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Involvement of protein phosphorylation in activation of Ca2+ efflux from sarcoplasmic reticulum.

ABSTRACT: Preincubation of sarcoplasmic reticulum (SR) membranes with a combination of ATP and NaF resulted in inhibition of Ca2+ accumulation and stimulation of Ca(2+)-ATPase and Ca2+ efflux. Under the same conditions, the activity of the SR phosphoprotein phosphatase was inhibited and the phosphorylation of two polypeptides with apparent molecular masses of 160 and 150 kDa was obtained. The effect of ATP is specific, since the ATP analogue adenosine 5'-[beta gamma-imido]triphosphate did not replace for ATP. In the absence of NaF, ATP was ineffective. The phosphorylation of the 160 kDa and/or 150 kDa proteins and the stimulation of Ca2+ efflux are clearly related. The phosphorylation of both proteins and the increase in Ca2+ efflux show a similar dependence on the concentration of ATP. The level of protein phosphorylation and the stimulation of Ca2+ efflux were also controlled by the NaF concentration which inhibits the phosphatase and of net Ca2+ accumulation, as well as for the stimulation of phosphorylation of both polypeptides. Quantitative analysis revealed a linear correlation between these three activities. Dicyclohexylcarbodi-imide, which inhibited Ca2+ efflux, also inhibited the phosphorylation of the two polypeptides. These results suggest the involvement of the phosphorylation/dephosphorylation of 160 kDa and/or 150 kDa polypeptides in the activation of Ca2+ release from SR membranes.

SUBMITTER: Gechtman Z

PROVIDER: S-EPMC1151148 | biostudies-other | 1991 May

REPOSITORIES: biostudies-other

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Project description:Basal phosphorylation of sarcoplasmic reticulum (SR) Ca(2+) proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca(2+) releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca(2+) sparks. We tested the hypothesis that in a physiologic Ca(2+) milieu, and independent of increased Ca(2+) influx, an increase in basal phosphorylation of SR Ca(2+) cycling proteins will convert stochastic Ca(2+) sparks into periodic, high-power Ca(2+) signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca(2+) release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca(2+)], prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca(2+) releases of low power and low amplitude, as assessed by confocal Ca(2+) imaging and spectral analysis. When the kinetics of Ca(2+) pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, "clock-like" local Ca(2+) releases, partially synchronized in space and time (Ca(2+) wavelets), emerged, and the ensemble of these rhythmic local Ca(2+) wavelets generated a periodic high-amplitude Ca(2+) signal. Thus, a Ca(2+) clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca(2+) cycling increases and spontaneous local Ca(2+) release becomes partially synchronized. This unleashed Ca(2+) clock that emerges in a physiological Ca(2+) milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature of novel bio-pacemaker designs.

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Involvement of protein phosphorylation in activation of Ca2+ efflux from sarcoplasmic reticulum.

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