Channels In Planar Lipid Bilayers Research Articles

Drosophila Mpv17 forms an ion channel and regulates energy metabolism

Mutations in MPV17 are a major contributor to mitochondrial DNA (mtDNA) depletion syndromes, a group of inherited genetic conditions due to mtDNA instability. To investigate the role of MPV17 in mtDNA maintenance, we generated and characterized a Drosophila melanogaster Mpv17 (dMpv17) KO model showing that the absence of dMpv17 caused profound mtDNA depletion in the fat body but not in other tissues, increased glycolytic flux and reduced lifespan in starvation. Accordingly, the expression of key genes of glycogenolysis and glycolysis was upregulated in dMpv17 KO flies. In addition, we demonstrated that dMpv17 formed a channel in planar lipid bilayers at physiological ionic conditions, and its electrophysiological hallmarks were affected by pathological mutations. Importantly, the reconstituted channel translocated uridine but not orotate across the membrane. Our results indicate that dMpv17 forms a channel involved in translocation of key metabolites and highlight the importance of dMpv17 in energy homeostasis and mitochondrial function.

Furosemide blocks the anion channel caused by Staphylococcus aureus α-hemolysin

To investigate the effect of furosemide on Staphylococcus aureus α-hemolysin (α-HL) channel in planar lipid bilayers by electrophysiological characterization and molecular docking studies. Planar lipid bilayer membranes were prepared and α-HL (0.07 mg/mL) was added to the standard solution in cis compartment of the experimental chamber. All experiments were performed at room temperature using an Axopatch 200A amplifier in the voltage clamp mode. At pH 7.5, α-HL channels were usually in a high conductance ~4 nS and rarely switch to low conductance states. After the ion channel was incorporated in bilayer membrane, the furosemide was also added to the standard solution to the cis compartment. To docking studies, atomics coordinates for the α-HL heptameric channel was retrieved from PDB ID (7AHL) and the structure of furosemide was removed from the PubChem, their coordinates were built and minimized with Avogadro software. The molecular docking experiments were performed using the Dockthor online. The furosemide inhibited (P<0.05) conductance α-HL channel and it was a voltage-dependent manner. The two best docking solutions and the α-HL channel were evaluated, it was observed the connection mode with the highest affinity of interaction has a greater number of hydrogen bonding. The residues were 113 and 147 that form the remainders of the constriction α-HL channel. In conclusion, furosemide blocks ion currents in the constriction of channel caused by Staphylococcus aureus α-hemolysin.

Acylated and alkylated benzo(crown-ethers) form ion-dependent ion channels in biological membranes

Synthetic ion channels based on benzo(crown-ether) compounds have been previously reported to function as ion-selective channels in planar lipid bilayers, with hydrogen bonding networks implicated in the formation of self-aggregated complexes. Herein, we report the synthesis and characterization of two new families of benzo(crown-ether) compounds, termed monoacylated and monoalkylated benzo(crown-ethers) (MABCE), both of which lack hydrogen bond donors. Depending on the length of alkyl chain substituent and the size of macrocycle, MABCE compounds inhibit bacterial growth and transport ions across biological membranes. Single-channel recordings show that the activity is higher in the presence of K+ as compared with Na+; however, under bionic conditions, open channels do not exhibit any preference between the two ions. These findings reveal that the ionic preference of benzo(crown-ether) compounds is either due to the regulation of assembly of ion-conducting supramolecular complexes or its membrane insertion by cations, as opposed to ion-selective transport through these scaffolds. Furthermore, our data show that the H-bonding network is not needed to form these assemblies in the membrane.

Apolipoproteins L1-6 share key cation channel-regulating residues but have different membrane insertion and ion conductance properties

