Azolectin Liposomes Research Articles

“Force-From-Lipids” Dependence of the MscCG Mechanosensitive Channel Gating on Anionic Membranes

Mechanosensory transduction in Corynebacterium glutamicum plays a major role in glutamate efflux for industrial MSG, whose production depends on the activation of MscCG-type mechanosensitive channels. Dependence of the MscCG channel activation by membrane tension on the membrane lipid content has to date not been functionally characterized. Here, we report the MscCG channel patch clamp recording from liposomes fused with C. glutamicum membrane vesicles as well as from proteoliposomes containing the purified MscCG protein. Our recordings demonstrate that mechanosensitivity of MscCG channels depends significantly on the presence of negatively charged lipids in the proteoliposomes. MscCG channels in liposome preparations fused with native membrane vesicles exhibited the activation threshold similar to the channels recorded from C. glutamicum giant spheroplasts. In comparison, the activation threshold of the MscCG channels reconstituted into azolectin liposomes was higher than the activation threshold of E. coli MscL, which is gated by membrane tension close to the bilayer lytic tension. The spheroplast-like activation threshold was restored when the MscCG channels were reconstituted into liposomes made of E. coli polar lipid extract. In liposomes made of polar lipids mixed with synthetic phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, the activation threshold of MscCG was significantly reduced compared to the activation threshold recorded in azolectin liposomes, which suggests the importance of anionic lipids for the channel mechanosensitivity. Moreover, the micropipette aspiration technique combined with patch fluorometry demonstrated that membranes containing anionic phosphatidylglycerol are softer than membranes containing only polar non-anionic phosphatidylcholine and phosphatidylethanolamine. The difference in mechanosensitivity between C. glutamicum MscCG and canonical MscS of E. coli observed in proteoliposomes explains the evolutionary tuning of the force from lipids sensing in various bacterial membrane environments.

Effect of Membrane Environment on the Ligand-Binding Properties of the Terminal Oxidase Cytochrome bd-I from Escherichia coli.

Cytochrome bd-I is a terminal oxidase of the Escherichia coli respiratory chain. This integral membrane protein contains three redox-active prosthetic groups (hemes b558, b595, and d) and couples the electron transfer from quinol to molecular oxygen to the generation of proton motive force, as one of its important physiological functions. The study was aimed at examining the effect of the membrane environment on the ligand-binding properties of cytochrome bd-I by absorption spectroscopy. The membrane environment was found to modulate the ligand-binding characteristics of the hemoprotein in both oxidized and reduced states. Absorption changes upon the addition of exogenous ligands, such as cyanide or carbon monoxide (CO), to the detergent-solubilized enzyme were much more significant and heterogeneous than those observed with the membrane-bound enzyme. In the native membranes, both cyanide and CO interacted mainly with heme d. An additional ligand-binding site (heme b558) appeared in the isolated enzyme, as was evidenced by more pronounced changes in the absorption in the Soret band. This additional reactivity could also be detected after treatment of E. coli membranes with a detergent. The observed effect did not result from the enzyme denaturation, since reconstitution of the isolated enzyme into azolectin liposomes restored the ligand-binding pattern close to that observed for the intact membranes.

Membrane stiffness is one of the key determinants of E. coli MscS channel mechanosensitivity

