Regulation of mitochondrial free Ca2+ is critically important for cellular homeostasis. An increase in mitochondrial matrix free Ca2+ concentration ([Ca2+]m) predisposes mitochondria to opening of the permeability transition pore (mPTP). Opening of the pore can be delayed by cyclosporin A (CsA), possibly by inhibiting cyclophilin D (Cyp D), a key regulator of mPTP. Here, we report on a novel mechanism by which CsA delays mPTP opening by enhanced sequestration of matrix free Ca2+. Cardiac-isolated mitochondria were challenged with repetitive CaCl2 boluses under Na+-free buffer conditions with and without CsA. CsA significantly delayed mPTP opening primarily by promoting matrix Ca2+ sequestration, leading to sustained basal [Ca2+]m levels for an extended period. The preservation of basal [Ca2+]m during the CaCl2 pulse challenge was associated with normalized NADH, matrix pH (pHm), and mitochondrial membrane potential (ΔΨm). Notably, we found that in PO43− (Pi)-free buffer condition, the CsA-mediated buffering of [Ca2+]m was abrogated, and mitochondrial bioenergetics variables were concurrently compromised. In the presence of CsA, addition of Pi just before pore opening in the Pi-depleted condition reinstated the Ca2+ buffering system and rescued mitochondria from mPTP opening. This study shows that CsA promotes Pi-dependent mitochondrial Ca2+ sequestration to delay mPTP opening and, concomitantly, maintains mitochondrial function.

Nearly 2 decades since its discovery as one of the genes responsible for the Wolf-Hirschhorn Syndrome (WHS), the primary function of the leucine-zipper EF-hand containing transmembrane 1 (LETM1) protein in the inner mitochondrial membrane (IMM) or the mechanism by which it regulates mitochondrial Ca2+ handling is unresolved. Meanwhile, LETM1 has been associated with the regulation of fundamental cellular processes, such as development, cellular respiration and metabolism, and apoptosis. This mini-review summarizes the diversity of cellular functions impacted by LETM1 and highlights the multiple roles of LETM1 in health and disease.

Complex formation between hexokinase-II (HKII) and the mitochondrial VDAC1 is crucial to cell growth and survival. We hypothesize that HKII first inserts into the outer membrane of mitochondria (OMM) and then interacts with VDAC1 on the cytosolic leaflet of OMM to form a binary complex. To systematically investigate this process, we devised a hybrid approach. First, we describe membrane binding of HKII with molecular dynamics (MD) simulations employing a membrane mimetic model with enhanced lipid diffusion capturing membrane insertion of its H-anchor. The insertion depth of the H-anchor was then used to derive positional restraints in subsequent millisecond-scale Brownian dynamics (BD) simulations to preserve the membrane-bound pose of HKII during the formation of the HKII/VDAC1 binary complex. Multiple BD-derived structural models for the complex were further refined and their structural stability probed with additional MD simulations, resulting in one stable complex. A major feature in the complex is the partial (not complete) blockade of VDAC1’s permeation pathway, a result supported by our comparative electrophysiological measurements of the channel in the presence and absence of HKII. We also show how VDAC1 phosphorylation disrupts HKII binding, a feature that is verified by our electrophysiology recordings and has implications in mitochondria-mediated cell death.

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Supplemental Digital Content is Available in the Text.Targeting intrinsically disordered regions facilitates discovery of T-type/calcium channels 3.2 (CaV3.2) inhibitory peptides. Adeno-associated virus–mediated expression of prototypic CaV3.2iPA1 and 2 in dorsal root ganglia primary sensory neurons in vivo produces sustained inhibition of calcium channel current conducted by CaV3.2/T-type channels and attenuates stimulated and spontaneous pain in rats with neuropathic pain.