Single-channel Behavior Research Articles

Ion Conduction in a Shaker Potassium Channel Mutant Having an Unusually High Single Channel Conductance

K-channels are endowed of a highly K+ selective pore but are very diverse in single channel conductance, ranging from 3 to 300 pS. Because the selectivity filter is so conserved among K-channels, the origin of this diversity must be searched for in other parts of the pore. We studied the single-channel behavior of the Shaker K-channel with the P475D point mutation located at its internal entrance. In 100 mM K+ solution, this variant has a unitary conductance 8-10 fold larger than wild type (Sukhareva et al.

Simulating complex ion channel kinetics with IonChannelLab

In silico simulation based on Markov chains is a powerful way to describe and predict the activity of many transport proteins including ion channels. However, modeling and simulation using realistic models of voltage- or ligand-gated ion channels exposed to a wide range of experimental conditions require building complex kinetic schemes and solving complicated differential equations. To circumvent these problems, we developed IonChannelLab a software tool that includes a user-friendly Graphical User Interface and a simulation library. This program supports channels with Ohmic or Goldman-Hodgkin-Katz behavior and can simulate the time-course of ionic and gating currents, single channel behavior and steady-state conditions. The program allows the simulation of experiments where voltage, ligand and ionic concentration are varied independently or simultaneously.

FKBP12 is a High Affinity, Reversible Activator of RyR2, and FKBP12.6 Antagonises Its Actions

FKBP12.6 binds tightly to RyR2 and evidence suggests that it plays a vital physiological role in regulating channel activity. Moreover, changes in FKBP12.6/RyR2 interactions have been implicated in heart failure. Controversy exists, however, as to how FKBP12.6 affects the single-channel behaviour of RyR2. Furthermore, although higher levels of FKBP12 than FKBP12.6 are present in cardiac cells, the effects of FKBP12 on RyR2 are virtually unresearched. We have therefore compared the effects of FKBP12 and FKBP12.6 on the single-channel function of sheep RyR2 incorporated into bilayers under voltage-clamp conditions. We find that FKBP12 increases RyR2 open probability (Po) in a dose-dependant, reversible manner with an EC50 of 51 nM. In the presence of 10 μM cytosolic Ca2+, physiological levels of FKBP12 (3 μM) increased Po from 0.187±0.051 in controls to 0.657±0.111 (SEM; n=14; P<0.001). In contrast, under identical experimental conditions, FKBP12.6 did not significantly increase or decrease RyR2 Po, however, it was able to antagonise the actions of FKBP12, shifting the EC50 value for FKBP12 to 4 μM. Our experiments demonstrate that FKBP12 has high affinity for RyR2 and that at physiological concentrations (1-3 μM) is an effective activator of the channel thereby suggesting that FKBP12 may have a more important role in cardiac excitation-contraction coupling than previously thought. We hypothesise that FKBP12.6 is a very low efficacy (but high affinity) partial agonist of RyR2 and that the balance between the effects of FKBP12 and FKBP12.6 is crucial for normal EC-coupling in cardiac cells. Supported by the British Heart Foundation

FKBP12 and FKBP12.6 Exert Opposing Actions on the Single-Channel Behaviour of Both RyR1 and RyR2

It is widely believed, that in striated muscle, FKBP12.6 selectively binds to RyR2 but that FKBP12 is the binding partner for RyR1. Few studies have addressed whether FKBP12 can modulate RyR2 function or whether FKBP12.6 can affect RyR1. Our recent single-channel studies show that FKBP12 activates RyR2 with nanomolar affinity but because binding is rapidly reversible, this interaction is unlikely to be detected by Western blot analysis. We have therefore compared how FKBP12 and FKBP12.6 affect the single-channel properties of RyR1 and RyR2 reconstituted into bilayers. Using Ca2+ as the permeant ion, cytosolic addition of 500 nM FKBP12 significantly decreased the open probability (Po) of RyR1 from 0.021 ± 0.005 (SEM; n = 4) in controls to 0.001 ± 0.001 (SEM; n = 4; P<0.05). This effect was irreversible after perfusing away the FKBP12. In contrast, 200 nM and 1 μM FKBP12.6 increased RyR1 Po. This effect was also irreversible after washout of FKBP12.6 but could be antagonised by addition of FKBP12. In comparison, FKBP12 is a reversible activator of RyR2 whereas FKBP12.6 has little intrinsic action itself but can antagonise FKBP12-induced activation. The presence or absence of FKBP12/12.6 did not lead to the appearance of sub-conductance gating states in RyR2 but, after FK-506 treatment or addition of FKBP12.6 to RyR1, sub-conductance states were frequently observed. Since FKBP12 activates RyR2 but is antagonised by FKBP12.6, and FKBP12.6 activates RyR1 but is antagonised by FKBP12, the ratio of FKBP12/FKBP12.6 levels in the cytoplasm will be critical in determining RyR activity in situ. Changes in the cytoplasmic FKBP12/FKBP12.6 ratio may be important in heart failure, where it has been suggested that less FKBP12.6 binds to RyR2.Supported by the British Heart Foundation