Mechanosensitive (MS) channels have an intimate relationship with membrane lipids that underlie their mechanosensitivity. Membrane lipids may influence channel activity by directly interacting with MS channels or by influencing the global properties of the membrane such as elastic area expansion modulus or bending rigidity. Previous work has implicated membrane stiffness as a potential determinant of the mechanosensitivity of E. coli (Ec)MscS. Here we systematically tested this hypothesis using patch fluorometry of azolectin liposomes doped with lipids of increasing elastic area expansion modulus. Increasing dioleoylphosphatidylethanolamine (DOPE) content of azolectin liposomes made it more difficult to activate EcMscS by membrane tension (i.e. increased gating threshold). This effect was exacerbated by stiffer forms of phosphatidylethanolamine such as the branched chain lipid diphytanoylphosphoethanolamine (DPhPE) or the fully saturated lipid distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). Furthermore, a comparison of the branched chain lipid diphytanoylphosphocholine (DPhPC) to the stiffer DPhPE indicated again that it was harder to activate EcMscS in the presence of the stiffer DPhPE. We show that these effects are not due to changes in membrane bending rigidity as the membrane tension threshold of EcMscS in membranes doped with PC18:1 and PC18:3 remained the same, despite a two-fold difference in their bending rigidity. We also show that after prolonged pressure application sudden removal of force in softer membranes caused a rebound reactivation of EcMscS and we discuss the relevance of this phenomenon to bacterial osmoregulation. Collectively, our data suggests that membrane stiffness (elastic area expansion modulus) is one of the key determinants of the mechanosensitivity of EcMscS.

The Role of the C-Terminal Domain on the Gating Properties of Corynebacterium Glutamicum Mechanosensitive Channel MscCG

The gram-positive soil bacterium Corynebacterium glutamicum exports a large amount of glutamate through the mechanosensitive channel MscCG. This process is utilized for the world industrial glutamate production. MscCG belongs to a subfamily of bacterial MscS-like channels, which function in osmoregulation, however this channel shows structural and functional differences compared to MscS. To understand the role of the carboxyl terminal domain of MscCG, the chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in E. coli spheroplasts characterized by a lower activation threshold and slow channel closing compared to MscS. The chimeric channel MscS-(C-MscCG) was also successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for the Corynebacterium glutamicum cells. This contribution to the gating hysteresis of the C-terminal domain of MscCG provides the channel with gating properties highly suitable for release of intracellular solutes.

The impact of the C-terminal domain on the gating properties of MscCG from Corynebacterium glutamicum

The mechanosensitive (MS) channel MscCG from the soil bacterium Corynebacterium glutamicum functions as a major glutamate exporter. MscCG belongs to a subfamily of the bacterial MscS-like channels, which play an important role in osmoregulation. To understand the structural and functional features of MscCG, we investigated the role of the carboxyl-terminal domain, whose relevance for the channel gating has been unknown. The chimeric channel MscS-(C-MscCG), which is a fusion protein between the carboxyl terminal domain of MscCG and the MscS channel, was examined by the patch clamp technique. We found that the chimeric channel exhibited MS channel activity in Escherichia coli spheroplasts characterized by a lower activation threshold and slow closing compared to MscS. The chimeric channel MscS-(C-MscCG) was successfully reconstituted into azolectin liposomes and exhibited gating hysteresis in a voltage-dependent manner, especially at high pipette voltages. Moreover, the channel remained open after releasing pipette pressure at membrane potentials physiologically relevant for C. glutamicum. This contribution to the gating hysteresis of the C-terminal domain of MscCG confers to the channel gating properties highly suitable for release of intracellular solutes.

Patch clamp characterization of the effect of cardiolipin on MscS of E. coli.

The bacterial mechanosensitive channels MscS and MscL are gated by an increase in membrane tension when the bacterium experiences hypoosmotic shock. It has been well established that membrane lipids modulate the mechanosensitivity and gating behavior of these channels. The focus of this study is a negatively charged phospholipid, cardiolipin, which has been shown to localize at curved regions of the bacterial cell, including the poles and the septum, and to have a strong preference for binding to membrane proteins. Here we characterize the effect of cardiolipin on MscS, the mechanosensitive channel of small conductance, using patch-clamp electrophysiology. We compare the gating kinetics and mechanosensitivity of the channel in both azolectin and mixtures of pure lipids DOPE/DOPC liposomes with and without cardiolipin. In azolectin liposomes, the addition of 10% cardiolipin abolishes hysteresis of MscS, but MscL remains largely unaffected, indicating that cardiolipin may stabilize the closed state of MscS. On the other hand, mixtures of DOPE/DOPC abolish the hysteresis gating of MscS even in the absence of cardiolipin, and the addition of cardiolipin increases the opening and closing thresholds of both MscS and MscL. In addition, we show that MscS gates more frequently when cardiolipin is present in both the azolectin and pure lipid systems; this dose-dependent effect ultimately destabilizes the open state of MscS and we consider the functional implications of this cardiolipin effect in the bacterial osmotic response. Our results show that cardiolipin modulates the mechanosensitivity and gating characteristics of MscS, indicating its important role in the physiology of bacterial cells.

