Voltage activation of heart inner mitochondrial membrane channels

The patch clamp records obtained from mitoplast membranes prepared in the presence of a calcium chelator generally lack channel activity. However, multiconductance channel (MCC) activity can be induced by membrane potentials above ±60mV [Kinnallyet al., Biochem. Biophys. Res. Commun. 176, 1183–1188 (1991)]. Once activated, the MCC activity persists at all voltages. The present report characterizes the activation by voltage of multiconductance channels of rat heart inner mitochondrial membranes using patch-clamping. In some membrane patches, the size of single current transitions progressively increases with time upon application of voltage. The inhibitor cyclosporin has also been found to decrease channel conductance in steps. The results suggest that voltage-induced effects which are inhibited by cyclosporin Aare likely to involve either an increase in effective pore diameter or the assembly of low-conductance units. In activated patches, we have found at high membrane potentials (e.g., 130 mV) changes in conductance as high as 5 nS occurring in large steps (up to 2.7 nS). These were generally preceded by a smaller transition. Similar results were obtained less frequently at lower voltages. These results can be explained on the assumption that once assembled the channels may act in unison.     

Selective effect of inhibitors on inner mitochondrial membrane channels.

The effect of amphiphilic cationic drugs on the channel activity of the mitochondrial inner membrane was examined with patch-clamp techniques. The therapeutic drugs amiodarone, propranolol and quinine reduced the probability of being open for the multiconductance channel (MCC) activity (levels from 30 pS to over 1 nS). While amiodarone decreased the probability of being open for the voltage dependent approximately 100 pS channel, it increased the conductance 42 +/- 20% (mean +/- SD, n = 6) with no significant change in mean open time. Similar results were obtained with propranolol. These data indicate that the approximately 100 pS channel is distinct from MCC activity.     

Calcium modulation of mitochondrial inner membrane channel activity.

Protocols were defined that enable patch-clamp studies of the approximately 107 pS voltage dependent channel and a class of activity we refer to as MCC (multiconductance channel) which is characterized by multiple levels and transitions as high as 1 to 1.5 nS. If free calcium was kept at 10(-7) M or lower during mitochondrial isolation, no activity was observed at low voltage (+/- 60 mV). If free calcium levels were higher, MCC activity was observed in about 96% of the patches. The observation of approximately 107 pS channel was enhanced from 2% to 68% of patches by washing isolated mitoplasts (mitochondria stripped of outer membrane) with EGTA. Increasing matrix calcium from 10(-9) to 10(-5) M decreased the probability of opening for the MCC and approximately 107 pS activities.     

Estimating the synaptic current in a multiconductance AMPA receptor model

Synaptic transmission starts after the presynaptic neuron has released diffusing neurotransmitters, leading to postsynaptic receptor activation and a postsynaptic current, mostly mediated by glutamatergic (AMPARs) receptors for excitatory neurons. Despite intense experimental and theoretical research, it is still unclear how factors such as the synaptic cleft geometry, the organization, the number and the multiconductance state of receptors, the geometry of postsynaptic density (PSD), and the neurotransmitter release location, shape the mean and the variance of the postsynaptic current and its plastic changes. To estimate the synaptic current amplitude and to account for the stochastic nature of synaptic transmission, we develop a semianalytical method in which we obtain a general expression for the coefficient of variation. The method uses the experimental data about the multiconductance channels. We find that PSD morphological changes can significantly modulate the synaptic current, which is maximally reliable (the coefficient of variation is minimal) for an optimal size of the PSD, that depends on the vesicular release active zone. We show that this optimal PSD size is due to nonlinear phenomena involving the receptor multibinding cooperativity. We conclude that changes in the PSD geometry can sustain a form of synaptic plasticity, independent of a change in the number of receptors.     

Regulation of the mechanosensitive cation channels by ATP and cAMP in leech neurons

Single-channel recordings were used to study the modulation of stretch-activated channels (SACs) by intracellular adenosine nucleotides in identified leech neurons. These channels exhibited two activity modes, spike-like (SL) and multiconductance (MC), displaying different polymodal activation. In the absence of mechanical stimulation, internal perfusion of excised patches with ATP induced robust and reversible activation of the MC but not of the SL mode. The ATP effect on channel activity was dose-dependent within a range of 1 microM-1 mM and was induced at different values of intracellular pH and Ca2+. The non-hydrolyzable ATP analog AMP-PNP, ATP without Mg2+ or ADP also effectively enhanced MC activity. Adenosine mimicked the effect of its nucleotides. At negative membrane potentials, both ATP and adenosine activated the channel. Moreover, ATP but not adenosine induced a flickering block. Addition of cAMP during maximal ATP activation completely and reversibly inhibited the channel, with activation and deactivation times of minutes. However, cAMP alone only induced a weak and rapid channel activation, without inhibitory effects. The expression of these channels in the growth cones of leech neurons, their permeability to Ca2+ and their sensitivity to intracellular cAMP are consistent with a role in the Ca2+ oscillations associated with cell growth.     