The human apolipoprotein L gene family encodes the apolipoprotein L1–6 (APOL1–6) proteins, which are effectors of the innate immune response to viruses, bacteria and protozoan parasites. Due to a high degree of similarity between APOL proteins, it is often assumed that they have similar functions to APOL1, which forms cation channels in planar lipid bilayers and membranes resulting in cytolytic activity. However, the channel properties of the remaining APOL proteins have not been reported. Here, we used transient overexpression and a planar lipid bilayer system to study the function of APOL proteins. By measuring lactate dehydrogenase release, we found that APOL1, APOL3, and APOL6 were cytolytic, whereas APOL2, APOL4, and APOL5 were not. Cells expressing APOL1 or APOL3, but not APOL6, developed a distinctive swollen morphology. In planar lipid bilayers, recombinant APOL1 and APOL2 required an acidic environment for the insertion of each protein into the membrane bilayer to form an ion conductance channel. In contrast, recombinant APOL3, APOL4, and APOL5 readily inserted into bilayers to form ion conductance at neutral pH, but required a positive voltage on the side of insertion. Despite these differences in membrane insertion properties, the ion conductances formed by APOL1-4 were similarly pH-dependent and cation-selective, consistent with conservation of the pore-lining region in each protein. Thus, despite structural conservation, the APOL proteins are functionally different. We propose that these proteins interact with different membranes and under different voltage and pH conditions within a cell to effect innate immunity to different microbial pathogens.

Combining in vitro translation with nanodisc technology and functional reconstitution of channels in planar lipid bilayers.

Experimental studies on membrane proteins have been recently enriched by two promising method developments: protocols for cell-free protein synthesis and the use of soluble nanoscale lipid bilayers, so called nanodiscs, as membrane mimics for keeping these proteins in a soluble form. Here, we show how the advantages of these techniques can be combined with the classical planar lipid bilayer method for a functional reconstitution of channel activity. The present data demonstrate that the combination of these methods offers a very rapid and reliable way of recording channel activity in different bilayer systems. This approach has additional advantages in that it strongly lowers the propensity of contamination from the expression system and allows the simultaneous reconstitution of thousands of channel proteins for macroscopic current measurements without compromising bilayer stability.

Ion channel reconstitution in lipid bilayers.

Incorporation of ion channels in planar lipid bilayers allows detecting and measuring ion channel activity in a well-controlled system. This technique provides critical information about ion channel kinetics, ion selectivity, gating mechanism, open probability, unitary conductance, subconductance states, voltage dependence, and burst opening events, particularly at the single molecule level. Planar lipid bilayers provide a unique controllable environment that enables maintaining specific regulatory components, including lipids, ligands, inhibitors, particular ions, and proteins, as well as the temperature that can modulate activity of many ion channels. Thus, this system provides explicit details about ion channel gating mechanism and enables identifying its particular regulatory molecules or components. This chapter will describe the planar lipid bilayer method using the example of a transient receptor potential (TRP) ion channel family member. The planar lipid bilayer electrophysiological approach has proven to be useful in studying intrinsic properties of TRP channels. This method is particularly valuable for our understanding of intrinsic temperature sensitivity of thermoreceptors such as TRP channels and direct effects of TRP channels agonists, antagonists, co-factors, and other modifiers.

Assessment of Endogenous and Exogenous Modulators of the TRPM7 Channel in Planar Lipid Bilayers

TRPM7 is ubiquitously expressed divalent cation-selective ion channel. The channel subunit contains the alpha-kinase domain, which is located at the C-terminus. TRPM7 channels are implicated in a variety of cellular processes, including cell proliferation, differentiation, survival, death, Mg2+ homeostasis, and oxidative stress. TRPM7 is essential for the early embryonic development as the knockout of its gene results in embryonic lethality of mice. Furthermore, TRPM7 plays important roles in thymopoiesis, kidney morphogenesis, cardiac rhythmicity and immune responses.

Identification of Novel and Natural High Affinity Peptide Inhibitors of KcsA by Phage-Display Reveals an Unexpected Mechanism of Pore Blockade

Peptide neurotoxins are powerful tools for research, diagnosis and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high-affinity and specificity. Here we describe isolation of toxins for one such orphan target, KcsA, a K+ channel that has been fundamental to delineating the structural basis for ion channel function. A phage display strategy is employed whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We focus here on two of nine toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (Ki) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a Ki of 0.5 nM while Shaker, Kv1.2 and Kv1.3 channels are blocked over 200-fold less well. In contrast, HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2 and Kv1.3 channels with Ki of 1 to 4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys21 and Tyr22, are essential for high affinity binding. Unexpectedly, K+ ions traversing the channel from the inside confer voltage-sensitivity to the Hui1 off-rate via Arg23, indicating that Lys21 is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors.

