Patch-Clamp Analysis of Mitochondrial Ca2+ Uniporter in Different Tissues

Abstract

The mitochondrial Ca2+ uptake has been recognized as a central player in cellular pathophysiology for decades. Pathways of Ca2+ transport across the inner mitochondrial membrane (IMM) were investigated indirectly with biochemical techniques and most recently by applying the patch clamp technique to mitoplasts isolated from mammalian systems. Patch-clamp recording of the IMM have proved to provide an unambiguous picture of mitochondrial Ca2+ uptake under strictly controlled conditions. In this study, we evaluated and compared the biophysical properties of mitochondrial Ca2+ uptake in different mouse tissues. Freshly isolated mitoplasts from mouse heart, skeletal muscle, liver, kidney, spleen and brown adipose tissue were patch clamped in the whole-mitoplast configuration. Voltage step and ramp protocols covering the whole range of physiological potentials were applied to elicit the inwardly rectifying Ca2+current sensitive to RuR known as MCU. The distribution of MCU current densities between tissues was as follows: spleen > brown adipose tissue ≈ skeletal muscle > kidney > liver >> heart. Interestingly, MCU current density in heart was about 10 times smaller than in skeletal muscle. Our results support the view of a differential tissue activity of MCU, which can be explained by either a different distribution of MCU channel and/or by a different expression of its regulatory subunits confering various modes of physiological regulation. Further on, in order to investigate the putative direct contribution of Uncoupling Protein Isoform 2 and 3 (UCP2/3) to the uniporter, we investigated MCU properties in wild type (WT) mouse tissues where these two proteins have been found to have a major role and compared its properties in the correspondent tissues of UCP2 and UCP3 KO mice. We found no significant differences in calcium currents of any of the WT tissues studied compared to their UCP2/3 KO counterparts.