Functional reconstitution of mammalian 'chloride intracellular channels' CLIC1, CLIC4 and CLIC5 reveals differential regulation by cytoskeletal actin

Chloride intracellular channels (CLICs) are soluble, signal peptide-less proteins that are distantly related to Omega-type glutathione-S-transferases. Although some CLICs bypass the classical secretory pathway and autoinsert into cell membranes to form ion channels, their cellular roles remain unclear. Many CLICs are strongly associated with cytoskeletal proteins, but the role of these associations is not known. In this study, we incorporated purified, recombinant mammalian CLIC1, CLIC4 and (for the first time) CLIC5 into planar lipid bilayers, and tested the hypothesis that the channels are regulated by actin. CLIC5 formed multiconductance channels that were almost equally permeable to Na(+), K(+) and Cl(-), suggesting that the 'CLIC' nomenclature may need to be revised. CLIC1 and CLIC5, but not CLIC4, were strongly and reversibly inhibited (or inactivated) by 'cytosolic' F-actin in the absence of any other protein. This inhibition effect on channels could be reversed by using cytochalasin to disrupt the F-actin. We suggest that actin-regulated membrane CLICs could modify solute transport at key stages during cellular events such as apoptosis, cell and organelle division and fusion, cell-volume regulation, and cell movement.     

Regulation of Synaptic Transmission by Mitochondrial Ion Channels

Mitochondria are abundant within neuronal presynaptic terminals, where they provide energy for sustained neurotransmitter secretion. Injection of Bcl-xL protein into squid giant presynaptic terminal potentiates neurotransmitter release, while a naturally occurring, proteolytic fragment of BCL-xL causes rundown of synaptic function. The cleaved form of BCL-xL generates large, multiconductance ion channel activity in synaptic mitochondrial outer membranes. A rapid onset of synaptic rundown can also be produced by depriving the synapse of oxygen, and hypoxia also induces large channel activity in mitochondrial outer membranes. Channel activity induced by cleaved BCL-xL or by hypoxia is attenuated by NADH, an inhibitor of the voltage-dependent anion channel (VDAC) of mitochondrial outer membranes. Finally, the large conductances elicited by hypoxia are prevented by the addition of a protease inhibitor that prevents cleavage of BCL-xL. The opposing activities of BCL-xL and its proteolytic fragment may regulate the release of ATP from mitochondria during synaptic transmission.     

Regulation of the mechanosensitive cation channels by ATP and cAMP in leech neurons

Single-channel recordings were used to study the modulation of stretch-activated channels (SACs) by intracellular adenosine nucleotides in identified leech neurons. These channels exhibited two activity modes, spike-like (SL) and multiconductance (MC), displaying different polymodal activation. In the absence of mechanical stimulation, internal perfusion of excised patches with ATP induced robust and reversible activation of the MC but not of the SL mode. The ATP effect on channel activity was dose-dependent within a range of 1 μM-1 mM and was induced at different values of intracellular pH and Ca²⁺. The non-hydrolyzable ATP analog AMP-PNP, ATP without Mg²⁺ or ADP also effectively enhanced MC activity. Adenosine mimicked the effect of its nucleotides. At negative membrane potentials, both ATP and adenosine activated the channel. Moreover, ATP but not adenosine induced a flickering block. Addition of cAMP during maximal ATP activation completely and reversibly inhibited the channel, with activation and deactivation times of minutes. However, cAMP alone only induced a weak and rapid channel activation, without inhibitory effects. The expression of these channels in the growth cones of leech neurons, their permeability to Ca²⁺ and their sensitivity to intracellular cAMP are consistent with a role in the Ca²⁺ oscillations associated with cell growth.     

ATP and ADP modulate a cation channel formed by Hsc70 in acidic phospholipid membranes

Heat shock proteins are molecular chaperones that participate in different cellular processes, particularly the folding and translocation of polypeptides across membranes. In this regard, members of the Hsp70 family of heat shock proteins have been observed in close proximity to cellular membranes. In this study, the direct interaction between Hsc70, which is constitutively expressed in cells, and lipid membranes was investigated. Recombinant Hsc70 was incorporated into artificial lipid bilayers, and a transmembrane ion flow was detected, suggesting the incorporation of an ion pathway. This ion flow was very stable and occurred in well defined, multilevel discrete electrical current events, indicating the formation of a multiconductance ion channel. The Hsc70 channel activity is ATP-dependent and is reversibly blocked by ADP. This channel has cationic selectivity. Thus, Hsc70 can directly interact with lipid membranes to create functionally stable ATP-dependent cationic pathways.     