Psalmotoxin-1 Docking to Human Acid-sensing Ion Channel-1

Acid-sensing ion channel-1 (ASIC-1) is a proton-gated ion channel implicated in nociception and neuronal death during ischemia. Recently the first crystal structure of a chicken ASIC was obtained. Expanding upon this work, homology models of the human ASICs were constructed and evaluated. Energy-minimized structures were tested for validity by in silico docking of the models to psalmotoxin-1, which potently inhibits ASIC-1 and not other members of the family. The data are consistent with prior radioligand binding and functional assays while also explaining the selectivity of PcTX-1 for homomeric hASIC-1a. Binding energy calculations suggest that the toxin and channel create a complex that is more stable than the channel alone. The binding is dominated by the coulombic contributions, which account for why the toxin-channel interaction is not observed at low pH. The computational data were experimentally verified with single channel and whole-cell electrophysiological studies. These validated models should allow for the rational design of specific and potent peptidomimetic compounds that may be useful for the treatment of pain or ischemic stroke.

Assembly of the Mitochondrial Apoptosis-induced Channel, MAC

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.

Mutant Ryanodine Receptor-dependent Calcium Leak, RyR2 Open Probability, Calcium Sparks And Cardiac Arrhythmogenesis

Ca2+ leak from the sarcoplasmic reticulum (SR) represents a major mechanism underlying arrhythmic disease in the heart. Nevertheless the links between cardiac ryanodine receptor (RyR2) single channel behavior, Ca2+ sparks, and Ca2+ waves remains surprisingly enigmatic. Here we investigate the relationship between a known missense mutation in RyR2 (R2474S) and Ca2+ sparks. R2474S has been shown to cause catecholaminergic polymorphic ventricular tachycardia (CPVT) in humans and knockin mice under conditions of stress and catecholamine exposure. Heterozygous CPVT-mutant RyR2-R2474S/WT (RS/WT) or wild-type (WT) channels were isolated from the hearts of 1) resting, control mice or 2) mice which underwent exercise stress testing (‘stressed’). Using the lipid bilayer method (cytosolic [Ca2+]cis = 150 nM to approximate diastolic concentrations), RyR2-RS/WT from ‘stressed’ mice showed a significant gain-of-function defect as evidenced by increased open probability (Po = 0.134±0.008, n=7) versus WT (0.018±0.008, n=7; P<0.05). When isolated cardiac myocytes were pre-treated with isoproterenol (1 μM) to mimic catecholaminergic stress and following 1 Hz field pacing, fluo-4-AM loaded RS/WT cells showed a significantly increased spark rate (341.8 ±115.7% vs. 108.1±44.6%) when compared to WT cells. These findings suggest that changes in RyR2 Po, Ca2+ sparks and arrhythmogenesis are linked mechanistically. How the ensemble of findings are interrelated dynamically, however, is model-dependent and this modeling will be presented. These findings suggest that our approach to the investigation of Ca2+ dependent arrhythmogenesis broadens understanding of molecular cardiac defects in disease and lays the foundation for the development and testing of novel therapeutic agents.

