Shaped apertures in photoresist films enhance the lifetime and mechanical stability of suspended lipid bilayers.

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 µm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 µm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.

Transmembrane and extramembrane contributions to membrane protein thermal stability: studies with the NaChBac sodium channel.

The thermal stabilities of the extramembranous and transmembranous regions of the bacterial voltage-gated sodium channel NaChBac have been characterised using thermal-melt synchrotron radiation circular dichroism (SRCD) spectroscopy. A series of constructs, ranging from the full-length protein containing both the C-terminal cytoplasmic and the transmembranous domains, to proteins with decreasing amounts of the cytoplasmic domain, were examined in order to separately define the roles of these two types of domains in the stability and processes of unfolding of a membrane protein. The sensitivity of the SRCD measurements over a wide range of wavelengths and temperatures has meant that subtle but reproducible conformational changes could be detected with accuracy. The residues in the C-terminal extramembranous domain were highly susceptible to thermal denaturation, but for the most part the transmembrane residues were not thermally-labile and retained their helical character even at very elevated temperatures. The process of thermal unfolding involved an initial irreversible unfolding of the highly labile distal extramembranous C-terminal helical region, which was accompanied by a reversible unfolding of a small number of helical residues in the transmembrane domain. This was then followed by the irreversible unfolding of a limited number of additional transmembrane helical residues at greatly elevated temperatures. Hence this study has been able to determine the different contributions and roles of the transmembrane and extramembrane residues in the processes of thermal denaturation of this multipass integral membrane protein.

Synchrotron radiation circular dichroism spectroscopy-defined structure of the C-terminal domain of NaChBac and its role in channel assembly.

Extramembranous domains play important roles in the structure and function of membrane proteins, contributing to protein stability, forming association domains, and binding ancillary subunits and ligands. However, these domains are generally flexible, making them difficult or unsuitable targets for obtaining high-resolution X-ray and NMR structural information. In this study we show that the highly sensitive method of synchrotron radiation circular dichroism (SRCD) spectroscopy can be used as a powerful tool to investigate the structure of the extramembranous C-terminal domain (CTD) of the prokaryotic voltage-gated sodium channel (Na(V)) from Bacillus halodurans, NaChBac. Sequence analyses predict its CTD will consist of an unordered region followed by an alpha-helix, which has a propensity to form a multimeric coiled-coil motif, and which could form an association domain in the homotetrameric NaChBac channel. By creating a number of shortened constructs we have shown experimentally that the CTD does indeed contain a stretch of approximately 20 alpha-helical residues preceded by a nonhelical region adjacent to the final transmembrane segment and that the efficiency of assembly of channels in the membrane progressively decreases as the CTD residues are removed. Analyses of the CTDs of 32 putative prokaryotic Na(V) sequences suggest that a CTD helical bundle is a structural feature conserved throughout the bacterial sodium channel family.

The T1-tetramerisation domain of Kv1.2 rescues expression and preserves function of a truncated NaChBac sodium channel.

Cytoplasmic domains frequently promote functional assembly of multimeric ion channels. To investigate structural determinants of this process, we generated the 'T1-chimera' construct of the NaChBac sodium channel by truncating its C-terminal domain and splicing the T1-tetramerisation domain of the Kv1.2 channel to the N terminus. Purified T1-chimera channels were tetrameric, conducted Na+ when reconstituted into proteoliposomes, and were functionally blocked by the drug mibefradil. Both the T1-chimera and full-length NaChBac had comparable expression levels in the membrane, whereas a NaChBac mutant lacking a cytoplasmic domain had greatly reduced membrane expression. Our findings support a model whereby bringing the transmembrane regions into close proximity enables their tetramerisation. This phenomenon is found with other channels, and thus, our findings substantiate this as a common assembly mechanism.

G219S mutagenesis as a means of stabilizing conformational flexibility in the bacterial sodium channel NaChBac.

