pH regulates cation selectivity of poly-(R)-3-hydroxybutyrate/polyphosphate channels from E. coli in planar lipid bilayers

Poly-(R)-3-hydroxybutyrate/polyphosphate (PHB/polyP) complexes, whether isolated from the plasma membranes of bacteria or prepared from the synthetic polymers, form ion channels in planar lipid bilayers that are highly selective for Ca(2+) over Na(+) at physiological pH. This preference for divalent over monovalent cations is attributed to a high density of negative charge along the polyP backbone and the higher binding energies of divalent cations. Here we modify the charge density of polyP by varying the pH, and observe the effect on cation selectivity. PHB/polyP complexes, isolated from E. coli, were incorporated into planar lipid bilayers, and unitary current-voltage relations were determined as a function of pH. When Ca(2+) was the sole permeant cation, conductance diminished steadily from 97 +/- 6 pS at pH 7.4 to 47 +/- 3 pS at pH 5.5. However, in asymmetric solutions of Ca(2+) and Na(+), there was a moderate increase in conductance from 98 +/- 4 at pH 7.4 to 129 +/- 4 pS at pH 6.5, and a substantially larger increase to 178 +/- 6 pS at pH 5.6, signifying an increase in Na(+) permeability or disorganization of channel structure. Reversal potentials point to a sharp decrease in preference for Ca(2+) over Na(+) over a relatively small decrease in pH. Ca(2+) was strongly favored over Na(+) at physiological pH, but the channels became nonselective near the pK(2) of phosphate (approximately 6.8), and displayed weak selectivity for Na(+) over Ca(2+) at acidic pH. Evidently, PHB/polyP complexes are versatile ion carriers whose selectivity may be modulated by small adjustments of the local pH. The results may be relevant to the physiological function of PHB/polyP channels in bacteria and the role of PHB and polyP in the Streptomyces lividans potassium channel.     

Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA

Here we present functional evidence for involvement of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP) in ion conduction and selection at the intracellular side of the Streptomyces lividans potassium channel, KcsA. At < or = 25 degrees C, KcsA forms channels in planar bilayers that display signal characteristics of PHB/polyP channels at the intracellular side; i.e., a preference for divalent Mg(2+) cations at pH 7.2, and a preference for monovalent K+ cations at pH 6.8. Between 25 and 26 degrees C, KcsA undergoes a transition to a new conformation in which the channel exhibits high selectivity for K+, regardless of solution pH. This suggests that basic residues of the C-terminal polypeptides have moved closer to the polyP end unit, reducing its negative charge. The data support a supramolecular structure for KcsA in which influx of ions is prevented by the selectivity pore, whereas efflux of K+ is governed by a conductive core of PHB/polyP in partnership with the C-terminal polypeptide strands.     

A high-conductance mode of a poly-3-hydroxybutyrate/calcium/polyphosphate channel isolated from competent Escherichia coli cells

Reconstitution into planar lipid bilayers of a poly-3-hydroxybutyrate/calcium/polyphosphate (PHB/Ca(2+)/polyP) complex from Escherichia coli membranes yields cationic-selective, 100 pS channels (Das, S., Lengweiler, U.D., Seebach, D. and Reusch, R.N. (1997) Proof for a non-proteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate. Proc. Natl. Acad. Sci. USA 94, 9075-9079). Here, we report that this complex can also form larger, weakly selective pores, with a maximal conductance ranging from 250pS to 1nS in different experiments (symmetric 150mM KCl). Single channels were inhibited by lanthanum (IC(50)=42+/-4microM, means+/-S.E.M.) with an unusually high Hill coefficient (8.4+/-1.2). Transition to low-conductance states (<250pS) was favored by increased membrane polarization (/V/ >or=50mV). High conductance states (>250pS) may reflect conformations important for genetic transformability, or "competence", of the bacterial cells, which requires the presence of the PHB/Ca(2+)/polyP complex in the membrane.     