Patch Clamp Characterisaton of the Effect of Cardiolipin on the Bacterial Mechanosensitive Channels of Small (MscS) and Large (MscL) Conductance

The bacterial mechanosensitive channels MscS and MscL respond to membrane tension by opening when the bacterium experiences hypoosmotic shock conditions to prevent cell lysis [1]. Environmental factors such as cholesterol [2] and cations/anions [3] also affect the gating behaviour of these channels. We have previously shown that addition of the negatively charged lipid cardiolipin to POPE/POPC membranes cause rapid and flickery behaviour of MscS [4]. Here, in an expanded study to include MscL, we compare the gating kinetics and pressure sensitivitiy of the channels with and without cardiolipin in both azolectin and mixtures of pure lipids DOPE/DOPC. In azolectin liposomes, mixtures of 10% cardiolipin abolish hysteresis of MscS, but MscL remains largely unaffected, indicating it may stabilise the closed state of MscS. Compared to the azolectin, mixtures of DOPE/DOPC abolish the hysteresis gating of MscS even in the absence of cardiolipin and addition of cardiolipin increases the opening and closing thresholds of both MscS and MscL. These results suggest that cardiolipin shows a significant effect on the mechanosensitive gating of both MscS and MscL.

Gigaseal mechanics: creep of the gigaseal under the action of pressure, adhesion, and voltage.

Patch clamping depends on a tight seal between the cell membrane and the glass of the pipet. Why does the seal have such high electric resistance? Why does the patch adhere so strongly to the glass? Even under the action of strong hydrostatic, adhesion, and electrical forces, it creeps at a very low velocity. To explore possible explanations, we examined two physical models for the structure of the seal zone and the adhesion forces and two respective mechanisms of patch creep and electric conductivity. There is saline between the membrane and glass in the seal, and the flow of this solution under hydrostatic pressure or electroosmosis should drag a patch. There is a second possibility: the lipid core of the membrane is liquid and should be able to flow, with the inner monolayer slipping over the outer one. Both mechanisms predict the creep velocity as a function of the properties of the seal and the membrane, the pipet geometry, and the driving force. These model predictions are compared with experimental data for azolectin liposomes with added cholesterol or proteins. It turns out that to obtain experimentally observed creep velocities, a simple viscous flow in the seal zone requires ∼10 Pa·s viscosity; it is unclear what structure might provide that because that viscosity alone severely constrains the electric resistance of the gigaseal. Possibly, it is the fluid bilayer that allows the motion. The two models provide an estimate of the adhesion energy of the membrane to the glass and membrane’s electric characteristics through the comparison between the velocities of pressure-, adhesion-, and voltage-driven creep.

Energetics of gating MscS by membrane tension in azolectin liposomes and giant spheroplasts

Mechanosensitive (MS) ion channels are molecular sensors that detect and transduce signals across prokaryotic and eukaryotic cell membranes arising from external mechanical stimuli or osmotic gradients. They play an integral role in mechanosensory responses including touch, hearing, and proprioception by opening or closing in order to facilitate or prevent the flow of ions and organic osmolytes. In this study we use a linear force model of MS channel gating to determine the gating membrane tension (γ) and the gating area change (ΔA) associated with the energetics of MscS channel gating in giant spheroplasts and azolectin liposomes. Analysis of Boltzmann distribution functions describing the dependence of MscS channel gating on membrane tension indicated that the gating area change (ΔA) was the same for MscS channels recorded in both preparations. The comparison of the membrane tension (γ) gating the channel, however, showed a significant difference between the MscS channel activities in these two preparations.