Testosterone is a Highly Potent and Specific Agonist of TRPM8

The role of the TRPM8 channel in the peripheral nervous system as a major receptor for cold temperatures has been well established. Less certainty was gained about the role of the channel beyond the nervous system. Particularly, the presence and high expression levels of TRPM8 in the prostate epithelium were not well understood. Recently, we identified that the prevalent male hormone, testosterone, is a highly potent endogenous agonist of TRPM8 channels. Picomolar concentrations of testosterone elicited rapid Ca2+-uptake in cells expressing TRPM8, including prostate epithelial cells and neurons. Furthermore, testosterone evoked openings of the purified TRPM8 channel in planar lipid bilayers that were promptly inhibited with TRPM8 antagonists, or the purified androgen receptor (AR) protein. In the bilayers, TRPM8 activity was induced by both, permeable testosterone, as well as an impermeable analog, testosterone covalently conjugated to BSA. Interestingly, BSA-testosterone evoked openings of TRPM8 only into a small conductance state, with a mean slope conductance of ∼7 pS, in comparison to ∼37 pS obtained with the permeable analog. Furthermore, the prevalent female hormones, estradiol and progesterone, had no effect on the channel at picomolar or nanomolar concentrations, and only exerted openings at micromolar concentrations, but even then these steroids could open TRPM8 only into a small conductance state of ∼8 pS. These results indicate that binding of steroids to the extracellular site induces conformational changes of the channel resulting in a narrow permeation path. Together, our results demonstrate that testosterone is not only a highly potent but also a highly specific agonist of TRPM8.

Stimulation-dependent gating of TRPM3 channel in planar lipid bilayers.

The transient receptor potential melastatin (TRPM)-3 channel is critical for various physiologic processes. In somatosensory neurons, TRPM3 has been implicated in temperature perception and inflammatory hyperalgesia, whereas in pancreatic β-cells the channel has been linked to glucose-induced insulin release. As a typical representative of the TRP family, TRPM3 is highly polymodal. In cells, it is activated by heat and chemical agonists, including pregnenolone sulfate (PS) and nifedipine (Nif). To define the nuances of TRPM3 channel activity and its modulators, we succeeded in incorporating the TRPM3 protein into planar lipid bilayers. We found that phosphatidylinositol-4,5-bisphosphate (PIP2) or clotrimazole is necessary for channel opening by PS. Unlike PS, the presence of Nif alone sufficed to induce TRPM3 activity and demonstrated distinct gating behavior. We also performed an extensive thermodynamic analysis of TRPM3 activation and found that TRPM3 exhibited slight temperature sensitivity in the bilayers. In the absence of other agonists TRPM3 channels remained closed upon heat-induced stimulation, but opened in the presence of PIP2, although with only a low open-probability profile. Together, our results elucidate the details peculiar to TRPM3 channel function in an isolated system. We confirmed its direct gating by PS and PIP2, but found a lack of the strong intrinsic temperature sensitivity common to other thermosensitive TRP channels.

Abstract 5005: Androgen-mediated genomic and non-genomic regulation of TRPM8 in prostate cancer

Abstract Transient receptor potential melastatin 8 (TRPM8) is a cold-sensing and Ca2+ permeable channel protein. The TRPM8 mRNA is overexpressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Thus, androgens, which are at the crossroads of several signaling pathways, appear to be associated with TRPM8-mediated Ca2+ -dependent signaling. Genomic regulation; From the chromatin-immunoprecipitation (ChIP) analysis, we observed that an androgen response element (ARE) mediates androgen regulation of trpm8. Although TRPM8 mRNA is expressed at high levels in prostate cancer (PC) cells, we found that the TRPM8 protein undergoes UBA1-mediated ubiquitination and degradation. PYR-41, a potent inhibitor of initial enzyme in the ubiquitination cascade, UBA1 increased TRPM8 activity on the plasma membrane of LNCaP cells. Further, PYR-41-mediated PMTRPM8 activity was accompanied by enhanced activation of p53 and Caspase-9. In addition, we found that the promoter region of trpm8 possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. Our findings support previous studies that suggest the fine balance between androgen receptor (AR) and p53 expression that regulates androgen-dependent growth of PC. Non-genomic regulation; Furthermore, using immunofluorescence, calcium-imaging and planar lipid bilayers, we identified that TRPM8 channel is functionally associated with androgens in the prostate. Most importantly, we report that testosterone can directly activate TRPM8 channel at low picomolar concentrations and resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Our data demonstrated that testosterone is an endogenous and highly potent agonist of TRPM8 channel. In light of these observations our data of non-genomic effect of testosterone on PMTRPM8 may support a strategy for targeting the androgen-TRPM8-AR interaction or rescuing PMTRPM8 expression as a new therapeutic application for PC. Note: This abstract was not presented at the meeting. Citation Format: Swapna Asuthkar, Eleonora Zakharian. Androgen-mediated genomic and non-genomic regulation of TRPM8 in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5005. doi:10.1158/1538-7445.AM2015-5005

Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: Relevance to trypanosome lysis

Apolipoprotein L-1 (APOL1), the trypanolytic factor of human serum, can lyse several African trypanosome species including Trypanosoma brucei brucei, but not the human-infective pathogens T. brucei rhodesiense and T. brucei gambiense, which are resistant to lysis by human serum. Lysis follows the uptake of APOL1 into acidic endosomes and is apparently caused by colloid-osmotic swelling due to an increased ion permeability of the plasma membrane. Here we demonstrate that nanogram quantities of full-length recombinant APOL1 induce ideally cation-selective macroscopic conductances in planar lipid bilayers. The conductances were highly sensitive to pH: their induction required acidic pH (pH 5.3), but their magnitude could be increased 3,000-fold upon alkalinization of the milieu (pK(a) = 7.1). We show that this phenomenon can be attributed to the association of APOL1 with the bilayer at acidic pH, followed by the opening of APOL1-induced cation-selective channels upon pH neutralization. Furthermore, the conductance increase at neutral pH (but not membrane association at acidic pH) was prevented by the interaction of APOL1 with the serum resistance-associated protein, which is produced by T. brucei rhodesiense and prevents trypanosome lysis by APOL1. These data are consistent with a model of lysis that involves endocytic recycling of APOL1 and the formation of cation-selective channels, at neutral pH, in the parasite plasma membrane.

Regulation of the TRPM3 Channel in Planar Lipid Bilayers

Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective cation channel that can be activated with nifedipine and pregnenolone sulfate (PS). The role of TRPM3 in nociceptive neurons has been linked to the heat sensation and development of heat hyperalgesia during inflammation. However, the functional characterization of TRPM3 and its specific agonists remain poorly understood. To investigate biophysical and pharmacological properties of TRPM3 we aimed to incorporate the purified channel in planar lipid bilayers. TRPM3 was purified from HEK-293 cells using immunoprecipitation technique. The single channel activity was then examined under various conditions. We investigated TRPM3 activity with different agonists. Application of nifedipine (1-10 µM) resulted in dose-dependent increase of open probability of the channel. These results suggest that nifedipine can alone activate TRPM3. Unlike nifedipine, addition to the bilayers of PS, did not induce the channel openings alone and required co-application of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or clotrimazole. TRPM3 demonstrated outward rectification upon activation with PS/PI(4,5)P2, but linear current-voltage relationship with PS/clotrimazole. In both conditions, TRPM3 tended to inactivate at higher voltages (100-120 mV) and with increased concentrations of PS. Interestingly, in the absence of any agonists TRPM3 exhibited basal channel activity that sustained for several minutes. Previously TRPM3 was proposed to serve as a temperature sensor. We tested temperature sensitivity of TRPM3 and found that in the framework of lipid bilayers the channel did not exhibit any temperature-induced activation in the absence of agonists. In the presence of nifedipine TRPM3 demonstrated weak temperature dependence with Q10of 1.5 at +100 mV and 1.7 at −100 mV. These results indicate that TRPM3 is unlikely to serve as a temperature sensor alone and may involve some alternative molecular mechanisms for temperature sensation.

The TRPM8 Protein Is a Testosterone Receptor: II. FUNCTIONAL EVIDENCE FOR AN IONOTROPIC EFFECT OF TESTOSTERONE ON TRPM8

Testosterone is a key steroid hormone in the development of male reproductive tissues and the regulation of the central nervous system. The rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual drive, aggressiveness, and fear conditioning. However, the currently identified testosterone receptor(s) is not believed to underlie the fast signaling, suggesting an orphan pathway. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions. Using cultured and primary cell lines along with the purified TRPM8 protein, we demonstrate that testosterone directly activates TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, PC3 prostate cancer cells, dorsal root ganglion neurons, and hippocampal neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited a cooling sensation. Our data conclusively demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits.