Validating models of the IP3 receptor: reproduction of experimental data with stochastic simulations

Event Abstract Back to Event Validating models of the IP3 receptor: reproduction of experimental data with stochastic simulations Katri Hituri1*, Stefan Wils2, Marja-Leena Linne1 and Erik De Schutter2 1 Tampere University of Technology, Finland 2 Okinawa Inst. of Science and Technology, Japan Transient rises in cytosolic calcium (Ca2+) concentration play a crucial role in initiating long-term depression (LTD) of synaptic activity, an electrophysiological phenomenon related to learning of motor functions in cerebellar Purkinje cells (PC). Ca2+ release from endoplasmic reticulum (ER) is particularly important in LTD. In PCs, the release is mediated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) that are highly expressed in dendritic spines. The small volume of the spine, as well as the small number of molecules involved, increase stochasticity (randomness) in the biochemical processes. Thus deterministic simulations of IP3R activation may not produce realistic results under all conditions [1]. In this work the behavior of two IP3R models [3,4] is compared to experimental data. The ultimate goal of the work is to find a model that is simple but enough yet succeeds in reproducing experimental data satisfactorily. The models were simulated with the stochastic simulation software STEPS. STEPS (STochastic Engine for Pathway Simulation) is a tool for full stochastic simulation of reaction kinetics, as well as diffusion, of molecules in three dimension. STEPS extends the stochastic simulation algorithm (SSA) described by Gillespie [2]. There exist a couple of studies on characterizing the single-channel behavior of the cerebellar IP3Rs in lipid bilayer. However, the data presented in the articles is quite limited. Information on mean open times of the IP3R, as well as some distributions of the open time times, related to a limited number of concentrations of IP3 and Ca2+, can be obtained from literature. The selected models were simulated to reproduce similar data. The IP3R model of Doi et al. (2005) was found to reproduce the available experimental data more accurately than the model of Fraiman and Dawson (2004). This indicates that the model of Doi et al. would be better choice for modeling IP3Rs in cerebellar PC spines. The results of the present work also indicate that the models for IP3R used in this work might need to be refined structurally and functionally more or less to reproduce correct time series behavior of IP3Rs. This would facilitate a better model to simulate the processes related to LTD induction and information storage in cerebellum.

A Quantitative Description of KcsA Gating II: Single-Channel Currents

The kinetic transitions of proton-activated WT KcsA and the noninactivating E71A mutant were studied at the single-channel level in purified, liposome-reconstituted preparations. Single-channel currents were recorded using patch-clamp techniques under nonstationary and steady-state conditions. Maximum-likelihood analyses reveal that the key influence of acidic pH is to increase the frequency of bursting without an effect on the intraburst open and closed dwell times, consistent with the finding from macroscopic currents that protons promote activation without a significant effect on inactivation. However, in steady-conditions of pH, voltage not only alters the burst frequency but also affects their properties, such as the frequency of the flickers and the dwell times of the closed and open states. This is to be expected if voltage modulates pathways connecting open and inactivated states. Upon opening, KcsA can enter at least two closed states that are not part of the activation pathway. The frequency and duration of these closed states was found to be voltage dependent and therefore these are likely to represent short-lived inactivated states. Single-channel recordings of WT KcsA also show varying propensity for the presence of subconductance states. The probability of occurrence of these states did not show clear modulation by voltage or pH and their origin remains unclear and a focus for further investigation. A kinetic model is proposed to describe the gating events in KcsA that recapitulates its macroscopic and single-channel behavior. The model has been constrained by the single-channel analyses presented in this work along with data from macroscopic currents in the preceding paper.

A Quantitative Description of KcsA Gating I: Macroscopic Currents

The prokaryotic K+ channel KcsA is activated by intracellular protons and its gating is modulated by transmembrane voltage. Typically, KcsA functions have been studied under steady-state conditions, using macroscopic Rb+-flux experiments and single-channel current measurements. These studies have provided limited insights into the gating kinetics of KcsA due to its low open probability, uncertainties in the number of channels in the patch, and a very strong intrinsic kinetic variability. In this work, we have carried out a detailed analysis of KcsA gating under nonstationary conditions by examining the influence of pH and voltage on the activation, deactivation, and slow-inactivation gating events. We find that activation and deactivation gating of KcsA are predominantly modulated by pH without a significant effect of voltage. Activation gating showed sigmoidal pH dependence with a pKa of ∼4.2 and a Hill coefficient of ∼2. In the sustained presence of proton, KcsA undergoes a time-dependent decay of conductance. This inactivation process is pH independent but is modulated by voltage and the nature of permeant ion. Recovery from inactivation occurs via deactivation and also appears to be voltage dependent. We further find that inactivation in KcsA is not entirely a property of the open-conducting channel but can also occur from partially “activated” closed states. The time course of onset and recovery of the inactivation process from these pre-open closed states appears to be different from the open-state inactivation, suggesting the presence of multiple inactivated states with diverse kinetic pathways. This information has been analyzed together with a detailed study of KcsA single-channel behavior (in the accompanying paper) in the framework of a kinetic model. Taken together our data constitutes the first quantitative description of KcsA gating.