Patch-Clamp Characterization of the MscS-like Mechanosensitive Channel from Silicibacter pomeroyi

Based on sequence similarity, the sp7 gene product, MscSP, of the sulfur-compound-decomposing Gram-negative marine bacterium Silicibacter pomeroyi belongs to the family of MscS-type mechanosensitive channels. To investigate MscSP channel properties, we measured its response to membrane tension using the patch-clamp technique on either a heterologous expression system using giant spheroplasts of MJF465 Escherichia coli strain (devoid of mechanosensitive channels MscL, MscS, and MscK), or on purified MscSP protein reconstituted in azolectin liposomes. These experiments showed typical pressure-dependent gating properties of a stretch-activated channel with a current/voltage plot indicating a rectifying behavior and weak preference for anions similar to the MscS channel of E. coli. However, the MscSP channel exhibited functional differences with respect to conductance and desensitization behavior, with the most striking difference between the two channels being the lack of inactivation in MscSP compared with MscS. This seems to result from the fact that although MscSP has a Gly in an equivalent position to MscS (G113), a position that is critical for inactivation, MscSP has a Glu residue instead of an Asn in a position that was recently shown to allosterically influence MscS inactivation, N117. To our knowledge, this study describes the first electrophysiological characterization of an MscS-like channel from a marine bacterium belonging to sulfur-degrading α-proteobacteria.

The Selectivity of MscS is Determined by the Cytoplasmic Domain

The mechanosensitive channel of small conductance (MscS) is a heptameric pressure sensitive channel expressed in the inner membrane of E. coli. This channel possesses three transmembrane helices and a large water-filled cytoplasmic cage which comprises more than 50% of the total protein. This cytoplasmic domain is conserved throughout the MscS channel family and in MscS has been shown to be a dynamic structure with an active role in channel gating. It has also been suggested that the cytoplasmic domain determines the weak anion selectivity exhibited by MscS. In order to address this question this study reconstituted wild type MscS and single residue mutants of the cytoplasmic vestibule in azolectin liposomes (protein:lipid 1:10000). Patch clamp recordings of these channels were then performed in the presence of asymmetric solutions of KCl (600/200 mM) and BaCl2 (50/200 mM). The MscS mutants studied were R184E, R185E, E187R and E227A. Both E187R and E227A mutants show reduced selectivity characterised by a lower anion-cation permeability ratio. These residues are likely to determine selectivity by binding cations. From these data it is clear that charged residues in the cytoplasmic domain of MscS determine its selectivity. This is interesting because unlike K+, Na+ and Ca2+ channels the selectivity of MscS is not determined by residues in the pore region but residues situated in the large water-filled cytoplasmic domain.

A Mechanosensitive Channel (MscS) with Multiple Conducting States Shows Stronger Selectivity for Anions in the Presence of Divalent Cations

The mechanosensitive channel of small conductance (MscS) in E. coli plays an integral role in osmoprotection[1]. The channel gates in response to an increase in membrane tension as a result of hypo-osmotic shock and acts as an ‘emergency release valve’ preventing cell lysis. Very little is known about the permeation of divalent cations through MscS. This was addressed by reconstituting MscS into azolectin liposomes (protein:lipid - 1:10,000) using the dehydration-rehydration method as previously described[2]. Single-channel recordings were performed in symmetrical 100 mM BaCl2, CaCl2, and MgCl2. At least 5 distinct conducting states were present at +50 mV pipette potential. A further increase in pipette potential to +70 mV resulted in 8 distinct conducting states which were conserved up to the highest voltage tested (+150 mV). It is not known whether these sub-conducting states are physiologically relevant. In addition to these sub-conducting states the substitution of K+ by Ba2+, Ca2+ and Mg2+ increased MscS rectification at negative pipette potentials. The rectification increased as the hydrated ionic radius of the major permeating cation increased (Mg2+ = Ca2+ = Ba2+ > Li+>Na+>K+>Rb+>Cs+). It was hypothesised that divalent cations interact and screen electronegative charges surrounding the portals of the cytoplasmic domain of MscS. If this were the case then divalent cations would increase the anion selectivity of MscS. In line with this prediction in asymmetric BaCl2 the calculated permeability ratio was PCl/PBa = ∼11.5 as opposed to PCl/PK = ∼3.5 in asymmetric KCl. The significance of the effects of divalent ions on MscS gating and ion selectivity is unknown at present, however they may give us a new insight into the global gating mechanism of MscS and importance of calcium in osmoregulation of bacterial cells.

