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...
Introduction: Multi-cellular organisms eliminate damaged cells as part of the normal cell life cy... more Introduction: Multi-cellular organisms eliminate damaged cells as part of the normal cell life cycle by a regulated process of programmed cell death known as apoptosis. In mammals, apoptosis is regulated by the Bcl-2 gene family, which controls loss of outer mitochondrial membrane (OMM) permeability. Two pro-apoptotic Bcl-2 members, Bax and Bak, oligomerize on the OMM forming Mitochondrial Apoptosis Induced Channel (MAC), from which death signals are released, representing the commitment step in apoptosis. Bax and Bak’s role in the induction of apoptosis is well documented. However, recent research suggests Bax and Bak may also play a housekeeping role in mitochondrial morphology. Bax/Bak knockout cells show short, fragmented mitochondria when compared to wildtype cells. (Youle and Karbowski, 2011). L63E cells (Bax/Bak knockouts where Bax is reintroduced with a BH3 domain mutation) result in exposure of the protein’s N-terminal end. With this mutation, Bax still translocates to the ...
Molecular Biology of Mitochondrial Transport Systems, 1994
The presence of channels in the outer mitochondrial membrane has been recognized for some time. I... more The presence of channels in the outer mitochondrial membrane has been recognized for some time. In particular the voltage dependent anion channel (VDAC1 or mitochondrial porin) has been studied most frequently after reconstitution in planar bilayers (e.g. Schein et al., 1976; Colombini, 1979; Benz, 1985). In contrast early information on the inner membrane was primarily derived from permeability studies carried out with mitochondrial suspensions. The use of patch-clamping which allowed the study of channels directly in their native or near-native environment is a recent development. Correlations between these two kinds of studies are now underway. Although a good deal of new information is now available, its interpretation and evaluation in terms of nature and significance of channels to mitochondrial function is far from clear.
All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in thecyt... more All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in thecytoplasm and imported into the organelle. Water-filled channels are integral to the process oftranslocating proteins since channels can provide an aqueous pathway through the hydrophobicenvironment of the membrane. The MCC (multiple conductance channel) and PSC(peptide-sensitive channel) are two high-conductance channels previously identified inelectrophysiological studies
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...
Publisher Summary This chapter describes the third class of membrane transporters, comprised of c... more Publisher Summary 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.
Mitochondria are double membrane organelles whose primary function is to fulfill the energy requi... more Mitochondria are double membrane organelles whose primary function is to fulfill the energy requirements of the cell. This function requires the net transport of large amounts of materials including substrates and adenine nucleotides as well as the import of mitochondrial proteins across the outer and inner membranes. One pathway for the flow of materials is through ion channels. The generally accepted view is that the outer membrane does not provide a permeability barrier for small molecules. This low-selectivity is due, at least in part, to the presence of a large channel called VDAC (voltage dependent anion-selective channel) which is presumed to remain open in situ (Colombini et al., 1979, 1989). The inner membrane, on the other hand, is the site of oxidative phosphorylation and is presumed to require a high electrical resistance for efficient energy coupling. It was therefore somewhat surprising when patch-clamp techniques revealed a variety of channels in the inner membrane as shown in Table 1. This apparent paradox is resolved by keeping in mind that, like in the cell membranes of neurons and muscle, the opening of the different mitochondrial channels is subject to regulation by physiological effectors such as voltage, pH, NADH or divalent cations.
Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from m... more Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane of Neurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.
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...
Introduction: Multi-cellular organisms eliminate damaged cells as part of the normal cell life cy... more Introduction: Multi-cellular organisms eliminate damaged cells as part of the normal cell life cycle by a regulated process of programmed cell death known as apoptosis. In mammals, apoptosis is regulated by the Bcl-2 gene family, which controls loss of outer mitochondrial membrane (OMM) permeability. Two pro-apoptotic Bcl-2 members, Bax and Bak, oligomerize on the OMM forming Mitochondrial Apoptosis Induced Channel (MAC), from which death signals are released, representing the commitment step in apoptosis. Bax and Bak’s role in the induction of apoptosis is well documented. However, recent research suggests Bax and Bak may also play a housekeeping role in mitochondrial morphology. Bax/Bak knockout cells show short, fragmented mitochondria when compared to wildtype cells. (Youle and Karbowski, 2011). L63E cells (Bax/Bak knockouts where Bax is reintroduced with a BH3 domain mutation) result in exposure of the protein’s N-terminal end. With this mutation, Bax still translocates to the ...
Molecular Biology of Mitochondrial Transport Systems, 1994
The presence of channels in the outer mitochondrial membrane has been recognized for some time. I... more The presence of channels in the outer mitochondrial membrane has been recognized for some time. In particular the voltage dependent anion channel (VDAC1 or mitochondrial porin) has been studied most frequently after reconstitution in planar bilayers (e.g. Schein et al., 1976; Colombini, 1979; Benz, 1985). In contrast early information on the inner membrane was primarily derived from permeability studies carried out with mitochondrial suspensions. The use of patch-clamping which allowed the study of channels directly in their native or near-native environment is a recent development. Correlations between these two kinds of studies are now underway. Although a good deal of new information is now available, its interpretation and evaluation in terms of nature and significance of channels to mitochondrial function is far from clear.
All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in thecyt... more All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in thecytoplasm and imported into the organelle. Water-filled channels are integral to the process oftranslocating proteins since channels can provide an aqueous pathway through the hydrophobicenvironment of the membrane. The MCC (multiple conductance channel) and PSC(peptide-sensitive channel) are two high-conductance channels previously identified inelectrophysiological studies
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...
Publisher Summary This chapter describes the third class of membrane transporters, comprised of c... more Publisher Summary 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.
Mitochondria are double membrane organelles whose primary function is to fulfill the energy requi... more Mitochondria are double membrane organelles whose primary function is to fulfill the energy requirements of the cell. This function requires the net transport of large amounts of materials including substrates and adenine nucleotides as well as the import of mitochondrial proteins across the outer and inner membranes. One pathway for the flow of materials is through ion channels. The generally accepted view is that the outer membrane does not provide a permeability barrier for small molecules. This low-selectivity is due, at least in part, to the presence of a large channel called VDAC (voltage dependent anion-selective channel) which is presumed to remain open in situ (Colombini et al., 1979, 1989). The inner membrane, on the other hand, is the site of oxidative phosphorylation and is presumed to require a high electrical resistance for efficient energy coupling. It was therefore somewhat surprising when patch-clamp techniques revealed a variety of channels in the inner membrane as shown in Table 1. This apparent paradox is resolved by keeping in mind that, like in the cell membranes of neurons and muscle, the opening of the different mitochondrial channels is subject to regulation by physiological effectors such as voltage, pH, NADH or divalent cations.
Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from m... more Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane of Neurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.
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Papers by Kathleen Kinnally