Outer membrane protein A of Escherichia coli forms temperature-sensitive channels in planar lipid bilayers

The temperature dependence of single-channel conductance and open probability for outer membrane protein A (OmpA) of Escherichia coli were examined in planar lipid bilayers. OmpA formed two interconvertible conductance states, small channels, 36-140 pS, between 15 and 37 degrees C, and large channels, 115-373 pS, between 21 and 39 degrees C. Increasing temperatures had strong effects on open probabilities and on the ratio of large to small channels, particularly between 22 and 34 degrees C, which effected sharp increases in average conductance. The data infer that OmpA is a flexible temperature-sensitive protein that exists as a small pore structure at lower temperatures, but refolds into a large pore at higher temperatures.     

Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli.

The lipidic polymer, poly-3-hydroxybutyrate (PHB), is found in the plasma membranes of Escherichia col complexed to calcium polyphosphate (CaPPi). The composition, location, and putative structure of the polymer salt complexes led Reusch and Sadoff (1988) to propose that the complexes function as Ca2+ channels. Here we use bilayer patch-clamp techniques to demonstrate that voltage-activated Ca2+ channels composed of PHB and CaPPi are in the plasma membranes of E. coli. Single channel calcium currents were observed in vesicles of plasma membranes incorporated into planar bilayers of synthetic 1-palmitoyl, 2-oleoyl phosphatidylcholine. The channels were extracted from cells and incorporated into bilayers, where they displayed many of the signal characteristics of protein Ca2+ channels: voltage-activated selective for divalent over monovalent cations, permeant to Ca2+, manner by La3+, Co2+, Cd2+, and Mg2+, in that order. The channel-active extract, purified by size exclusion chromatography, was found to contain only PHB and CaPPi. This composition was confirmed by the observation of comparable single channel currents with complexes reconstituted from synthetic CaPPi and PHB, isolated from E. coli. This is the first report of a biological non-proteinaceous calcium channel. We suggest that poly-3-hydroxybutyrate/calcium polyphosphate complexes are evolutionary antecedents of protein Ca2+ channels.     

Gating Kinetics of E. coli Poly-3-Hydroxybutyrate/Polyphosphate Channels in Planar Bilayer Membranes

Nonproteinaceous calcium channel complexes from Escherichia coli, composed of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP), exhibit two distinct gating modes (modes 1 and 2) in planar lipid bilayers. Here we report the kinetic characterization of the channel in mode 2, a mode characterized by two well-defined conductance levels, a fully open state (87 ± 3 pS), and a major subconductance state (56 ± 2 pS). Other subconductance states and full closures are rare (<0.5% of total time). Several kinetic properties of the channel showed asymmetric voltage-dependence indicating an asymmetry in the channel structure. Accordingly, single channels responded to potential change in one of two mirror-image patterns, postulated to arise from opposite orientations of the asymmetrical channel complex in the bilayer. The fraction of time spent in each conductance level was strongly voltage-sensitive. For channels reported in this study, presumably all oriented in the same direction, residence time in the fully open state increased as clamping potentials became more positive whereas residence time in the major subconductance state increased at more negative potentials. Analysis of open time distributions revealed existence of two kinetically distinct states for each level. The shorter time constants for both conductance states exhibited weak voltage-sensitivity; however, the longer time constants were strongly voltage-sensitive. A kinetic scheme, consistent with the complex voltage dependence of the channel, is proposed. Received: 1 February 1999/Revised: 2 April 1999     

Transmembrane ion transport by polyphosphate/poly-(R)-3-hydroxybutyrate complexes

