Distinct intracellular sAC-cAMP domains regulate ER Ca2+ signaling and OXPHOS function

Abstract

cAMP regulates a wide variety of physiological functions in mammals. This single second messenger can regulate multiple, seemingly disparate functions within independently regulated cell compartments. We have previously identified one such compartment inside the matrix of the mitochondria, where soluble adenylyl cyclase (sAC) regulates oxidative phosphorylation (OXPHOS). We now show that sAC knockout fibroblasts have a defect in OXPHOS activity and attempt to compensate for this defect by increasing OXPHOS proteins. Importantly, sAC knockout cells also exhibit decreased probability of endoplasmic reticulum (ER) Ca release associated with diminished phosphorylation of the inositol 3-phosphate receptor. Restoring sAC expression exclusively in the mitochondrial matrix rescues OXPHOS activity and reduces mitochondrial biogenesis, indicating that these phenotypes are regulated by intramitochondrial sAC. In contrast, Ca release from the ER is only rescued when sAC expression is restored throughout the cell. Thus, we show that functionally distinct, sAC-defined, intracellular cAMP signaling domains regulate metabolism and Ca signaling.

Soluble adenylyl cyclase‐mediated cAMP signaling and the putative role of PKA and EPAC in cerebral mitochondrial function

“…Intramitochondrial cAMP signaling and its downstream effects have been studied in either acutely isolated mitochondria or cell lines. Working in cell lines provides the advantage of being able to express sensors or doing knock out/knock down or overexpression of proteins of interest (e.g., sAC KO cells and HeLa cells expressing a mitochondrial cAMP sensor (Di Benedetto et al, ; Valsecchi et al, )). However, one concern when investigating mitochondria is that cell lines rely more heavily on glycolytic metabolism (the Warburg effect and/or the Crabtree effect), and thus their mitochondrial regulation must be expected to differ compared to what is occurring in the brain and other organs highly depending on oxidative metabolism (Diaz‐Ruiz, Rigoulet, & Devin, ; Phillips et al, ; Semenza, ).…”

“…All of these factors were normalized when sAC was expressed in the mitochondria. Cytosolic sAC KO caused changes in IP 3 R signaling which led to changes in ER Ca 2+ release (Valsecchi et al, 2017).…”

“…All of these factors were normalized when sAC was expressed in the mitochondria. Cytosolic sAC KO caused changes in IP 3 R signaling which led to changes in ER Ca 2+ release (Valsecchi et al, ). Recently, in a pharmacological characterization of the sAC inhibitors 2‐OHE, KH7 and bithionol, we showed that inhibition of sAC in cerebral cortical‐isolated mitochondria caused decreased ATP production, respiration and membrane potential thus confirming the presence of the sAC pathway in brain mitochondria (Jakobsen et al, ).…”

Non-canonical regulation of glycogenolysis and the Warburg phenotype by soluble adenylyl cyclase

“…We next investigated the mechanism underlying the disparity of cAMP effects derived from sAC and from tmACs on glycogen. sAC has been shown to signal via both Epac (Flacke, Flacke et al, 2013, Onodera, Nam et al, 2014) and PKA (Acin-Perez et al, 2009, Valsecchi, Konrad et al, 2017). Since tmAC-derived cAMP regulates glycogenolysis via PKA, we hypothesized that sAC-derived cAMP uses a different cAMP effector, namely Epac1 or Epac2.…”

An improved method for isolation of mitochondria from cell lines that enables reconstitution of calcium-dependent processes

“…The Ca 2+ uptake into the mitochondrial matrix is regulated via the calcium sensor proteins “MICU1” and “MICU2” that control MCU gating [4]. Recently, it has been reported that soluble adenylyl cyclase protein kinase A (sAC-PKA) signaling inside mitochondria regulates ATP production and it also regulates mitochondrial Ca 2+ uptake [5, 6]. More recently, several G-protein coupled receptors that can use calcium as second messengers, such as cannabinoid receptors CB-1 [7], melatonin MT1 receptors [8], GABA B receptors [9], and 5-HT receptors 5- HTR4 [10] have been identified in the mitochondrial membranes.…”