"Mechanism of Inhibition of the Plasma Membrane Calcium Pump by IP3-kinase"
Pharmacology & Physiology Program
B.Sc. 2002, Catholic University of Chile
Thesis Advisor: Andrew Thomas, Ph.D.
Department of Pharmacology & Physiology
Friday, June 8, 2012
1:00 P.M., MSB Room H-609
Inositol (1,4,5)P3 3-kinase (IP3K) belongs to one of the 6 evolutionarily conserved kinases responsible for the synthesis of inositol phosphates. Three mammalian isoforms of this enzyme have so far been cloned and isolated: IP3KA, IP3KB and IP3KC. They catalyze the formation of inositol (1,3,4,5)-tetrakisphosphate (IP4) by selectively phosphorylating Ins(1,4,5)P3 at the 3-OH position on the inositol ring. Since its discovery, IP4 was proposed as a potential second messenger, but studies trying to establish its physiological function, in particular regarding its effects on Ca2+ signaling, have been controversial.
Ca2+ extrusion by the plasma membrane Ca2+ ATPase (PMCA) is, in many cells, the main mechanism contributing to the return of the cytosolic free Ca2+ concentration ([Ca2+]c) to baseline during the declining phase of Ca2+ signals. Due to its high affinity for Ca2+, PMCA can also modulate the level of this ion according to tissue- and cell-specific demands, for which it needs multiple levels of control. Previous results from our laboratory have shown that overexpression of IP3 3-kinase causes inhibition of PMCA activity. The main goal of the present work was to understand the mechanisms whereby the inhibition of PMCA takes place. By transiently transfecting CHO cells with IP3K isoforms fused with GFP and measuring Ca2+ fluxes with digital imaging fluorescence microscopy we observed that IP3K overexpressing cells present a higher steady-state [Ca2+]c and the Ca2+ decay rate is slower than in non-transfected cells. This effect, which was also observed in Hela cells and hepatocytes, depends on the expression level of IP3K and is similarly produced by IP3K isoforms A and B. The mechanism of PMCA inhibition is explained by the catalytic activity of IP3K, because a mutant enzyme that cannot form IP4 failed to reproduce this effect. In addition, we observed that the retardation of Ca2+ extrusion depends on the time interval elapsed between IP3 phosphorylation by IP3K and PMCA activation. These results, along with the observation that PMCA inhibition was significantly lower during a second Ca2+ pulse, argue against a non-enzymatic activity of IP3K. We also confirmed that the enzymatic mechanism does not depend on the reduction of IP3, since cells transfected with IP3 5-phosphatase do not present inhibition of the Ca2+ elimination rate. Our results support the hypothesis that IP4 is responsible for the inhibition of PMCA activity. CHO cells treated with Li+, which causes an increase in the hormone-induced accumulation of several inositol phosphates, but not in the accumulation of IP4, did not slow down Ca2+ extrusion. Furthermore, treatment with Li+ of cells overexpressing IP3K did not amplify the inhibitory effect on PMCA. In addition, in experiments where we prevented the IP4 accumulation produced by IP3K by simultaneous co-transfection with 5-phosphatase, the inhibitory effect on PMCA was significantly attenuated. Finally, experiments performed with membrane vesicle preparations indicate that, at least in those subcellular systems, IP4 does not exert a direct action on PMCA and does not interfere with the CaM- or PIP2-dependent PMCA activation. These data suggest that IP4 exerts the inhibitory effect on PMCA indirectly, through a cellular factor that is probably lost or inactivated in our preparations.
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