The heterogeneity of ion channels in chromaffin granule membranes

Chromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic ⁸⁶Rb⁺ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 ± 10 pS, 220 ± 8 pS, 152 ± 8 pS and 13 ± 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 ± 8 pS and 31 ± 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.     

Ion channel activity of the BH3 only Bcl-2 family member, BID.

BID is a member of the BH3-only subgroup of Bcl-2 family proteins that displays pro-apoptotic activity. The NH(2)-terminal region of BID contains a caspase-8 (Casp-8) cleavage site and the cleaved form of BID translocates to mitochondrial membranes where it is a potent inducer of cytochrome c release. Secondary structure and fold predictions suggest that BID has a high degree of alpha-helical content and structural similarity to Bcl-X(L), which itself is highly similar to bacterial pore-forming toxins. Moreover, circular dichroism analysis confirmed a high alpha-helical content of BID. Amino-terminal truncated BIDDelta1-55, mimicking the Casp-8-cleaved molecule, formed channels in planar bilayers at neutral pH and in liposomes at acidic pH. In contrast, full-length BID displayed channel activity only at nonphysiological pH 4.0 (but not at neutral pH) in planar bilayers and failed to form channels in liposomes even under acidic conditions. On a single channel level, BIDDelta1-55 channels were voltage-gated and exhibited multiconductance behavior at neutral pH. When full-length BID was cleaved by Casp-8, it too demonstrated channel activity similar to that seen with BIDDelta1-55. Thus, BID appears to share structural and functional similarity with other Bcl-2 family proteins known to have channel-forming activity, but its activity exhibits a novel form of activation: proteolytic cleavage.     

Can membrane excitation be described without a membrane capacity?

A simplified model of excitation is introduced in which the membrane capacity is ignored. It is shown that: (1) Threshold, action potentials, and strength-duration relation can be reproduced by a membrane without a capacity, even for a very simplified model. (2) The delayed build up of the sodium conductance can mimic a membrane capacity. (3) A constant potential stimulus can be used to reveal the influence of the membrane capacity, eventually combined with a feed back mechanism which reduces the effect of the capacity. (4) The effect of the membrane capacity depends on the ratio between the membrane time constant and the time constant for the fast conductance changes.     

From coiled tubules to a secretory canaliculus: a new model for membrane transformation and acid secretion by gastric parietal cells

The acid-secreting gastric parietal cell has a unique secretory membrane system. This membrane system exists in an inactive (non-secreting) and an active (secreting) form. The current accepted model to explain the transformation events associated with the conversion of the non-secreting membrane to the secreting membrane, and vice versa, invokes membrane recycling of elongated vesicle structures. However, recent studies employing cryopreparation have shown that the non-secreting membrane in these cells is actually a complex network of helically coiled tubules. Here, we present an alternative model to explain how the membrane in parietal cells is activated to secrete HCl.     

Negative co-operativity in clustered receptors as a possible basis for membrane action

A model for the interaction of a membrane receptor with a stimulating ligand is presented. It is assumed that (a) the receptor macromolecules are embedded in the membrane as a close packed two-dimensional cluster and (b) strong negative co-operative interaction occurs among the receptor molecules. The model explains the existence of (a) strong membrane stimulation by fractional ligand occupancy of the receptor; (b) the absence of positively co-operative binding curves for ligand to membrane receptors and (c) it provides a molecular explanation for the existence of “spare receptor”.     

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates.     

Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease

Background

The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system.

Results

In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability.

Conclusions

It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.     

New insights into the structure and hydration of a stratum corneum lipid model membrane by neutron diffraction

The structure and hydration of a stratum corneum (SC) lipid model membrane composed of N-(-hydroxyoctadecanoyl)-phytosphingosine (CER6)/cholesterol (Ch)/palmitic acid (PA)/cholesterol sulfate (ChS) were characterized by neutron diffraction. The neutron scattering length density across the SC lipid model membrane was calculated from measured diffraction peak intensities. The internal membrane structure and water distribution function across the bilayer were determined. The low hydration of the intermembrane space is a major feature of the SC lipid model membrane. The thickness of the water layer in the SC lipid model membrane is about 1 Å at full hydration. For the composition 55% CER6/25% Ch/15% PA/5% ChS, in a partly dehydrated state (60% humidity) and at 32°C, the lamellar repeat distance and the membrane thickness have the same value of 45.6 Å . The hydrophobic region of the membrane has a thickness of 31.2 Å . A decrease of the Ch content increases the membrane thickness. The water diffusion through the SC lipid model multilamellar membrane is a considerably slow process relative to that through phospholipid membranes. In excess water, the membrane hydration follows an exponential law with two characteristic times of 93 and 44 min. At 81°C and 97% humidity, the membrane separates into two phases with repeat distances of 45.8 and 40.5 Å . Possible conformations of CER6 molecules in the dry and hydrated multilayers are discussed.     

