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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.