden cardiac death. Large‐conductance, Ca2+‐activated K+ (BK) channels mediate repolarization of many cell types, but they are not expressed in the plasma membrane of cardiomyocytes. We hypothesized that introduction of BK channels may shorten APD in cardiomyocytes as a potential therapy for LQTS. HL‐1 atrial myocytes were transiently transfected with BK plasmids bicistronically expressing hBK and mCherry or control plasmids expressing only mCherry. Expression of the hBK transgene was confirmed by Western blot, immunocytochemistry and by detecting iberiotoxin (Ibtx)‐sensitive BK current in patch‐clamped cells. Western blots showed that the expression of other ion channels including KV4.3, KV7.1, NaV1.5, and CaV1.2 did not significantly change after BK transfection, whereas KV11.1 was down‐regulated by 20% (n=5). APD at 50% repolarization was 3.7±0.5 ms in BK‐transfected cells compared to 6.3±0.6 ms in control‐transfected cells. Ibtx (100 nmol/L) restored the shortened APD in hBK‐transfected cells to 7.9±1.5 ms (n=5–14). Thus, BK channels can exert a powerful repolarizing influence in HL‐1 myocytes, and may be a useful therapy to reverse LQTS. AHA SDG 0830060N (SWR) and NIH R01HL093526 (NJR)