TRPM8 is an Ionotropic Testosterone Receptor

Testosterone is a key steroid hormone in the development of male reproductive tissues and in the regulation of the central nervous system. Rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual behavior, aggressiveness, or fear conditioning. However, currently identified testosterone receptor(s) are not believed to underlie the fast signaling. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions.TRPM8 is highly expressed in the prostate epithelial cells. Using an immunohistochemistry approach, we detected a colocalization pattern of TRPM8 with endogenous androgens on human prostate tissues obtained from healthy individuals and patients with prostate cancer. Co-immunoprecipitation experiments performed on cultured prostate epithelial cells, prostate cancer cells, and HEK-293 cells stably expressing TRPM8, further confirmed direct binding of testosterone to TRPM8.Using cultured and primary cell lines and the purified TRPM8 protein we demonstrate that testosterone can directly activate TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, prostate cancer cells PC3, dorsal root ganglion (DRG) neurons, and hippocampus neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited cooling sensation, implying testosterone-induced activation of TRPM8. Additionally, animal studies showed altered sniffing behavior and increased blood concentration of testosterone in the TRPM8 knockout mice, suggesting the existence of a feedback loop mechanism in the absence of the receptor. Our data demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well-established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits.

Role of β-hydroxybutyrate, its polymer poly-β-hydroxybutyrate and inorganic polyphosphate in mammalian health and disease.

We provide a comprehensive review of the role of β-hydroxybutyrate (β-OHB), its linear polymer poly-β-hydroxybutyrate (PHB), and inorganic polyphosphate (polyP) in mammalian health and disease. β-OHB is a metabolic intermediate that constitutes 70% of ketone bodies produced during ketosis. Although ketosis has been generally considered as an unfavorable pathological state (e.g., diabetic ketoacidosis in type-1 diabetes mellitus), it has been suggested that induction of mild hyperketonemia may have certain therapeutic benefits. β-OHB is synthesized in the liver from acetyl-CoA by β-OHB dehydrogenase and can be used as alternative energy source. Elevated levels of PHB are associated with pathological states. In humans, short-chain, complexed PHB (cPHB) is found in a wide variety of tissues and in atherosclerotic plaques. Plasma cPHB concentrations correlate strongly with atherogenic lipid profiles, and PHB tissue levels are elevated in type-1 diabetic animals. However, little is known about mechanisms of PHB action especially in the heart. In contrast to β-OHB, PHB is a water-insoluble, amphiphilic polymer that has high intrinsic viscosity and salt-solvating properties. cPHB can form non-specific ion channels in planar lipid bilayers and liposomes. PHB can form complexes with polyP and Ca2+ which increases membrane permeability. The biological roles played by polyP, a ubiquitous phosphate polymer with ATP-like bonds, have been most extensively studied in prokaryotes, however polyP has recently been linked to a variety of functions in mammalian cells, including blood coagulation, regulation of enzyme activity in cancer cells, cell proliferation, apoptosis and mitochondrial ion transport and energy metabolism. Recent evidence suggests that polyP is a potent activator of the mitochondrial permeability transition pore in cardiomyocytes and may represent a hitherto unrecognized key structural and functional component of the mitochondrial membrane system.

Canavalia ensiformis urease, Jaburetox and derived peptides form ion channels in planar lipid bilayers

Ureases catalyze the hydrolysis of urea into NH3 and CO2. They are synthesized by plants, fungi and bacteria but not by animals. Ureases display biological activities unrelated to their enzymatic activity, i.e., platelet and neutrophil activation, fungus inhibition and insecticidal effect. Urease from Canavalia ensiformis (jack bean) is toxic to several hemipteran and coleopteran insects. Jaburetox is an insecticidal fragment derived from jack bean urease. Among other effects, Jaburetox has been shown to interact with lipid vesicles. In this work, the ion channel activity of C. ensiformis urease, Jaburetox and three deletion mutants of Jaburetox (one lacking the N-terminal region, one lacking the C-terminal region and one missing the central β-hairpin) were tested on planar lipid bilayers. All proteins formed well resolved, highly cation-selective channels exhibiting two conducting states whose conductance ranges were 7–18pS and 32–79pS, respectively. Urease and the N-terminal mutant of Jaburetox were more active at negative potentials, while the channels of the other peptides did not display voltage-dependence. This is the first direct demonstration of the capacity of C. ensiformis urease and Jaburetox to permeabilize membranes through an ion channel-based mechanism, which may be a crucial step of their diverse biological activities, including host defense.