The NaChBac sodium channel from Bacillus halodurans is a homologue of eukaryotic voltage-gated sodium channels. It can be solubilized in a range of detergents and consists of four identical subunits assembled as a tetramer. Sodium channels are relatively flexible molecules, adopting different conformations in their closed, open and inactivated states. This study aimed to design and construct a mutant version of the NaChBac protein that would insert into membranes and retain its folded conformation, but which would have enhanced stability when subjected to thermal stress. Modelling studies suggested a G219S mutant would have decreased conformational flexibility due to the removal of the glycine hinge around the proposed gating region, thereby imparting increased resistance to unfolding. The mutant expressed in Escherichia coli and purified in the detergent dodecyl maltoside was compared to wildtype NaChBac prepared in a similar manner. The mutant was incorporated into the membrane fraction and had a nearly identical secondary structure to the wildtype protein. When the thermal unfolding of the G219S mutant was examined by circular dichroism spectroscopy, it was shown to not only have a Tm approximately 10 degrees C higher than the wildtype, but also in its unfolded state it retained more ordered helical structure than did the wildtype protein. Hence the G219S mutant was shown to be, as designed, more thermally stable.

Importance of direct interactions with lipids for the function of the mechanosensitive channel MscL.

We have studied the effects of lipid structure on the function of the mechanosensitive channel of large conductance (MscL) from Escherichia coli to determine whether effects follow from direct interaction between the lipids and protein or whether they follow indirectly from changes in the curvature stress in the membrane. The G22C mutant of MscL was reconstituted into sealed vesicles containing the fluorescent molecule calcein, and the release of calcein from the vesicles was measured following opening of the channel by reaction with [2-(triethylammonium)ethyl] methanethiosulfonate (MTSET), which introduces five positive charges into the region of the pore constriction. The presence of anionic lipids in the vesicle membrane changed the rates and amplitudes of calcein release, the effects not correlating with calculated changes in lipid spontaneous curvature. Mutation of charged residues in the Arg-104, Lys-105, Lys-106 cluster removed high-affinity binding of anionic lipids to MscL, and the presence of anionic lipid no longer affected calcein flux through MscL. Changing the zwitterionic lipid from phosphatidylcholine to phosphatidylethanolamine resulted in a large decrease in the rate of calcein release, the change in rate varying linearly with lipid composition, as expected if spontaneous curvature affected the rate of release. However, rates of release of calcein measured in the presence of phosphatidylethanolamine- N-methyl and phosphatidylethanolamine- N, N-dimethyl did not fit the correlation between rate and curvature established for the phosphatidylcholine/phosphatidylethanolamine mixtures. Rather, the effects of zwitterionic lipid headgroup on calcein flux suggested that what was important was the presence of a proton in the headgroup, able to take part in hydrogen bonding to MscL. We conclude that the function of MscL is likely to be modulated by direct interaction with the surrounding, annular phospholipids that contact the protein in the membrane.

Screening ion-channel ligand interactions with passive pumping in a microfluidic bilayer lipid membrane chip.

We describe a scalable artificial bilayer lipid membrane platform for rapid electrophysiological screening of ion channels and transporters. A passive pumping method is used to flow microliter volumes of ligand solution across a suspended bilayer within a microfluidic chip. Bilayers are stable at flow rates up to ∼0.5 µl/min. Phospholipid bilayers are formed across a photolithographically defined aperture made in a dry film resist within the microfluidic chip. Bilayers are stable for many days and the low shunt capacitance of the thin film support gives low-noise high-quality single ion channel recording. Dose-dependent transient blocking of α-hemolysin with ß-cyclodextrin (ß-CD) and polyethylene glycol is demonstrated and dose-dependent blocking studies of the KcsA potassium channel with tetraethylammonium show the potential for determining IC50 values. The assays are fast (30 min for a complete IC50 curve) and simple and require very small amounts of compounds (100 µg in 15 µl). The technology can be scaled so that multiple bilayers can be addressed, providing a screening platform for ion channels, transporters, and nanopores.

Characterization of the Prokaryotic Sodium Channel NavSp Pore with a Microfluidic Bilayer Platform.

This paper describes the use of a newly-developed micro-chip bilayer platform to examine the electrophysiological properties of the prokaryotic voltage-gated sodium channel pore (Na(v)Sp) from Silicibacter pomeroyi. The platform allows up to 6 bilayers to be analysed simultaneously. Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips. The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds. Dose-dependent modulation of the opening probability by the channel blocking drug nifedipine was measured and its IC50 determined.

Differential lipid dependence of the function of bacterial sodium channels.

The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp) by cumulative (22)Na(+) uptake into proteoliposomes containing a 3∶1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different "guest" glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively), whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed) and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains.

Anionic phospholipids affect the rate and extent of flux through the mechanosensitive channel of large conductance MscL.

