Faculty Bibliography

Apoptosis is an elemental form of programmed cell death; it is fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Apoptosis is also involved in many pathologies including cancer, neurodegenerative diseases, aging, and infarcts. This cell death program is tightly regulated by Bcl-2 family proteins by controlling the formation of the mitochondrial apoptosis-induced channel or MAC. Assembly of MAC corresponds to permeabilization of the mitochondrial outer membrane, which is the so called commitment step of apoptosis. MAC provides the pathway through the mitochondrial outer membrane for the release of cytochrome c and other pro-apoptotic factors from the intermembrane space. While overexpression of anti-apoptotic Bcl-2 eliminates MAC activity, oligomers of the pro-apoptotic members Bax and/or Bak are essential structural component(s) of MAC. Assembly of MAC from Bax or Bak was monitored in real time by directly patch-clamping mitochondria with micropipettes containing the sentinel tBid, a direct activator of Bax and Bak. Herein, a variety of high affinity inhibitors of MAC (iMAC) that may prove to be crucial tools in mechanistic studies have recently been identified. This review focuses on characterization of MAC activity, its regulation by Bcl-2 family proteins, and a discussion of how MAC can be pharmacologically turned on or off depending on the pathology to be treated.

Ion channels located in the outer and inner mitochondrial membranes are key regulators of cellular signaling for life and death. Permeabilization of mitochondrial membranes is one of the most critical steps in the progression of several cell death pathways. The mitochondrial apoptosis-induced channel (MAC) and the mitochondrial permeability transition pore (mPTP) play major roles in these processes. Here, the most recent progress and current perspectives about the roles of MAC and mPTP in mitochondrial membrane permeabilization during cell death are presented. The crosstalk signaling of MAC and mPTP formation/activation mediated by cytosolic Ca(2+) signaling, Bcl-2 family proteins, and other mitochondrial ion channels is also discussed. Understanding the mechanisms that regulate opening and closing of MAC and mPTP has revealed new therapeutic targets that potentially could control cell death in pathologies such as cancer, ischemia/reperfusion injuries, and neurodegenerative diseases.

Ca(2+) channels that underlie mitochondrial Ca(2+) transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that mitochondrial Ca(2+) uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca(2+) transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca(2+) overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca(2+) channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca(2+) channels.

The study of mitochondrial ion channels changed our perception of these double-wrapped organelles from being just the power house of a cell to the guardian of a cell's fate. Mitochondria communicate with the cell through these special channels. Most of the time, the message is encoded by ion flow across the mitochondrial outer and inner membranes. Potassium, sodium, calcium, protons, nucleotides, and proteins traverse the mitochondrial membranes in an exquisitely regulated manner to control a myriad of processes, from respiration and mitochondrial morphology to cell proliferation and cell death. This review is an update on both well established and putative mitochondrial channels regarding their composition, function, regulation, and therapeutic potential.

Coordination of Ca(2+) signaling among cells contributes to synchronization of salivary gland cell function. However, mechanisms that underlie this signaling remain elusive. Here, intercellular Ca(2+) waves (ICW) in submandibular gland cells were investigated using Fura-2 fluorescence imaging. Mechanical stimulation of single cells induced ICW propagation from the stimulated cells through approximately 7 layers of cells or approximately 120microm. Our findings indicate that an extracellular ATP-dependent pathway is involved because the purinergic receptor antagonist suramin and the ATP hydrolyzing enzyme apyrase blocked ICW propagation. However, the gap junction uncoupler oleamide had no effect. ATP is released from mechanically stimulated cells possibly through opening of mechanosensitive maxi-anion channels, and does not appear to be directly linked to cytosolic Ca(2+). The ICW is propagated by diffusing ATP, which activates purinergic receptors in neighboring cells. This purinergic signaling induces a Ca(2+) transient that is dependent on Ca(2+) release via IP(3) receptors in the ER and store operated Ca(2+) entry (SOCE). Finally, inhibition of mitochondrial Ca(2+) uptake modified ICW indicating an important role of these organelles in this phenomenon. These studies increase our understanding of purinergic receptor signaling in salivary gland cells, and its role as a coordination mechanism of Ca(2+) signals induced by mechanical stimulation.