Influence of cholesterol on the formation of palmitate/Ca2+-activated pores in mitochondria and liposomes

The influenc of cholesterol on the formation of the mitochondrial cyclosporin A (CsA)-insensitive palmitate/Ca(2+)-activated pore has been studied. It has been established that increasing the cholesterol level in mitochondrial membranes results in an increase in the of rate of mitochondrial swelling induced by palmitic acid (> or = 20 microM) and Ca2+ (30 microM). This effect is not related to changes in the functional activity of organelles since cholesterol did not influence the mitochondrial respiration in different metabolic states. At the same time, it was shown that the palmitate/Ca(2+)-induced permeabilization of cholesterol-containing azolectin liposomes was Stronger than that of azolectin liposomes. It was found that, in the liposomal membrane, the Ca(2+)-induced phase separation of palmitic acid into distinct membrane domains takes place. The presence of cholesterol in membranes increases the extent of segregation.

Rapid And Efficient Co-reconstitution Of Bacterial Mechanosensitive Ion Channels Of Small And Large Conductance Into Liposomes

Bacterial mechanosensitive (MS) channels protect bacterial cells from osmotic shock, acting as emergency relief valves (1,2). E. coli has three such channels, the MS channel of large conductance (MscL), the MS channel of small conductance (MscS) and the MS channel of mini conductance (MscM). Both MscL and MscS have been extensively studied using the patch-clamp technique in giant spheroplasts (3,4). However, only MscL incorporates efficiently in liposomes (2,5). Here we report the first example of co-reconstitution of both MscS and MscL into azolectin liposomes. We also report reconstitution efficiencies of both proteins into liposomes of different lipid composition and using different incorporation methods.View Large Image | View Hi-Res Image | Download PowerPoint Slide1. Martinac, B., J. Cell Sci., 117, 2449-2460 (2004).2. Perozo, E., Nat. Rev. Mol. Biol., 7, 109-119 (2006).3. Martinac, B., Buechner, M., Delcour, A.H., Adler, J., Kung, C. PNAS, 84, 2297-2301 (1987).4. Booth, I.R., Edwards, M.D., Black, S., Schumann, U., Miller, S. Nat. Rev. Microbiol., 5, 431-440 (2007).5. Hase, C.C., Le Dain, A.C., Martinac, B., J Biol. Chem, 270, 18329-18334 (1995).Supported by the Australian Research Council and National Health & Medical Research Council.

The effects of parabens on the mechanosensitive channels of E. coli

Parabens are alkyl esters of p-hydroxybenzoic acid used as preservatives in a wide range of food, pharmaceutical, and cosmetic products. Despite their common use for over 50 years, their mechanism of action is still unclear. In this study we examined the effects of ethyl and propyl paraben, on gating of the E. coli mechanosensitive channel of large conductance (MscL) reconstituted into azolectin liposomes. We found that propyl and ethyl paraben spontaneously activate MscL. Moreover, the addition of propyl paraben caused an increase in MscL activity and the lowering of p(1/2), the pressure at which the MscL was opened 50% of the time, the DeltaG(o), the free energy required to open the MscL, and the parameter alpha, which describes the channel sensitivity to pressure. In addition, in silico studies showed that propyl paraben binds to the channel gate of the MscL. The mechanosensitive channel of small conductance was also found to be spontaneously activated by parabens. In summary, our study indicates that one of the previously unidentified mechanisms of action of parabens as antimicrobial agents is via an interaction with the mechanosensitive channels to upset the osmotic gradients in bacteria.

