Papers by Kathleen Kinnally
eLife, 2013
A critical event in ischemia-based cell death is the opening of the mitochondrial permeability tr... more A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane res...
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J Biol Chem, 1996
The functional relationship between the adenine nucleotide translocator (ANT) and the mitochondri... more The functional relationship between the adenine nucleotide translocator (ANT) and the mitochondrial multiple conductance channel (MCC) was investigated using patch-clamp techniques. MCC activity with the same conductance, ion selectivity, voltage dependence, and peptide sensitivity could be reconstituted from inner membrane fractions derived from mitochondria of ANT-deficient and wild-type Saccharomyces cerevisiae. In addition, the MCC activity of mouse kidney mitoplasts was unaffected by carboxyatractyloside, a known inhibitor of ANT and inducer of a permeability transition. These results suggest that MCC activity is independent of ANT.
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Biochemical and Biophysical Research Communications, Feb 28, 1991
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This chapter describes the third class of membrane transporters, comprised of channels or pores, ... more This chapter describes the third class of membrane transporters, comprised of channels or pores, entities that define water-filled transmembrane pathways of varying size and selectivity. Numerous channel activities have been detected by application of bilayer and patch-clamp methodologies to mitochondrial membranes. In the outer membrane, the main channel activity is that of the porin, voltage-dependent anion channels (VDAC), and a voltage-gated large conductance channel. It is likely that this channel represents the primary permeability pathway through the outer membrane for metabolites and ions. The modulation of the VDAC channel by endogenous effectors suggests a regulatory role for outer membrane permeability in mitochondrial metabolism. A second large-conductance outer-membrane channel activity has been detected and may be involved in protein import. Five inner-membrane channel activities have been described based on patch-clamp studies of mitoplasts. The first discovered was mCS, a voltage-dependent 100pS anion channel activity that co-isolates with the ATP synthase. The multi conductance channel (MCC) activity has a maximum conductance twice that of VDAC and appears to correspond to the calcium- and cyclosporin-sensitive permeability transition pore (PTP) deduced from swelling studies. There are several indications that MCC and VDAC interact at a drug-receptor complex located at intermembrane contact sites. Three low-conductance channel activities have been detected in the mitochondrial inner membrane: a glibenclamide- and adenosine triphosphate (ATP)-sensitive K+ channel, and two pH-sensitive channel activities with opposite ion selectivity. The inner membrane ion channels appear to be highly regulated and are not expected to be open continuously under normal conditions.
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Journal of Bioenergetics and Biomembranes, 2015
Mitochondrial Apoptotic Channel inhibitors or iMACs are di-bromocarbazole derivatives with anti-a... more Mitochondrial Apoptotic Channel inhibitors or iMACs are di-bromocarbazole derivatives with anti-apoptotic function which have been tested and validated in several mouse models of brain injury and neurodegeneration. Owing to the increased therapeutic potential of these compounds, we sought to expand our knowledge of their mechanism of action. We investigated the kinetics of MAC inhibition in mitochondria from wild type, Bak, and Bax knockout cell lines using patch clamp electrophysiology, fluorescence microscopy, ELISA, and semiquantitative western blot analyses. Our results show that iMACs work through at least two mechanisms: 1) by blocking relocation of the cytoplasmic Bax protein to mitochondria and 2) by disassembling Bax and Bak oligomers in the mitochondrial outer membrane. iMACs exert comparable effects on channel conductance of Bax or Bak and similarly affect cytochrome c release from Bax or Bak-containing mitochondria. Interestingly, wild type mitochondria were more susceptible to inhibition than the Bak or Bax knockouts. Western blot analysis showed that wild type mitochondria had lower steady state levels of Bak in the absence of apoptotic stimulation.
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Structure and Dynamics of Confined Polymers, 2002
Protein translocation across membranes is a fundamental cellular process that is especially vital... more Protein translocation across membranes is a fundamental cellular process that is especially vital for mitochondrial biogenesis. More than 95% of all mitochondrial proteins must be imported from the cytoplasm. The protein-translocating channels of mitochondria have many identical properties including conductance, voltage dependence and regulation by synthetic peptides whose sequences mimic the targeting domains of precursors. Viewing protein translocation across membranes as movement of polymers through confined pathways has provided a novel perspective of channel function. These considerations suggest the observed flickering of the channels in the presence of targeting peptides represents an interaction of the peptides with the channel rather than translocation events.