Similarity in calcium channel activity of annexin V and matrix vesicles in planar lipid bilayers.

Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation in growing bone, are rich in annexin V. When MVs are fused with planar phospholipid bilayers, a multiconductance Ca2+ channel is formed, with activity essentially identical to that observed when annexin V is delivered to the bilayer with phosphatidylserine liposomes. Ca2+ currents through this channel, from either MV or annexin V liposomes, are blocked by Zn2+, as is Ca2+ uptake by MV incubated in synthetic cartilage lymph. Blockage by Zn2+ was most effective when applied to the side containing the MV or liposomes. ATP and GTP differentially modulated the activity of this channel: ATP increased the amplitude of the current and the number of conductance states; GTP dramatically reduced the number of events and conductance states, leading to well-defined Ca2+ channel activity from either MV or the annexin V liposomes. In the distinctive effects of ATP, GTP, and Zn2+ on the Ca2+ channel activity observed in both the MV and the liposome systems, the common factor was the presence of annexin V. From this we conclude that Ca2+ entry into MV results from the presence of annexin V in these membrane-enclosed structures.     

An implicit membrane generalized born theory for the study of structure, stability, and interactions of membrane proteins

Exploiting recent developments in generalized Born (GB) electrostatics theory, we have reformulated the calculation of the self-electrostatic solvation energy to account for the influence of biological membranes. Consistent with continuum Poisson-Boltzmann (PB) electrostatics, the membrane is approximated as an solvent-inaccessible infinite planar low-dielectric slab. The present membrane GB model closely reproduces the PB electrostatic solvation energy profile across the membrane. The nonpolar contribution to the solvation energy is taken to be proportional to the solvent-exposed surface area (SA) with a phenomenological surface tension coefficient. The proposed membrane GB/SA model requires minor modifications of the pre-existing GB model and appears to be quite efficient. By combining this implicit model for the solvent/bilayer environment with advanced computational sampling methods, like replica-exchange molecular dynamics, we are able to fold and assemble helical membrane peptides. We examine the reliability of this model and approach by applications to three membrane peptides: melittin from bee venom, the transmembrane domain of the M2 protein from Influenza A (M2-TMP), and the transmembrane domain of glycophorin A (GpA). In the context of these proteins, we explore the role of biological membranes (represented as a low-dielectric medium) in affecting the conformational changes in melittin, the tilt of transmembrane peptides with respect to the membrane normal (M2-TMP), helix-to-helix interactions in membranes (GpA), and the prediction of the configuration of transmembrane helical bundles (GpA). The present method is found to perform well in each of these cases and is anticipated to be useful in the study of folding and assembly of membrane proteins as well as in structure refinement and modeling of membrane proteins where a limited number of experimental observables are available.     

Accelerating membrane insertion of peripheral proteins with a novel membrane mimetic model

Characterizing atomic details of membrane binding of peripheral membrane proteins by molecular dynamics (MD) has been significantly hindered by the slow dynamics of membrane reorganization associated with the phenomena. To expedite lateral diffusion of lipid molecules without sacrificing the atomic details of such interactions, we have developed a novel membrane representation, to our knowledge, termed the highly mobile membrane-mimetic (HMMM) model to study binding and insertion of various molecular species into the membrane. The HMMM model takes advantage of an organic solvent layer to represent the hydrophobic core of the membrane and short-tailed phospholipids for the headgroup region. We demonstrate that using these components, bilayer structures are formed spontaneously and rapidly, regardless of the initial position and orientation of the lipids. In the HMMM membrane, lipid molecules exhibit one to two orders of magnitude enhancement in lateral diffusion. At the same time, the membrane atomic density profile of the headgroup region produced by the HMMM model is essentially identical to those obtained for full-membrane models, indicating the faithful representation of the membrane surface by the model. We demonstrate the efficiency of the model in capturing spontaneous binding and insertion of peripheral proteins by using the membrane anchor (γ-carboxyglutamic-acid-rich domain; GLA domain) of human coagulation factor VII as a test model. Achieving full insertion of the GLA domain consistently in 10 independent unbiased simulations within short simulation times clearly indicates the robustness of the HMMM model in capturing membrane association of peripheral proteins very efficiently and reproducibly. The HMMM model will provide significant improvements to the current all-atom models by accelerating lipid dynamics to examine protein-membrane interactions more efficiently.