Contribution of a newly found NCLX protein to mitochondrial and cytosolic calcium signaling

The present BSF grant deals with molecular and cellular mechanisms of mitochondrial sodium-calcium exchanger (NCXL), which has been discovered in 2010 in Israeli (Palty et al., PNAS USA 107:436-441, 2010). This breakthrough finding provided new opportunities for elucidating fundamental events in the cell related to neurodegenerative, cardiovascular and diabetes. The essence of this BSF grant was to take an advantage on molecular and biochemical tools (developed in the two Israeli laboratories, Khananshvili and Sekler) in conjunction with advanced biophysical and cellular techniques (the Lederer laboratory, USA). The primary goal was to resolve the mitochondrial targeting and regulation of NCLX in neuronal, muscle and non-excitable tissues.

A close collaboration between the Khananshvili and Lederer laboratories lead to important finding that Ca2+ binding regulatory domains (CBD1 and CBD2) of NCX1-3, play a key role in regulating cardiomyocyte function under normal and arrhythmia conditions (e.g., ischemia or acidosis). This collaborative work has been published in JBC (Boyman L, Hagen BM, Giladi M, Hiller R, Lederer WJ, Khananshvili D, J Biol Chem 286:28811-28820, 2011). In line with previous findings, the Khananshvili laboratory has hypothesized that the N-terminal sequence may determine mitochondrial targeting of NCLX. To test this working hypothesis, the Khananshvili laboratory explored fluorescence GFP technology by preparing the N-terminal truncated NCLX (ΔN-NCLX-GFP). Computer-aided analysis of NCLX clearly demonstrated that the N-terminal sequence of 35 amino acids has α-helix structure meaning that this signal sequence might govern mitochondrial targeting of NCLX. We have demonstrated that the truncated ΔN-NCLX-GFP protein is targeted to the cell membrane of neuroblsatoma and smooth muscle cells, whereas the full length NCLX-GFP was shown is co-localized with mitochontrial markers detected by confocal microscopy. Moreover, without the N-terminal signal sequence NCLX reaches the plasma membrane and not mitochondria. The Sekler laboratory provided that the N-terminal truncated NCLX (ΔN-NCLX-GFP) can mediate the Ca2+-entry mode of Na+/Ca2+ exchange, which can potentially trigger pathophysiological conditions.

The Sekler laboratory has found that NCLX regulates not only the mitochondrial Ca2+ homeostasis but also the Ca2+-entry into the cell through the regulation of voltage-dependent Ca2+ channels. These findings have been published (Nita I, Hershfinkel M, Ozeri E, Lewis E, Khananshvili D, Rutter GA, Israel Sekler, PloS One, 7:e46649, 2012). The significance of these findings is that NCLX limits the Ca2+-dependent secretion of insulin. Previous studies using the mitochondrial exchanger inhibitor CGP-37157 suggested that the inhibition of the exchanger led to enhanced ATP production and insulin secretion. The Lederer laboratory explored the state-of-the-art biophysical approaches for measuring intra-mitochondrial Ca2+([Ca2+]mito) changes with a goal of resolving the underlying mechanisms of NCLX function. This project was conducted by Dr. Boyman (a former PhD student in the Khananshvili laboratory), who is presently a postdoctoral fellow in the Lederer laboratory. By using the genetically encoded Ca2+ indicator "Mitycam", the [Ca2+]mito(matrix mitochondrial [Ca2+]) was measured in isolated ventricular myocytes. The cytosolic [Ca2+]i transients and [Ca2+]mito were simultaneously measure in patched cardiomyocytes to find any possible correlations between these two parameters. A major finding is that in electrically paced isolated cardiomyocytes the cytosolic [Ca2+]i transients are not associated with [Ca2+]mito transients, although the [Ca2+]mito gradually increases in time. These findings in conjunction with computational simulations support a notion that NCLX-mediated Ca2+ extrusion from the mitochondria is so fast that [Ca2+]mito transients cannot be seen under the given experimental conditions. These very important results were summarized a joint paper (Boyman L, Williams GSB, Khananshvili D, Sekler I and Lederer WJ. J Mol Cell Cardiology 2013, 59:205-213).

In conclusion, the present BSF grant has elucidated the molecular and cellular mechanisms underlying the function and regulation of mitochondrial NCLX in excitable and non-excitable tissues. These mechanisms could be related to pathophysiological conditions implemented in cardiovascular, neurodegenerative and diabetes diseases. Selective pharmacologic targeting (downregulation) of mitochondrial NCLX could be feasible approach for beneficial handling of relevant diseases.