Characterization of a Cyanobacterial Outer Membrane Protein: An E. Coli Tolc Homologue from Synechocystis Sp. Pcc 6803

E. coli TolC (tolerance to colicins) represents an interesting class of outer membrane (OM) proteins, as it has an α-helical periplasmic tunnel and β-barrel membrane region, providing a conduit for export of metabolites and xenobiotics from cell interior to exterior, and import of colicin E1 (1). A TolC homologue, Slr1270 from Synechocystis 6803, cloned and expressed in E. coli, has > 40% similarity and ∼16% identity to E. coli and Pseudomonas counterparts, and has a similar domain organization. Homology modeling using Pseudomonas OprM as template modeled 93% of Slr1270 sequence. The 1581bp slr1270 gene was cloned and overexpressed in E. coli. Protein from inclusion bodies, refolded through step-wise dialysis showed major bands at ∼55kDa and >150 kDa on SDS-PAGE corresponding to the monomer and trimer respectively and ∼300 kDa on CN-PAGE. Purified protein displays a far-UV CD spectrum characteristic of E. coli TolC with > 50% α-helix, and formed channels in planar lipid bilayers with a characteristic single channel conductance of ∼50 pS in 0.1M NaCl. The intact protein mass spectrum (LC-MS) with a major peak at 54,489 probably represents a mixture of two species, the TolC product with 1-40 removed and an intact 6-His tag (calculated mass 54,457.1 Da), and a product with 1-38 removed and a 5-His tag (calculated mass 54,490.2 Da) after a single carboxypeptidase event. The small peak at 54,638 Da probably corresponds to TolC product with 1-38 removed and an intact 6-His tag (calculated mass 54,627.3 Da). Peptides 39-76 and 41-76 were recovered from trypsin digests confirming N-terminal heterogeneity. (1) Zakharov, S. D. et al. 2012. Pathways of Colicin Import: Utilization of BtuB, OmpF Porin, and the TolC Drug Export Protein, Biochem. Soc. Trans., 40, 1463-1468.

Tau Protein Forms Ion Channels

Tau is a microtubule associated protein that is intrinsically disordered and accumulates in amyloid fibril deposits alone and in combination with other amyloid forming proteins. We have found that Tau 441 can form ion- permeable channels in planar lipid bilayers under conditions of acidic pH. The channels are irreversibly associated with the membrane and exhibit heterodisperse conductances. Acidic phospholipids enhanced channel activity. The channels are only weakly selective for cations. Zn++ does not block the channels. These channels show significant, but incomplete, similarity to the amyloid channels formed by the Alzheimer peptide Abeta, the diabetes associated peptide IAPP, the prion related PrP106-126 ,and other amyloid channel peptides. Channel forming activity may play a pathogenic role in Tau associated diseases such as Lewy Body Dementia, by causing ion leakage in target cell membranes.

The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit

Mitochondrial calcium uniporter (MCU) channel is responsible for Ruthenium Red-sensitive mitochondrial calcium uptake. Here, we demonstrate MCU oligomerization by immunoprecipitation and Förster resonance energy transfer (FRET) and characterize a novel protein (MCUb) with two predicted transmembrane domains, 50% sequence similarity and a different expression profile from MCU. Based on computational modelling, MCUb includes critical amino-acid substitutions in the pore region and indeed MCUb does not form a calcium-permeable channel in planar lipid bilayers. In HeLa cells, MCUb is inserted into the oligomer and exerts a dominant-negative effect, reducing the [Ca(2+)]mt increases evoked by agonist stimulation. Accordingly, in vitro co-expression of MCUb with MCU drastically reduces the probability of observing channel activity in planar lipid bilayer experiments. These data unveil the structural complexity of MCU and demonstrate a novel regulatory mechanism, based on the inclusion of dominant-negative subunits in a multimeric channel, that underlies the fine control of the physiologically and pathologically relevant process of mitochondrial calcium homeostasis.