Formation of palmitic acid/Ca2+ complexes in the mitochondrial membrane: a possible role in the cyclosporin-insensitive permeability transition.

A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter. With time, the potential is restored. Evidence has been obtained indicating that the specific content of mitochondrial lipids would favor the PA/Ca2+ -induced permeabilization of the membrane. In experiments with liposomes, the PA/Ca2+ -induced membrane permeabilization was larger for liposomes formed from the mitochondrial lipids, as compared to the azolectin liposomes. Additionally, it has been found that in mitochondria of the TNF (tumor necrosis factor)-sensitive cells (WEHI-164 line), the content of PA is larger than in mitochondria of the TNF-insensitive cells (C6 line), with this difference being mainly provided by PA incorporated in phosphatidylethanolamine and especially, cardiolipin. The PA/Ca2+ -dependent mechanism of permeability transition in mitochondria might be related to some pathologies, e.g. myocardial ischemia. The heaviness of myocardial infarction of ischemic patients has been demonstrated to correlate directly with the content of PA in the human blood serum.

Reincorporated plasma membrane Ca2+-ATPase can mediate B-Type Ca2+ channels observed in native membrane of human red blood cells.

Recently, we reported indirect evidence that plasma membrane Ca2+-ATPase (PMCA) can mediate B-type Ca2+ channels of cardiac myocytes. In the present study, in order to bring more direct evidence, purified PMCA from human red blood cells (RBC) was reconstituted into giant azolectin liposomes amenable to the patch-clamp technique. Purified RBC PMCA was used because it is available pure in larger quantity than cardiac PMCA. The presence of B-type Ca2+ channels was first investigated in native membranes of human RBC. They were detected and share the characteristics of cardiac myocytes. They spontaneously appeared in scarce short bursts of activity, they were activated by chlorpromazine (CPZ) with an EC50 of 149 mmole/l or 1 mmole/l vanadate, and then switched off by 10 mmole/l eosin or dose-dependently blocked by 1-5 mmole/l ATP. Independent of membrane potential, the channel gating exhibited complex patterns of many conductance levels, with three most often observed conductance levels of 22, 47 and 80 pS. The activation by vanadate suggests that these channels could play a role in the influx of extracellular Ca2+ involved in the vanadate-induced Gardos effect. In PMCA-reconstituted proteoliposomes, nearly half of the ATPase activity was retained and clear "channel-like" openings of Ba2+- or Ca2+-conducting channels were detected. Channel activity could be spontaneously present, lasting the patch lifetime or, when previously quiescent, activity could be induced by application of 50 mmole/l CPZ only in presence of 25 U/ml calmodulin (CaM), or by application of 1 mmole/l vanadate alone. Eosin (10 mmole/l) and ATP (5 mmole/l) significantly reduced spontaneous activity. Channel gating characteristics were similar to those of RBC, with main conductance levels of 21, 40 and 72 pS. The lack of direct activation by CPZ alone might be attributed to a purification-induced modification or absence of unidentified regulatory component(s) of PMCA. Despite a few differences in results between RBC and reincorporated PMCA, most probably attributable to the decrease in ATPase activity following the procedure of reincorporation, the present experimental conditions appear to reveal a channel-mode of the PMCA that shares many similarities with the B-type Ca2+ channel.

Voltage-dependent Porin-like Ion Channels in the Archaeon Haloferax volcanii

Membrane vesicles isolated from the cell envelope of the archaebacterium Haloferax volcanii were either reconstituted in giant liposomes and examined by the patch-clamp technique or were fused into planar lipid bilayers. In addition, cell envelope proteins were solubilized by detergent and reconstituted in azolectin liposomes, which were then fused into planar lipid bilayers. Independently of the technique used the predominant channel activity encountered exhibited the following characteristics. Channels were open at all voltages in the range approximately -120 to +120 mV and exhibited frequent fast transitions to closed levels of different amplitudes. At voltages greater than 120 mV the channels tended to close in a manner characterized by large, slow transitions of variable amplitudes. The tendency to close at high membrane potentials was much stronger at one polarity. The channel gating pattern was complex exhibiting a range of subconductances of 10-300 picosiemens in symmetric 100 mM KCl. These electrophysiological characteristics are comparable with those of bacterial and mitochondrial porins, suggesting that the archaeal channels may belong to the general class of porin channels. Some channels showed preference for K+, whereas the others preferred Cl-, suggesting the existence of at least two types of porin-like channels in H. volcanii.