Transmembrane ion transport, a critical process in providing energy for cell functions, is carried out by pore-forming macromolecules capable of discriminating among very similar ions and responding to changes in membrane potential. It is widely regarded that ion channels are exclusively proteins, relatively late arrivals in cell evolution. Here we discuss the formation of ion-selective, voltage-activated channels by complexes of two simple homopolymers, namely, inorganic polyphosphates (polyPs) and poly-(R)-3-hydroxybutyrates (PHBs), derived from phosphate and acetate, respectively. Each has unique molecular characteristics that facilitate ion selection, solvation, and transport. Complexes of the two polymers, isolated from bacterial plasma membranes or prepared from the synthetic polymers, form voltage-dependent, Ca2+-selective channels in planar lipid bilayers that are selective for divalent over monovalent cations, permeant to Ca2+, Sr2+, and Ba2+, and blocked by transition metal cations in a concentration-dependent manner. Recently, both polyP and PHB have been found to be components of ion-conducting proteins: namely, the human erythrocyte Ca2+-ATPase pump and the Streptomyces lividans potassium channel. The contribution of polyP and PHB to ion selection and/or transport in these proteins is yet unknown, but their presence gives rise to the hypothesis that these and other ion transporters are supramolecular structures in which proteins, polyP, and PHB cooperate in forming well-regulated and specific cation transfer systems.     

Inorganic Polyphosphate Modulates TRPM8 Channels

Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms complexes and modulates activities of many proteins including ion channels. Here we investigated the role of polyP in the function of the transient receptor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp and fluorescent calcium measurements we demonstrate that enzymatic breakdown of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in human embryonic kidney and F-11 neuronal cells expressing TRPM8. We demonstrate that the TRPM8 channel protein is associated with polyP. Furthermore, addition of scPPX1 altered the voltage-dependence and blocked the activity of the purified TRPM8 channels reconstituted into planar lipid bilayers, where the activity of the channel was initiated by cold and menthol in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P₂). The biochemical analysis of the TRPM8 protein also uncovered the presence of poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with polyP. We conclude that the TRPM8 protein forms a stable complex with polyP and its presence is essential for normal channel activity.     

Voltage and temperature dependence of single K+ channels isolated from canine cardiac sarcoplasmic reticulum.

The temperature and voltage dependence of gating and conductance of sarcoplasmic reticulum K+ channels (S-R K+) isolated from adult canine hearts were studied using the reconstituted bilayer technique. Fusion of vesicles from this preparation frequently resulted in the incorporation of a single channel. Only bilayers into which a single S-R K+ channel had fused were studied. The three conductance states of the channel, fully open (O2), substate conductance (O1), and closed (C) were studied as a function of voltage (-50 to +50 mV) and temperature (16 to 37 degrees C). Permeation through the O1 state showed the same temperature dependence as the O2 state corresponding to an enthalpy of permeation of 4.1-4.2 kcal/mol, which is similar to that for K+ diffusion through water. As expected, increased temperature increased the frequency of gating transitions and shortened the average dwell time spent in any conductance state. Over the range of 25 to 37 degrees C, the average dwell time spent in the O1, O2, and C states decreased by 44 +/- 11, 36 +/- 13, and 78 +/- 7% (n = 3 to 4 channels), respectively. The ratio of probabilities between the various conductance states was not strongly temperature sensitive. Analysis of the voltage dependence of this channel was carried out at 37 degrees C and revealed that the dwell times of the O1 and O2 states were voltage insensitive and the probability ratio (PO2:PO1) was approximately 7 and was voltage insensitive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of polyphosphate in regulation of the Streptomyces lividans KcsA channel

We examine the hypotheses that the Streptomyces lividans potassium channel KcsA is gated at neutral pH by the electrochemical potential, and that its selectivity and conductance are governed at the cytoplasmic face by interactions between the KcsA polypeptides and a core molecule of inorganic polyphosphate (polyP). The four polypeptides of KcsA are postulated to surround the end unit of the polyP molecule with a collar of eight arginines, thereby modulating the negative charge of the polyP end unit and increasing its preference for binding monovalent cations. Here we show that KcsA channels can be activated in planar lipid bilayers at pH 7.4 by the chemical potential alone. Moreover, one or both of the C-terminal arginines are replaced with residues of progressively lower basicity-lysine, histidine, valine, asparagine-and the effects of these mutations on conductance and selectivity for K⁺ over Mg²⁺ is tested in planar bilayers as a function of Mg²⁺ concentration and pH. As the basicity of the C-terminal residues decreases, Mg²⁺ block increases, and Mg²⁺ becomes permeant when medium pH is greater than the pI of the C-terminal residues. The results uphold the premise that polyP and the C-terminal arginines are decisive elements in KcsA channel regulation.     

Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous,PHB-polyphosphate Ca2+ channels in the membrane

PHB(polyP) complexes bind calcium and form calcium channels in the cytoplasmic membrane in Escherichia coli and are likely to be important in Ca(2+) homeostasis in this organism. E. coli N43, which lacks the AcrA component of a major multidrug resistance pump, was shown to be defective in calcium handling, with an inability to maintain submicromolar levels of free Ca(2+) in the cytoplasm. Therefore, using an N-phenyl-1-napthylamine (NPN)-dependent fluorescence assay, we measured temperature-dependent phase transitions in the membranes of intact cells. These transitions specifically depend on the presence of PHB(Ca(2+)polyP) complexes. PHB(Ca(2+)polyP) channel complexes, particularly in stationary phase cultures, were detected in wild-type strains; however, in contrast, isogenic acrA(-) strains had greatly reduced amounts of the complexes. This indicates that the AcrAB transporter may have a novel, hitherto undetected physiological role, either directly in the membrane assembly of the PHB complexes or the transport of a component of the membrane, which is essential for assembly of the complexes into the membrane. In other experiments, we showed that the particular defective calcium handling detected in N43 was not due to the absence of AcrA but to other unknown factors in this strain.     

Kinetics of folding of Escherichia coli OmpA from narrow to large pore conformation in a planar bilayer

The outer membrane protein of Escherichia coli, OmpA, is currently alleged to adopt two native conformations: a major two-domain conformer in which 171 N-terminal residues form a narrow eight beta-barrel pore and 154 C-terminal residues are in the periplasm and a minor one-domain conformer in which all 325 residues create a large pore. However, recent studies in planar bilayers indicate the conformation of OmpA is temperature-sensitive and that increasing temperature converts narrow pores to large pores. Here we examine the reversibility and kinetics of this transition for single OmpA molecules in planar bilayers of diphytanoylphosphatidylcholine (DPhPC). We find that the transition is irreversible. When temperatures are decreased, large pores close down, and when temperatures are stabilized they reopen in the large pore conformation, with gradually increasing open time. Large pores are converted to narrow pores only by denaturing agents. The transition from narrow to large pores requires temperatures >or= 26 degrees C and is a biphasic process with rates that rise steeply with temperature. The first phase, a flickering stepwise transition from a low-conductance to a high-conductance state requires approximately 7 h at 26 degrees C but only approximately 13 min at 42 degrees C, signifying an activation energy of 139 +/- 12 kJ/mol. This is followed by a gradual increase in conductance and open probability, interpreted as optimization of the large pore structure. The results indicate that the two-domain structure is a partially folded intermediate that is kinetically stable at lower temperatures and that mature fully folded OmpA is a large pore.     

Involvement of the C-terminal part of Pseudomonas fluorescens OprF in the modulation of its pore-forming properties