Awaking TIM22, a Dynamic Ligand-gated Channel for Protein Insertion in the Mitochondrial Inner Membrane

Aqueous channels are at the core of the translocase of the outer membrane (TOM) and the translocase of the inner membrane for the transport of preproteins (TIM23), the translocases mediating the transport of proteins across the outer and inner mitochondrial membranes. Yet, the existence of a channel associated to the translocase of the inner membrane for the insertion of multitopic protein (TIM22) complex has been arguable, as its function relates to the insertion of multispanning proteins into the inner membrane. For the first time, we report conditions for detecting a channel activity associated to the TIM22 translocase in organelle, i.e. intact mitoplasts. An internal signal peptide in the intermembrane space of mitochondria is a requisite to inducing this channel, which is otherwise silent. The channel showed slightly cationic and high conductance activity of 1000 pS with a predominant half-open substate. Despite their different composition, the channels of the three mitochondrial translocases were thus remarkably similar, in agreement with their common task as pores transiently trapping proteins en route to their final destination. The opening of the TIM22 channel was a step-up process depending on the signal peptide concentration. Interestingly, low membrane potentials kept the channel fully open, providing a threshold level of the peptide is present. Our results portray TIM22 as a dynamic channel solely active in the presence of its cargo proteins. In its fully open conformation, favored by the combined action of internal signal peptide and low membrane potential, the channel could embrace the in-transit protein. As insertion progressed and initial interaction with the signal peptide faded, the channel would close, sustaining its role as a shunt that places trapped proteins into the membrane.

Native TRPC7 Channel Activation by an Inositol Trisphosphate Receptor-dependent Mechanism

In DT40 B lymphocytes, Canonical Transient Receptor Potential 7 (TRPC7) functions as a diacylglycerol-activated non-selective cation channel. However, previous work indicated that the non-store-operated Ca2+ entry in this cell type depends upon inositol trisphosphate receptors (IP3R). With the cell-attached configuration oleyl-acetyl-glycerol (OAG) induced single channel activity (75 pS) that was not observed in TRPC7-/- cells but was rescued by expression of TRPC7 under conditions expected to produce relatively low levels of expression ((LowT7)TRPC7-/-). A DT40 cell line lacking IP3R(IP3R-/- cells) showed no OAG-induced single channel activity, but this activity was rescued by transient expression of an IP3R((IP3R)IP3R-/-). Single channel properties in (LowT7)TRPC7-/- or (IP3R)IP3R-/- DT40 cells were indistinguishable from one another and from wild-type cells. Thus, TRPC7 forms, or is part of, the channel underlying endogenous diacylglycerol-activated currents in DT40 B lymphocytes, and this activity of native TRPC7 requires IP3R. However, with conditions expected to produce greater expression levels, TRPC7 functioned independently of the presence of IP3R. This finding may serve to resolve previously conflicting reports from expression studies of TRPC channels.

Plastic and Lateral-Torsional Buckling Behavior of Single Cold-Formed Channels Connected Back-to-Back

This paper describes the results of a series of experiments on semi-rigid and inelastic joints, and lateral-torsional buckling of the eaves region of portal frames comprising cold-formed single channels bolted back to back at the eaves and apex joints. The tests demonstrate the considerable ductility that is achieved in the back to back bolted connections, which should be sufficient in many cases to accommodate plastic analysis of the portal frames. Variables in the tests include the number of bolts in the connection, the lengths between lateral restraints, the width of the channel flanges, and the strength of the channels. Adjacent column and rafter channels are oriented successively in opposite transverse directions to take advantage of the counterbalancing moments and forces in the back to back connections. This system is important in enhancing the lateral buckling strength of the channels. An innovative method is proposed for connecting each purlin to the web of the column or rafter through a cold-formed angle. Experiments have shown this system to be capable of restraining the frames from failing due to lateral-torsional buckling. This eliminates the idea of having fly bracings, which is normally done in practice to restrain torsional instability.