The mechanosensitive channel of large conductance MscL from Escherichia coli has been reconstituted into sealed vesicles, and the effects of lipid structure on the flux of the fluorescent molecule calcein through the open channel have been studied. The channel was opened by reaction of the G22C mutant of MscL with the reagent [2-(triethylammonium)ethyl]methanethiosulfonate (MTSET) which introduces five positive charges within the pore constriction. Flux through the channel was small when the lipid was phosphatidylcholine, but addition of the anionic lipids phosphatidylglycerol, phosphatidic acid, or cardiolipin up to 50 mol % resulted in increases in the amplitudes and rates of release of calcein. Similar effects were seen when either wild-type MscL or the G22C mutant was opened by osmotic pressure difference; rates of release of calcein were very slow in the absence of anionic lipid but increased with increasing concentrations of phosphatidylglycerol to 50 mol %. The observed partial release of trapped calcein following activation of MscL was attributed to the formation of a long-lived subconductance state of MscL following channel opening. Effects of anionic lipid were attributed to an increase in the rate of the transition from closed to fully open state and to a decrease in the rate of the transition from the fully open state to the subconductance state. Higher concentrations of anionic lipid led to a decrease in the rate and amplitude of release of calcein, possibly due to a decreased rate of flux through the open channel. In mixtures with anionic lipids, phosphatidylethanolamine resulted in lower rates and amplitude of release than phosphatidylcholine.

Different effects of lipid chain length on the two sides of a membrane and the lipid annulus of MscL.

Quenching of the fluorescence of Trp residues in a membrane protein by lipids with bromine-containing fatty acyl chains provides a powerful technique for measuring lipid-protein binding constants. Single Trp residues have been placed on the periplasmic and cytoplasmic sides of the mechanosensitive channel of large conductance MscL from Mycobacterium tuberculosis to measure, separately, lipid binding constants on the two faces of MscL. The chain-length dependence of lipid binding was found to be different on the two sides of MscL, the chain-length dependence being more marked on the cytoplasmic than on the periplasmic side. To determine if lipid binding constants are affected by the properties of the lipid molecules not in direct contact with MscL (the bulk lipid), the amount of bulk lipid present in the system was varied. The binding constant of the short-chain phospholipid didodecylphosphatidylcholine was found to be independent of the molar ratio of lipid/MscL pentamer over the range 500:1-50:1, suggesting that lipid binding constants are determined largely by the properties of the lipid molecules interacting directly with MscL. These results point to a model in which lipid molecules located on the transmembrane surface of a membrane protein (the annular lipid molecules), by playing a dominant role in the interaction between a membrane protein and the surrounding lipid bilayer, could effectively buffer the membrane protein from changes in the properties of the bulk lipid bilayer.

Fluorescence quenching methods to study lipid-protein interactions.

This unit describes how fluorescence quenching methods can be used to determine binding constants for phospholipids binding to intrinsic membrane proteins. Reconstitution of a Trp-containing intrinsic membrane protein with bromine-containing phospholipids leads to quenching of the Trp fluorescence of the protein; the extent of quenching depends on the strength of binding of the phospholipid to the protein. Protocols are included for the synthesis of bromine-containing phospholipids from phospholipids containing carbon-carbon double bonds in their fatty acyl chains and for the reconstitution of membrane proteins into bilayers containing bromine-containing phospholipids. Details are included on data analysis, including equations and software that can be used for fitting the fluorescence quenching data.

Heterogeneity in the binding of lipid molecules to the surface of a membrane protein: hot spots for anionic lipids on the mechanosensitive channel of large conductance MscL and effects on conformation.