Measurement of membrane potential and [Ca2+]i in cell ensembles: application to the study of glutamate taste in mice

We have studied the spectral properties of the voltage-sensitive dye, 1-(3-sulfonatopropyl)-4-[beta [2-(di-n-octylamino)-6-naphtyl]vinyl] pyridinium betaine (di-8-ANEPPS), and the Ca(2+)-sensitive dye, fura-2, in azolectin liposomes and in isolated taste buds from mouse. We find that the fluorescence excitation spectra of di-8-ANEPPS and fura-2 are largely nonoverlapping, allowing alternate ratio measurements of membrane potential and intracellular calcium ([Ca2+]i). There is a small spillover of di-8-ANEPPS fluorescence at the excitation wavelengths used for fura-2 (340 and 360 nm). However, voltage-induced changes in the fluorescence of di-8-ANEPPS, excited at the fura-2 wavelengths, are small. In addition, di-8-ANEPPS fluorescence is localized to the membrane, whereas fura-2 fluorescence is distributed throughout the cytoplasm. Because of this, the effect of spillover of di-8-ANEPPS fluorescence in the [Ca2+]i estimate is < 1%, under the appropriate conditions. We have applied this method to study of the responses of multiple taste cells within isolated taste buds. We show that membrane potential and [Ca2+]i can be measured alternately in isolated taste buds from mouse. Stimulation with glutamate and glutamate analogs indicates that taste cells express both metabotropic and ionotropic receptors. The data suggest that the receptors responding to 2-amino-4-phosphonobutyrate (L-AP4), presumably metabotropic L-glutamate receptors, do not mediate excitatory glutamate taste responses.

Ion Channels in the Chloroplast Envelope Membrane

Isolated chloroplast envelope membranes were fused with azolectin liposomes. Ion transport across the membrane of these liposomes was investigated by the patch-clamp technique and in planar bilayers. Our results show that the chloroplast envelope contains voltage-dependent anion- and cation-selective channels as well as anion- and cation-selective pores with high conductances. At least one of the high-conductance pores could be located in the chloroplast outer envelope membrane. The low-conductance chloride channel and the potassium channel showed complex gating behavior with subconductant states. Potassium channel gating was affected by monovalent and divalent cations as well as by millimolar concentrations of ATP. Low concentrations of Cs+ induced a flickering block. Voltage dependence of the open probability reveals that macroscopic currents of potassium channels are rectified with preferential potassium uptake into the chloroplast. Flux measurements and determinations of the stroma pH of intact chloroplasts confirm the presence of a potassium channel that is regulated by divalent cations (Mg2+) and by ATP. The fully open potassium channel revealed a conductance of lambda approximately equal to 100 pS in asymmetric KCl (250/20 mM KCl), and the fully open chloride channel revealed a conductance of lambda approximately equal to 60 ps in 100 mM Tris/HCl. One high-conductance channel, mainly active at holding potentials > 60 mV, was slightly selective for glutamate anions (PK+/PGlu- approximately equal to 2) and revealed fast voltage-dependent gating. This high-conductance channel had a conductance of lambda approximately equal to 540 pS (in 250/20 mM potassium glutamate) and was closed most of the time. A second type of high-conductance channel, mainly open and active at holding potentials below 30 mV, was slightly selective for cations (PGlu-/PK+ approximately equal to 2) with a conductance of lambda approximately equal to 1.14 nS (in 250/20 mM potassium glutamate).