Large-conductance cholesterol–amphotericin B channels in reconstituted lipid bilayers

The antimycotic activity of amphotericin B (AmB) depends on its ability to make complexes sterols to form ion channels that cause membrane leakage. To study this phenomenon, surface pressure (pi) as a function of surface area (A) and pi-A hysteresis were measured in monolayers of AmB-cholesterol mixtures on the water-air interface. The most stable monolayers were produced from molecules of AmB and cholesterol with 2:1 stoichiometry. At this ratio, AmB and cholesterol interact to form ion channels in lipid bilayers with millisecond dwell times and conductances of 4-400 pS. The AmB-cholesterol complexes assemble in three, four, etc., subunit aggregates to form ion channels of diverse and large-conductances. Their I-V characteristics were linear over a range of +/-200 mV. The channel currents were inhibited by the addition of tetraethylammonium (TEA), potassium channel blocker, to the cis-side of the membrane. Likewise, AmB-cholesterol complexes reconstituted in membrane-coated nanoporous silicon dioxide surfaces showed single channel behavior with large amplitudes at various voltages. Large-conductance ion channels show great promise for use in biosensors on solid supports.

Influence of agonist concentration on AMPA and kainate channels in CA1 pyramidal cells in rat hippocampal slices

We have determined the functional properties of single AMPA receptor (AMPAR) and kainate receptor channels present in CA1 cells in hippocampal slices, to shed light on the relationship between single-channel behaviour and synaptic currents in these cells. To derive basic properties of AMPA and kainate channels activated by their excitatory transmitter, we examined outside-out patches exposed to glutamate. The kainate agonist SYM 2081, was used to confirm the presence of kainate receptors. Channels activated by glutamate or SYM 2081 exhibited conductance levels of 2-20 pS. Properties of single channels depended on the glutamate or AMPA concentration used. We observed a marked increase in mean channel conductance (gamma) from gamma = 6.9, to 11.2 pS, when glutamate was increased from 10 mum to 10 mm. The kinetic behaviour of AMPAR channels was also influenced by agonist concentration, with an increase in 'bursty' events at higher concentrations. In contrast, kainate channels were characterized by brief openings without bursts. Consistent with the view that 'bursty' events arose from AMPARs, these openings decreased in the presence of the AMPAR blocker GYKI 53655. Furthermore, our experiments revealed a concentration-dependent increase in the number of conductance states during an individual AMPAR opening; AMPAR channels displayed up to four distinct levels. Our results are consistent with the view that the AMPAR channel conductance depends on the number of transmitter molecules bound in CA1 cells. We consider the implications of these findings for the change in EPSC properties during long-term potentiation (LTP).

Modeling of Single Noninactivating Na+ Channels: Evidence for Two Open and Several Fast Inactivated States

Voltage-gated Na+ channels play a fundamental role in the excitability of nerve and muscle cells. Defects in fast Na+ channel inactivation can cause hereditary muscle diseases with hyper- or hypoexcitability of the sarcolemma. To explore the kinetics and gating mechanisms of noninactivating muscle Na+ channels on a molecular level, we analyzed single channel currents from wild-type and five mutant Na+ channels. The mutations were localized in different protein regions which have been previously shown to be important for fast inactivation (D3-D4-linker, D3/S4-S5, D4/S4-S5, D4/S6) and exhibited distinct grades of defective fast inactivation with varying levels of persistent Na+ currents caused by late channel reopenings. Different gating schemes were fitted to the data using hidden Markov models with a correction for time interval omission and compared statistically. For all investigated channels including the wild-type, two open states were necessary to describe our data. Whereas one inactivated state was sufficient to fit the single channel behavior of wild-type channels, modeling the mutants with impaired fast inactivation revealed evidence for several inactivated states. We propose a single gating scheme with two open and three inactivated states to describe the behavior of all five examined mutants. This scheme provides a biological interpretation of the collected data, based on previous investigations in voltage-gated Na+ and K+ channels.