The major outer-membrane protein, OprF, from the psychrotrophic bacterium Pseudomonas fluorescens undergoes a reduction of its conductance value (from 250 pS to 80 pS) when the growth temperature is shifted from 28 degrees C to 8 degrees C. The involvement of changes in tertiary or quaternary structure in this behaviour, was implied by enzymatic digestion experiments in which OprFs purified from 8 degrees C and 28 degrees C cultures showed different accessibility to pronase. Resistant proteolytic fragments of 19 kDa, obtained from both OprF preparations, were identified as the N-terminal half of the native protein. These 19 kDa fragments induced ion channels in planar lipid bilayers with similar conductance values of 65-75 pS in 1 M NaCl, in contrast to the native proteins. Thus, the C-terminal part of the protein is required for the growth temperature-dependent modulation of OprF channel-forming properties. LPS was not detected on the proteolytic fragments while it was found in similar amounts on the native OprFs. These results suggest the LPS/porin association occurs through the C-terminal part of the porin. Radiolabelling experiments showed different phosphorylation levels of LPS for 8 degrees C and 28 degrees C cultures. Thus, in response to growth temperature, the structural modification of the LPS could be associated to the modulation of OprF pore size.     

Pore characteristics of nontypeable Haemophilus influenzae outer membrane protein P5 in planar lipid bilayers

The structure of outer membrane protein P5 of NTHi, a homolog of Escherichia coli OmpA, was investigated by observing its pore characteristics in planar lipid bilayers. Recombinant NTHi P5 was overexpressed in E. coli and purified using ionic detergent, LDS-P5, or nonionic detergent, OG-P5. LDS-P5 and OG-P5 could not be distinguished by their migration on SDS-PAGE gels; however, when incorporated into planar bilayers of DPhPC between symmetric aqueous solutions of 1 M KCl at 22 degrees C, LDS-P5 formed narrow pores (58 +/- 6 pS) with low open probability, whereas OG-P5 formed large pores (1.1 +/- 0.1 nS) with high open probability (0.99). LDS-P5 narrow pores were gradually and irreversibly transformed into large pores, indistinguishable from those formed by OG-P5, at temperatures >or=40 degrees C; the process took 4-6 h at 40 degrees C or 35-45 min at 42 degrees C. Large pores were stable to changes in temperatures; however, large pores were rapidly converted to narrow pores when exposed to LDS at room temperatures, indicating acute sensitivity of this conformer to ionic detergent. These studies suggest that narrow pores are partially denatured forms and support the premise that the native conformation of NTHi P5 is that of a large monomeric pore.     

The reduction in electroporation voltages by the addition of a surfactant to planar lipid bilayers.

The effects of a nonionic surfactant, octaethyleneglycol mono n-dodecyl ether (C12E8), on the electroporation of planar bilayer lipid membranes made of the synthetic lipid 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 100-450 mV) rectangular voltage pulses were used to electroporate the bilayers, followed by a prolonged, low-amplitude ( approximately 65 mV) voltage clamp to monitor the ensuing changes in transmembrane conductance. The electroporation thresholds of the membranes were found for rectangular voltage pulses of given durations. The strength-duration relationship was determined over a range from 10 micros to 10 s. The addition of C12E8 at concentrations of 0.1, 1, and 10 microM to the bath surrounding the membranes decreased the electroporation threshold monotonically with concentration for all durations (p < 0.0001). The decrease from control values ranged from 10% to 40%, depending on surfactant concentration and pulse duration. For a 10-micros pulse, the transmembrane conductance 150 micros after electroporation (G150) increased monotonically with the surfactant concentration (p = 0.007 for 10 microM C12E8). These findings suggest that C12E8 incorporates into POPC bilayers, allowing electroporation at lower intensities and/or shorter durations, and demonstrate that surfactants can be used to manipulate the electroporation threshold of lipid bilayers.     

Properties of ion channels formed by Staphylococcus aureus delta-toxin

The delta-toxin of Staphylococcus aureus has been investigated in terms of its potential to form ion channels in planar lipid bilayers formed at the tip of patch electrodes. Channel formation has been shown to occur for delta-toxin concentrations in the range 0.1 to 2.0 microM. In 0.5 M KCl, two major classes of channels were seen--'small' with conductances of 70-100 pS, and 'large' with a conductance of approx. 450 pS. Current-voltage relationships for lipid bilayers containing several delta-toxin channels revealed both voltage-dependent and independent components to channel gating. Reversal potential measurements showed the channels to be cation selective. In the presence of 3.0 M KCl, the channel gating kinetics were complex, with multiple open and closed states. The results are interpreted in terms of a model for the channel consisting of a hexameric cluster of alpha-helical delta-toxin molecules.     