We have introduced single Trp residues into the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and used fluorescence quenching by brominated phospholipids to detect the presence of a binding site of high affinity for anionic phospholipids. A cluster of three positively charged residues, Arg-98, Lys-99, and Lys-100, is located on the cytoplasmic side of MscL, in a position where they could interact with the headgroup of an anionic phospholipid. Single mutations of these charged residues in the Trp-containing mutant F80W results in a decreased affinity for phosphatidic acid. Single mutations of the charged residues also result in a significant shift in the fluorescence emission spectrum in dioleoylphosphatidylcholine [di(C18:1)PC] but smaller shifts in dioleoylphosphatidic acid [di(C18:1)PA], suggesting that single mutations result in a conformational change for the protein that is reversed by interaction with anionic phospholipids. This is consistent with the observation that single mutations of the charged residues do not result in a gain of function phenotype. In contrast, simultaneous mutation of all three charged residues results in a gain of function phenotype, and a shift in fluorescence emission spectrum in di(C18:1)PC not reversed in di(C18:1)PA. The gain of function mutant F80W:V21K also shows a shifted fluorescence emission spectrum in both di(C18:1)PC and di(C18:1)PA and binds di(C18:1)PC and di(C18:1)PA with equal affinity, suggesting that the conformational change caused by the V21K mutation results in a breakup of the cluster of three positive charges. Experiments with the Trp mutants L69W and Y87W allow us to measure lipid binding constants on the periplasmic and cytoplasmic sides of the membrane, respectively. On both sides of the membrane the affinity for di(C18:1)PC is equal to that for dioleoylphosphatidylethanolamine. On the periplasmic side of the membrane, there is no selectivity for anionic phospholipids. In contrast, quenching data for Y87W provides evidence for the existence of two lipid binding sites on the cytoplasmic side of the membrane close to the Trp residue at position 87, with binding to one of these sites showing a marked preference for anionic lipid over zwitterionic lipid, presumably involving the charged cluster Arg-98, Lys-99, and Lys-100.

Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL.

The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL) contains two transmembrane alpha-helices, of which the second (TM2) is lipid-exposed. The region of TM2 that spans the hydrocarbon core of the bilayer when MscL is reconstituted into bilayers of dioleoylphosphatidylcholine runs from Leu-69 to Leu-92, giving a hydrophobic thickness of ca. 25 A. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methyl-amino-7-nitroben-2-oxa-1,3-diazole [NBD] group; both fluorescence emission maxima and fluorescence lifetimes for the NBD group are sensitive to solvent dielectric constant over the range (2-40) thought to span the lipid headgroup region of a lipid bilayer. Changing phospholipid fatty acyl chain lengths from C14 and C24 results in no significant change for the fluorescence of the interfacial residues, suggesting very efficient hydrophobic matching between the protein and the surrounding lipid bilayer.

Lipid-protein interactions studied by introduction of a tryptophan residue: the mechanosensitive channel MscL.

Trp fluorescence spectroscopy is a powerful tool to study the structures of membrane proteins and their interactions with the surrounding lipid bilayer. Many membrane proteins contain more than one Trp residue, making analysis of the fluorescence data more complex. The mechanosensitive channels MscL's of Mycobacterium tuberculosis (TbMscL) and Escherichia coli (EcMscL) contain no Trp residues. We have therefore introduced single Trp residues into the transmembrane regions of TbMscL and EcMscL to give the Trp-containing mutants F80W-TbMscL and F93W-EcMscL, respectively, which we show are highly suitable for measurements of lipid binding constants. In vivo cell viability assays in E. coli show that introduction of the Trp residues does not block function of the channels. The Trp-containing mutants have been reconstituted into lipid bilayers by mixing in cholate followed by dilution to re-form membranes. Cross-linking experiments suggest that the proteins retain their pentameric structures in phosphatidylcholines with chain lengths between C14 and C24, phosphatidylserines, and phosphatidic acid. Quenching of Trp fluorescence by brominated phospholipids suggests that the Trp residue in F80W-TbMscL is more exposed to the lipid bilayer than the Trp residue in F93W-EcMscL. Binding constants for phosphatidylcholines change with changing fatty acyl chain length, the strongest interaction for both TbMscL and EcMscL being observed with a chain of length C16, corresponding to a bilayer of hydrophobic thickness ca. 24 A, compared to a hydrophobic thickness for TbMscL of about 26 A estimated from the crystal structure. Lipid binding constants change by only a factor of 1.5 in the chain length range from C12 to C24, much less than expected from theories of hydrophobic mismatch in which the protein is treated as a rigid body. It is concluded that MscL distorts to match changes in bilayer thickness. The binding constants for dioleoylphosphatidylethanolamine for both TbMscL and EcMscL relative to those for dioleoylphosphatidylcholine are close to 1. Quenching experiments suggest a single class of binding sites for phosphatidylserine, phosphatidylglycerol, and cardiolipin on TbMscL; binding constants are greater than those for phosphatidylcholine and decrease with increasing ionic strength, suggesting that charge interactions are important in binding these anionic phospholipids. Quenching experiments suggest two classes of lipid binding sites on TbMscL for phosphatidic acid, binding of phosphatidic acid being much less dependent on ionic strength than binding of phosphatidylserine.