Dichotomy between Mitochondrial Ca2+ Uptake and Martix [Ca2+] in MICU1 Deficient Cells

Abstract

Mitochondrial calcium uptake is central to cell physiology, but the underlying molecular mechanism remains elusive. Recently, MICU1 was identified as a mitochondrial protein required for Ca2+ uptake but dispensible for mitochondrial respiration or membrane potential generation. To study the specific role of MICU1 in mitochondrial Ca2+ uptake, we used wild type HeLa cells (Ctrl), MICU1 knockdown by 2 distinct shRNAs (KD) and MICU1 rescued cells (Rescue). First, we monitored the cytoplasmic and mitochondrial matrix [Ca2+] ([Ca2+]c and [Ca2+]m, respectively) during store-operated Ca2+ entry. Specifically, cells were preincubated in a Ca2+-free buffer containing thapsigargin (Tg), to deplete the ER Ca2+ store, and then Ca2+ was added back and the ensuing [Ca2+]c (fura2) and [Ca2+]m (mitochondria-targeted pericams) changes were measured. In both Ctrl and KD a [Ca2+]c increase occurred that was associated with a [Ca2+]m rise only in Ctrl. The Rescue behaved similarly to the Ctrl. Also, when Ca2+ was added to permeabilized cells, no [Ca2+]m increase appeared in the KD. These results confirm that the shortage of MICU1 causes suppression of the [Ca2+]m signal. Surprisingly, when the Ca2+ entry was followed by uncoupler addition to release the mitochondrial Ca2+ content, a huge [Ca2+]c increase appeared in KD, However, when the uncoupler was added prior to Ca2+ entry, no [Ca2+]c increase occurred. Thus, mitochondria in the KD also accumulated Ca2+ during Ca2+ entry. Mitochondrial Ca2+ uptake evoked by Ca2+ addition was also documented in permeabilized KD by measurement of ruthenium red sensitive 45Ca uptake and cytoplasmic Ca2+ clearance (fura2). Thus, Ca2+ is accumulated by MICU1 deficient mitochondria but it fails to result an increase in [Ca2+]m. We are currently investigating, whether suppression of the [Ca2+]m rise in the KD is due to enhanced matrix Ca2+-buffering.