K Channel Subconductance Levels Result from Heteromeric Pore Conformations

Voltage-gated K channels assemble from four identical subunits symmetrically arranged around a central permeation pathway. Each subunit harbors a voltage-sensing domain. The sigmoidal nature of the activation kinetics suggests that multiple sensors need to undergo a conformational change before the channel can open. Following activation, individual K channels alternate stochastically between two main permeation states, open and closed. This binary character of single channel behavior suggests the presence of a structure in the permeation pathway that can exist in only two conformations. However, single channel analysis of drk1 (Kv2.1) K channels demonstrated the existence of four additional, intermediate conductance levels. These short-lived subconductance levels are visited when the channel gate moves between the closed and fully open state. We have proposed that these sublevels arise from transient heteromeric pore conformations, in which some, but not all, subunits are in the “open” state. A minimal model based on this hypothesis relates specific subconductance states with the number of activated subunits (Chapman et al., 1997). To stringently test this hypothesis, we constructed a tandem dimer that links two K channel subunits with different activation thresholds. Activation of this dimer by strong depolarizations resulted in the characteristic binary open–close behavior. However, depolarizations to membrane potentials in between the activation thresholds of the two parents elicited highly unusual single channel gating, displaying frequent visits to two subconductance levels. The voltage dependence and kinetics of the small and large sublevels associate them with the activation of one and two subunits, respectively. The data therefore support the hypothesis that subconductance levels result from heteromeric pore conformations. In this model, both sensor movement and channel opening have a subunit basis and these processes are allosterically coupled.

The Role of the Mitochondrial Apoptosis Induced Channel MAC in Cytochrome c Release

Permeabilization of the mitochondrial outer membrane is a crucial event during apoptosis. It allows the release of proapoptotic factors, like cytochrome c, from the intermembrane space, and represents the commitment step in apoptosis. The mitochondrial apoptosis-induced channel, MAC, is a high-conductance channel that forms during early apoptosis and is the putative cytochrome c release channel. Unlike activation of the permeability transition pore, MAC formation occurs without loss of outer membrane integrity and depolarization. The single channel behavior and pharmacology of reconstituted MAC has been characterized with patch-clamp techniques. Furthermore, MAC's activity is compared to that detected in mitochondria inside the cells at the time cytochrome c is released. Finally, the regulation of MAC by the Bcl-2 family proteins and insights concerning its molecular composition are also discussed.

Fifty Years of Progress in Ion Channel Research

Fifty years ago, ion channels were but a reasonable hypothesis. I outline some major steps in transforming this idea from a plausible description of the biological assemblies responsible for controlling passive ion transport across membranes to established fact. Important electrophysiological, biochemical, molecular biological, structural, and theoretical tools are discussed in the context of the transition from studying whole cell preparations, containing many channels, to investigating single channel behavior. Six channel families are exemplified: the model peptide, gramicidin, the acetylcholine receptor, and the sodium, potassium, calcium, and chloride channels. Some questions of current interest are posed.

Oligomeric Bax Is a Component of the Putative CytochromecRelease Channel MAC, Mitochondrial Apoptosis-induced Channel

Bcl-2 family proteins regulate apoptosis, in part, by controlling formation of the mitochondrial apoptosis-induced channel (MAC), which is a putative cytochrome c release channel induced early in the intrinsic apoptotic pathway. This channel activity was never observed in Bcl-2-overexpressing cells. Furthermore, MAC appears when Bax translocates to mitochondria and cytochrome c is released in cells dying by intrinsic apoptosis. Bax is a component of MAC of staurosporine-treated HeLa cells because MAC activity is immunodepleted by Bax antibodies. MAC is preferentially associated with oligomeric, not monomeric, Bax. The single channel behavior of recombinant oligomeric Bax and MAC is similar. Both channel activities are modified by cytochrome c, consistent with entrance of this protein into the pore. The mean conductance of patches of mitochondria isolated after green fluorescent protein-Bax translocation is significantly higher than those from untreated cells, consistent with onset of MAC activity. In contrast, the mean conductance of patches of mitochondria indicates MAC activity is present in apoptotic cells deficient in Bax but absent in apoptotic cells deficient in both Bax and Bak. These findings indicate Bax is a component of MAC in staurosporine-treated HeLa cells and suggest Bax and Bak are functionally redundant as components of MAC.