Temperature dependence of Ca2+-activated K+ currents in the membrane of human erythrocytes

The currents through single Ca2+-activated K+ channels were studied in excised inside-out membrane patches of human erythrocytes. The effects of temperature on single-channel conductance, on channel gating and on activation by Ca2+ were investigated in the temperature range from 0 up to 47 degrees C. The single-channel conductance shows a continuous increase with increasing temperature; an Arrhenius plot of the conductance gives the activation energy of 29.6 +/- 0.4 kJ/mol. Reducing the temperature alters channel-gating kinetics which results in a significant increase of the probability of the channel being open (Po). The calcium dependence of Po is affected by temperature in different ways; the threshold concentration for activation by Ca2+ is not changed, the Ca2+ concentration of half-maximal channel activation is reduced from 2.1 mumol/l at 20 degrees C to 0.3 mumol/l at 0 degrees C, the saturation level of the dependence is reduced for temperatures higher then about 30 degrees C. The relevance of the obtained data for the interpretation of the results known from flux experiments on cells in suspensions is discussed.     

Estimation of the radius of the pores formed by the Bacillus thuringiensis Cry1C delta-endotoxin in planar lipid bilayers

Pore formation constitutes a key step in the mode of action of Bacillus thuringiensis delta-endotoxins and various activated Cry toxins have been shown to form ionic channels in receptor-free planar lipid bilayers at high concentrations. Multiple conductance levels have been observed with several toxins, suggesting that the channels result from the multimeric assembly of a variable number of toxin molecules. To test this possibility, the size of the channels formed by Cry1C was estimated with the non-electrolyte exclusion technique and polyethylene glycols of various molecular weights. In symmetrical 300 mM KCl solutions, Cry1C induced channel activity with 15 distinct conductance levels ranging from 21 to 246 pS and distributed in two main conductance populations. Both the smallest and largest conductance levels and the mean conductance values of both populations were systematically reduced in the presence of polyethylene glycols with hydrated radii of up to 1.05 nm, indicating that these solutes can penetrate the pores formed by the toxin. Larger polyethylene glycols had little effect on the conductance levels, indicating that they were excluded from the pores. Our results indicate that Cry1C forms clusters composed of a variable number of channels having a similar pore radius of between 1.0 and 1.3 nm and gating synchronously.     

Temperature dependence of K(+)-channel properties in human T lymphocytes.

We have used whole-cell patch clamp to determine the temperature dependence of the conductance and gating kinetics of the voltage-gated potassium channel in quiescent, human peripheral blood T lymphocytes. Threshold for activation, steady-state inactivation, and the reversal potential are the same at 22 degrees and 37 degrees C. However, the time-constants for activation, inactivation, deactivation, and release from inactivation are quite sensitive to temperature, changing by at least a factor of five in each case over this range of temperatures. The onset of cumulative inactivation at 22 degrees and 37 degrees C reflects the time-course of deactivation. Peak outward current is approximately twofold greater at 37 degrees C than at 22 degrees C; this increase is also manifest at the single channel level. Energies of activation for conductance, activation, inactivation, deactivation, and release from inactivation are 8.2, 22.1, 25.0, 36.2, and 42.2 kcal/mol, respectively. No new channels were observed at 37 degrees C, and there was no evidence for alteration of the K+ conductance by putative modulators at 22 or 37 degrees C.     

Outer membrane protein NmpC of Escherichia coli: pore-forming properties in black lipid bilayers.

The purified NmpC outer membrane protein from Escherichia coli, when incorporated into planar lipid bilayers, gave rise to channels with a single-channel conductance of 1.8 nS in 1 M KCl. This suggests that the NmpC protein is a porin.