The physiological and molecular role of NCLX in liver

Calcium ions flow into the mitochondria via the mitochondrial Ca2+ uniporter (MCU), powered by the steep mitochondrial membrane potential. In excitable cells Ca2+ is primarily extruded by the mitochondrial Na+/Ca2+ exchanger (NCLX), but the role of this exchanger in non-excitable cells, such as hepatocytes is still not fully understood. Previous studies proposed that mitochondrial Ca2+ efflux in the liver is primarily mediated by a H+/Ca2+ exchanger, and that Na+/ Ca2+ exchange has a minimal role. To determine the role of NCLX in the liver mitochondria, we recorded mitochondrial transients triggered by purinergic activation by ATP in Rhod-2 pre-loaded primary hepatocytes from WT and NCLX KO mice. The mitochondrial Ca2+ removal was Na+/Li+ dependent only in the WT, the same Na+/Li+ dependent Ca2+ removal we monitored in the digitonin-permeabilized hepatocytes. Our results led us to ask whether NCLX KO would influence oscillatory Ca2+ responses in hepatocytes triggered by low physiological concentrations of hormones. Consistent with previous studies hormone application triggered mitochondrial Ca2+ oscillations in WT hepatocytes. In contrast, oscillations were absent in NCLX KO hepatocytes. To further investigate the physiological role on NCLX we the performed a conditional KO model using double-floxed mice (NCLX lox/lox) with AAV virus expressing active or inactive mutant Cre-recombinase to yield NCLX double knockout (dKO) and NCLX wild-type (WT) hepatocytes. Taken together, our results indicate that NCLX is the primary Ca2+ extruder in hepatocytes and is essential for mediating the hormone-induced mitochondrial Ca2+ oscillations in these cells.