The death of one cell can precipitate the death of nearby cells in a process referred to as the bystander effect. We investigated whether mitochondrial apoptosis generated a bystander effect and, if so, by which pathway. Microinjection with cytochrome c mimicked function of the mitochondrial apoptosis-induced channel MAC and caused apoptosis of both target and nearby osteoblasts. This effect was suppressed by inhibiting gap junction intercellular communication. A bystander effect was also observed after exogenous expression of tBid, which facilitates MAC formation and cytochrome c release. Interestingly, in connexin-43 deficient osteoblasts, microinjection of cytochrome c induced apoptosis only in the target cell. These findings indicate that a death signal was generated downstream of MAC function and was transmitted through gap junctions to amplify apoptosis in neighboring cells. This concept may have implications in development of new therapeutic approaches.

MAC (mitochondrial apoptosis-induced channel) forms in the mitochondrial outer membrane and unleashes cytochrome c to orchestrate the execution of the cell. MAC opening is the commitment step of intrinsic apoptosis. Hence closure of MAC may prevent apoptosis. Compounds that blocked the release of fluorescein from liposomes by recombinant Bax were tested for their ability to directly close MAC and suppress apoptosis in FL5.12 cells. Low doses of these compounds (IC50 values ranged from 19 to 966 nM) irreversibly closed MAC. These compounds also blocked cytochrome c release and halted the onset of apoptotic markers normally induced by IL-3 (interleukin-3) deprivation or staurosporine. Our results reveal the tight link among MAC activity, cytochrome c release and apoptotic death, and indicate this mitochondrial channel is a promising therapeutic target.

Although Bcl-2 family proteins control intrinsic apoptosis, the mechanisms underlying this regulation are incompletely understood. Patch clamp studies of mitochondria isolated from cells deficient in one or both of the pro-apoptotic proteins Bax and Bak show that at least one of the proteins must be present for formation of the cytochrome c-translocating channel, mitochondrial apoptosis-induced channel (MAC), and that the single channel behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak with a time course of minutes and does not require VDAC1 or VDAC3. Mathematical analysis of the stepwise changes in conductance associated with MAC formation is consistent with pore assembly by a barrel-stave model. Assuming the staves are two transmembrane alpha-helices in Bax and Bak, mature MAC pores would typically contain approximately 9 monomers and have diameters of 5.5-6 nm. The mitochondrial permeability data are inconsistent with formation of lipidic pores capable of transporting megadalton-sized macromolecules as observed with recombinant Bax in liposomes.

There is excellent agreement between the electrophysiological properties and the structure of the mitochondrial outer membrane protein, VDAC, ex vivo. However, the inference that the well-defined canonical "open" state of the VDAC pore is the normal physiological state of the channel in vivo is being challenged by several lines of evidence. Knowing the atomic structure of the detergent solubilized protein, a long sought after goal, will not be sufficient to understand the functioning of this channel protein. In addition, detailed information about VDAC's topology in the outer membrane of intact mitochondria, and the structural changes that it undergoes in response to different stimuli in the cell will be needed to define its physiological functions and regulation.

The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial. Two mechanisms, involving opening of two different mitochondrial channels, have been proposed to be responsible for the permeabilization; the permeability transition pore (PTP) in the inner membrane and the mitochondrial apoptosis-induced channel (MAC) in the outer membrane. Opening of PTP would lead to matrix swelling, subsequent rupture of the outer membrane, and an unspecific release of intermembrane proteins into the cytosol. However, many believe PTP opening is a consequence of apoptosis and this channel is thought to principally play a role in necrosis, not apoptosis. Activation of MAC is exquisitely regulated by Bcl-2 family proteins, which are the sentinels of apoptosis. MAC provides specific pores in the outer membrane for the passage of intermembrane proteins, in particular cytochrome c, to the cytosol. The electrophysiological characteristics of MAC are very similar to Bax channels and depletion of Bax significantly diminishes MAC activity, suggesting that Bax is an essential constituent of MAC in some systems. The characteristics of various mitochondrial channels and Bax are compared. The involvement of MAC and PTP activities in apoptosis of disease and their pharmacology are discussed.