Differential lipid dependence of function of bacterial sodium channel homologues

The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in maintaining the hydrophobic/charged interactions necessary for structure, conformational flexibility and function. Despite the intimate relationship between ion channels and the membranes in which they are embedded, challenges resulting from the dynamic and complex nature of cellular membranes have limited our ability to address the functional role of these interactions. To directly assess lipid dependence of activity, we examined channel function ofthree purified bacterial sodium channel orthologues (NaChBac, NavMs, and NavSp) by cumulative 22Na+ uptake into proteoliposomes containing a 3:1 ratio of POPE and another glycerophospholipid (POPC, POPG, POPS, Cardiolipin (CL), POPA, or PI). We observed a unique lipid dependence for each homologue tested. Common to each was a low level of activity above background (uptake into protein free liposomes) when the second lipid was a zwitterionic lipid such as POPE and POPC. Maximal activity for full-length NaChBac and NavMs proteins was observed in POPE + POPG liposomes. On the other hand, full-length NavSp channels possessed a different lipid dependence, with maximal activity in liposomes containing POPE + PI. No strong lipid dependence was observed for pore-only constructs of NavMs or NavSp, that lacked the S1-S4 segments, suggesting that the lipid dependence of sodium channels may arise from their abilities to affect the voltage-sensing domains. The effect may be maximized by specific lipid-protein interactions that are uniquely favourable in each homologue, giving rise to differing lipid dependences

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 22Na+ 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

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 (NavSp) 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

Mutagenesis of the NaChBac sodium channel discloses a functional role for a conserved S6 asparagine

Asparagine is conserved in the S6 transmembrane segments of all voltage-gated sodium, calcium, and TRP channels identified to date. A broad spectrum of channelopathies including cardiac arrhythmias, epilepsy, muscle diseases, and pain disorders is associated with its mutation. To investigate its effects on sodium channel functional properties, we mutated the simple prokaryotic sodium channel NaChBac. Electrophysiological characterization of the N225D mutant reveals that this conservative substitution shifts the voltage-dependence of inactivation by 25 mV to more hyperpolarized potentials. The mutant also displays greater thermostability, as determined by synchrotron radiation circular dichroism spectroscopy studies of purified channels. Based on our analyses of high-resolution structures of NaChBac homologues, we suggest that the side-chain amine group of asparagine 225 forms one or more hydrogen bonds with different channel elements and that these interactions are important for normal channel function. The N225D mutation eliminates these hydrogen bonds and the structural consequences involve an enhanced channel inactivation

Interaction of a membrane protein with its surrounding lipid bilayer : studies with the mechanosensitive channel MscL

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Interaction of a membrane protein with its surrounding lipid bilayer : studies with the mechanosensitive channel MscL

mechanosensitive (MS) channels open in response to membrane stretch.  The most studied of the MS channels is the bacterial mechanosensitive ion channel of large conductance, MscL.  Interactions between the lipid bilayer and MscL are particularly important since MscL opens on increasing tension in model systems containing just lipid and MscL, so that membrane tension must be transduced directly from the lipid molecules to the protein.  Key to understanding how stretching of the lipid bilayer leads to opening of MscL is understanding how MscL interacts with the lipid bilayer.  Site directed mutagenesis and fluorescence spectroscopy were used to define how the MscL channel interacts with the lipid bilayer that surrounds it in the membrane.  Single Trp-containing mutants of Mycobacterium tuberculosis MscL were reconstituted into lipid bilayers of defined composition.  Fluorescence emission maxima were used to identify the transmembrane region of MscL in bilayers of dioleoylphosphatidylcholine (di(C18:1)PC) and the levels of fluorescence quenching by brominated lipid gave lipid binding constants for MscL in the vicinity of the Trp groups.  Introducing a lysine residue into the central pore of MscL generated a gain of function phenotype, thereby allowing the lipid-protein interactions of both the open and closed channel to be determined.  Preferential binding was observed with phosphatidylcholines possessing fatty acyl chains with a length of C16 for the closed channel and C14 for the open channel, suggesting the open channel has thinned by 4 Å.  All lipid head groups bind equally well to MscL on the periplasmic side of the membrane.  In contrast, anionic lipids bind with significantly greater affinity on the cytoplasmic side of MscL, with two classes of binding site involving the conserved charge cluster Arg-98, Lys-99 and Lys-100.  This binding site is broken up when the channel forms an open structure.</p

Chimeric bacterial-human NaV17 sodium channels expressed in E coli

Voltage-gated sodium channels selectively transport sodium ions across cellular membranes in response to changes in membrane potential. Prokaryotic voltage-gated sodium channels are homotetramers, each monomer containing six transmembrane helices (S1–S6), consisting of a voltage-sensing subdomain (S1–S4) and a pore-forming subdomain (S5–S6). In eukaryotes, sodium channels consist of a single polypeptide chain containing four similar domains, each with six transmembrane helices (S1–S6), which create pseudo-tetrameric channels. In humans, genetic diseases associated with the NaV1.7 sodium channel isoform include loss-of-function (i.e. channelopathy-associated indifference to pain), in addition to gain-of-function inherited painful neuropathies; hence, this channel is an important target for drug discovery. Expression of eukaryotic membrane proteins in E. coli is often a difficult task, resulting in cell death, no expression of the target protein, or proteins inserted into inclusion bodies. In order to enable the expression of crucial functional regions of eukaryotic sodium channels, we have developed a method for creating chimeric proteins with the N-terminal subdomain of a prokaryotic homologue, and the C-terminal subdomain of the eukaryotic protein of interest, thereby tricking the bacterial host into expressing a protein with functional regions of interest from the eukaryote. In this study we designed, constructed, expressed, and characterised a number of sodium channel chimeras containing the voltage sensor (S1–S4) from B. halodurans NaChBac and the pore regions (S5–S6) from domains II and III of human NaV1.7, including the S4–S5 linkers from either the bacterial or eukaryotic protein

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

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 Å, compared to a hydrophobic thickness for TbMscL of about 26 Å 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

The Nachbac Pore: creation and characterisation of a KcsA-like sodium channel

Voltage-gated sodium channels (VGSC) are integral membrane proteins responsible for the transient flux of sodium ions across cell membranes in response to changes in membrane potential. In humans as well as lower eukaryotes they are essential for homeostasis and normal functioning, and mutations in them are associated with a range of disease states. Although potassium channels, which are members of the same large family of voltage-gated channels have been well characterized, much less known about the structural features of sodium channels. For potassium ion channels, an important advance in understanding resulted from the determination of the three dimensional structure of the bacterial potassium channel KcsA, a simplified channel composed only of two transmembrane segments per subunit present in the tetrameric structure. In 2001, Ren et al found that bacteria also possess simplified versions of sodium channels, although in this case the individual subunits of all the homologues that have been identified thus far possess six transmembrane segments, which include both a pore-forming subdomain (S5-S6) and a voltage-sensing subdomain (S1-S4). Here we report on the creation of a smaller KcsA-like pore-only version of a sodium channel from the B. halodurans VGSC (pNaChBac), engineered to contain S5-S6 plus the C-terminal region of the NaChBac channel. The NaChBac pore has been expressed and purified from E. coli membranes, solubilised in detergent micelles, reconstituted into lipid vesicles and characterized for its secondary structure and thermal stability, as well as its electrophysiological properties from single-channel recordings, providing new insight into features of sodium channel structure and function