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Objectives: We previously screened synthetic organogold compounds for anti-tumor activity in oral... more Objectives: We previously screened synthetic organogold compounds for anti-tumor activity in oral carcinoma cells. Here we aimed to study these compounds’ mechanism of action. Based on our previous results, we hypothesized that the organogold compounds acted on mitochondria and induced opening of the mitochondrial apoptosis-induced channel (MAC) or the mitochondrial permeability transition pore (mPTP), which are located in the mitochondrial outer and inner membranes, respectively. Opening of either channel commonly results in release of factors from the mitochondrial intermembrane space into the cytosol, which causes cell death. Methods: Organogold compounds IV and V induced death in Bak-/-Bax-/- double-knockout (DKO) and parental mouse embryonic fibroblasts (MEF). Bax and Bak are integral components of MAC. The cytotoxicity of the two compounds on both cell lines was assessed using alamar blue viability assays. The compounds’ mechanism in causing cell death in the parental cells wa...
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Molecular Biology of Mitochondrial Transport Systems, 1994
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Journal of bioenergetics and biomembranes, 2000
All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in the cy... more All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in the cytoplasm and imported into the organelle. Water-filled channels are integral to the process of translocating proteins since channels can provide an aqueous pathway through the hydrophobic environment of the membrane. The MCC (multiple conductance channel) and PSC (peptide-sensitive channel) are two high-conductance channels previously identified in electrophysiological studies of mitochondrial membranes. MCC and PSC are the putative pores of the import complexes of the inner and outer membranes, respectively. The genetic, biochemical, and biophysical evidence regarding these assignments are summarized herein. These findings support the identification of MCC and PSC as the protein import channels of mitochondria.
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Cell Death and Disease, 2013
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Neurobiology, 1996
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Mitochondrion, 2012
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The Journal of Membrane Biology, 1991
Alkalinization of the matrix side of the mitochondrial inner membrane by pH shifts from 6.8 to 8.... more Alkalinization of the matrix side of the mitochondrial inner membrane by pH shifts from 6.8 to 8.3 caused a reversible increase in current of 3.2 +/- 0.2 pA (mean +/- SE, n = 21) at +/- 40 mV measured using patch-clamp techniques. The current increase was reversed in a graded fashion by the addition of Mg2+ as well as a reduction in pH. Detection of single-channel events was done at 0.5, 1 and 2 M KCl. The single-channel amplitude in 0.15 M KCl corresponds to approximately 15 pS. Reversal potentials derived from whole patch currents indicated that the inner mitochondrial membrane was primarily cation selective at pH 6.8 with a PK/PCl = 32 (n = 6). Treatment with alkaline pH (8.3) increased the current and anion permeability (PK/PCl = 16, n = 6). The membrane becomes completely cation selective when low concentrations (12 microM) of the drug propranolol are added. The amphiphilic drugs amiodarone (4 microM), propranolol (70 microM) and quinine (0.6 mM) blocked almost all of the current. The pH-dependent current was also inhibited by tributyltin. These results are consistent with the presence of two pathways in the inner mitochondrial membrane. One is cation selective and generally open and the other is anion selective and induced by alkaline pH. The alkaline pH-activated channel likely corresponds to the inner membrane anion channel postulated by others from suspension studies.
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Journal of Biological Chemistry, 2011
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Journal of Biological Chemistry, 1995
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Journal of Biological Chemistry, 2006
The TIM23 complex mediates import of preproteins into mitochondria, but little is known of the me... more The TIM23 complex mediates import of preproteins into mitochondria, but little is known of the mechanistic properties of this translocase. Here patch clamping reconstituted inner membranes allowed for first time insights into the structure and function of the preprotein translocase. Our findings indicate that the TIM23 channel has "twin pores" (two equal sized pores that cooperatively gate) thereby strikingly resembling TOM, the translocase of the outer membrane. Tim17p and Tim23p are homologues, but their functions differ. Tim23p acts as receptor for preproteins and may largely constitute the preprotein-conducting passageway. Conversely depletion of Tim17p induces a collapse of the twin pores into a single pore, whereas N terminus deletion or C terminus truncation results in variable sized pores that cooperatively gate. Further analysis of Tim17p mutants indicates that the N terminus is vital for both voltage sensing and protein sorting. These results suggest that although Tim23p is the main structural unit of the pore Tim17p is required for twin pore structure and provides the voltage gate for the TIM23 channel.
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FEBS Letters, 2003
Protein import into mitochondria is inhibited by protons (IC(50) pH 6.5). The channels of the imp... more Protein import into mitochondria is inhibited by protons (IC(50) pH 6.5). The channels of the import machinery were examined to further investigate this pH dependence. TOM and TIM23 are the protein translocation channels of the mitochondrial outer and inner membranes, respectively, and their single channel behaviors at various pHs were determined using patch-clamp techniques. While not identical, increasing H(+) concentration decreases the open probability of both TIM23 and TOM channels. The pattern of the pH dependences of protein import and channel properties suggests TIM23 open probability can limit import of nuclear-encoded proteins into the matrix of yeast mitochondria.
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FEBS Letters, 1998
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FEBS Letters, 2010
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Papers by Kathleen Kinnally