N-Methyl-d-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.
Ca2+/calmodulin-dependent protein kinase II (αCaMKII) is thought to exert its role in memory formation by autonomous Ca2+-independent persistent activity conferred by Thr286 autophosphorylation, allowing the enzyme to remain active even when intracellular [Ca2+] has returned to resting levels. Ca2+ sequestration-induced inhibition, caused by a burst of Thr305/306 autophosphorylation via calmodulin (CaM) dissociation from the Thr305/306 sites, is in conflict with this view. The processes of CaM binding, autophosphorylation, and inactivation are dissected to resolve this conflict. Upon Ca2+ withdrawal, CaM sequential domain dissociation is observed, starting with the rapid release of the first (presumed N-terminal) CaM lobe, thought to be bound at the Thr305/306 sites. The time courses of Thr305/306 autophosphorylation and inactivation, however, correlate with the slow dissociation of the second (presumed C-terminal) CaM lobe. Exposure of the Thr305/306 sites is thus not sufficient for their autophosphorylation. Moreover, Thr305/306 autophosphorylation and autoinactivation are shown to occur in the continuous presence of Ca2+ and bound Ca2+/CaM by time courses similar to those seen following Ca2+ sequestration. Our investigation of the activity and mechanisms of phospho-Thr286-αCaMKII thus shows time-dependent autoinactivation, irrespective of the continued presence of Ca2+ and CaM, allowing a very short, if any, time window for Ca2+/CaM-free phospho-Thr286-αCaMKII activity. Physiologically, the time-dependent autoinactivation mechanisms of phospho-Thr286-αCaMKII (t½ of ∼50 s at 37 °C) suggest a transient kinase activity of ∼1 min duration in the induction of long term potentiation and thus memory formation.
Our study unravels the cellular and molecular mechanisms of attenuation of P2XR-mediated preglomerular vasoconstriction that elevates glomerular susceptibility to harmful hypertensive pressures. This provides an important impetus towards understanding of the pathology of hypertensive renal injury.
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