Funktionsanalyse des Zinktransporters I (ZnT-1) mittels genetischer Deletion
Final Report Abstract
Starting point of this grant application was the observation that between 3 and 10 % of human proteins contain zinc. Very little was known about the regulation of the cytosolic zinc concentration. The human genome codes for two different zinc transporter families; a) ZnT family which lowers cytosolic zinc concentration and b) the Zip protein family which transports zinc from the extracellular space or vesicles to the cytosol. Our previous work showed that ZnT-1 blocks the LTCC in cardiac cells and in culture and in vivo where ZnT-1 expression was augmented following atrial rapid pacing. ZnT-1 expression was also augmented in patient with atrial fibrillation. Using both surface biotinylation and Total Internal Reflection Microscopy (TIRFM), we demonstrated a ZnT-1 dependent decrease in the surface expression of the pore forming subunit of the LTCC, the alpha1-subunit. ZnT-1 and the beta-subunit of the LTCC directly interact in HEK293 cells. These papers demonstrated that the ZnT-1-mediated decrease of the LTCC currents depends on the reduced surface expression of the alpha1-subunit. Iit was published that ZnT-1 interacts directly with Raf-1 kinase leading to downstream activation of MEK/MAPK signaling. In the following study we investigated the role of ZnT-1 in myocardial ischemia/reperfusion injury (I/R) and its underlying molecular mechanism/s. ZnT-1 confers cellular resistance to I/R injury through its ability to interact with Raf-1 and its ability to stimulate the Raf/MEK/MAPK signaling pathway. The function of ZnT-1 as a modulator of voltage sensitive calcium channel on the one hand and activator of the Ras/Raf/ERK system is apparent in the last part of the work in which we tested the regulatory activity of ZnT-1 on T-type calcium channel. In Xenopus oocytes expressing Cav3.1 or Cav3.2 channels, ZnT-1 enhanced the low threshold calcium currents (IcaT) to 182 ± 15 % and 167.95 ± 9.27 % of control, respectively (p<0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Co-expression of ZnT-1 and non-active Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1 induced augmentation of IcaT. Our findings indicate that ZnT-1 enhances the activity of T-type calcium channels through activation of Ras-ERK signaling leading to enhanced Cav3 surface expression. We suggest that ZnT-1 may become a target for the treatment of atrial fibrillation.
Publications
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(2007) Crosstalk between L-type calcium channels and ZnT-1, a new player in rate-dependent cardiac electrical remodeling. Cell Calcium. 42(1):71-82. Epub 2006 Dec 29
Beharier O, Etzion Y, Katz A, Friedman H, Tenbosh N, Zacharish S, Bereza S, Goshen U, Moran A
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(2008) Correlation between atrial ZnT-1 expression and atrial fibrillation in humans: a pilot study. J Cardiovasc Electrophysiol. 19(2):157-64. Epub 2007 Oct 24
Etzion Y, Ganiel A, Beharier O, Shalev A, Novack V, Volvich L, Abrahamov D, Matsa M, Sahar G, Moran A, Katz A
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(2009) Molecular basis for zinc transporter 1 action as an endogenous inhibitor of L-type calcium channels. J Biol Chem. 284(47):32434-43. Epub 2009 Sep 18
Levy S, Beharier O, Etzion Y, Mor M, Buzaglo L, Shaltiel L, Gheber LA, Kahn J, Muslin AJ, Katz A, Gitler D, Moran A
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(2010) The involvement of ZnT-1, a new modulator of cardiac L-type calcium channels, in atrial tachycardia remodeling. Ann N Y Acad Sci. 1188:87-95
Beharier O, Etzion Y, Levi S, Mor M, Mor M, Dror S, Kahn J, Katz A, Moran A
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(2012).ZnT-1 protects HL-1 cells from simulated ischemia-reperfusion through activation of Ras-ERK signaling. J Mol Med 90(2):127-38. Epub 2011 Dec 23
Beharier O, Dror S, Levy S, Kahn J, Mor M, Etzion S, Gitler D, Katz A, Muslin AJ, Moran A, Etzion Y
