A Drug Repositioning Approach Reveals that Streptococcus mutans Is Susceptible to a Diverse Range of Established Antimicrobials and Nonantibiotics.

Streptococcus mutans is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. An adenylate kinase-based assay was optimized for S. mutans to detect cell lysis when exposed to the Selleck library (Selleck Chemical, Houston, TX) of 853 FDA-approved drugs in, to our knowledge, the first high-throughput drug screen in S. mutans We found 126 drugs with activity against S. mutans planktonic cultures, and they were classified into six categories: antibacterials (61), antineoplastics (23), ion channel effectors (9), other antimicrobials (7), antifungals (6), and other (20). These drugs were also tested for activity against S. mutans biofilm cultures, and 24 compounds were found to inhibit biofilm formation, 6 killed preexisting biofilms, 84 exhibited biofilm inhibition and killing activity, and 12 had no activity against biofilms. The activities of 9 selected compounds that exhibited antimicrobial activity were further characterized for their activity against S. mutans planktonic and biofilm cultures. Together, our results suggest that S. mutans exhibits a susceptibility profile to a diverse array of established and novel antibacterials.

Tetanus neurotoxin utilizes two sequential membrane interactions for channel formation.

Tetanus neurotoxin (TeNT) causes neuroparalytic disease by entering the neuronal soma to block the release of neurotransmitters. However, the mechanism by which TeNT translocates its enzymatic domain (light chain) across endosomal membranes remains unclear. We found that TeNT and a truncated protein devoid of the receptor binding domain (TeNT-LHN) associated with membranes enriched in acidic phospholipids in a pH-dependent manner. Thus, in contrast to diphtheria toxin, the formation of a membrane-competent state of TeNT requires the membrane interface and is modulated by the bilayer composition. Channel formation is further enhanced by tethering of TeNT to the membrane through ganglioside co-receptors prior to acidification. Thus, TeNT channel formation can be resolved into two sequential steps: 1) interaction of the receptor binding domain (heavy chain receptor binding domain) with ganglioside co-receptors orients the translocation domain (heavy chain translocation domain) as the lumen of the endosome is acidified and 2) low pH, in conjunction with acidic lipids within the membrane drives the conformational changes in TeNT necessary for channel formation.

Monitoring charge flux to quantify unusual ligand-induced ion channel activity for use in biological nanopore-based sensors.

The utility of biological nanopores for the development of sensors has become a growing area of interest in analytical chemistry. Their emerging use in chemical analysis is a result of several ideal characteristics. First, they provide reproducible control over nanoscale pore sizes with an atomic level of precision. Second, they are amenable to resistive-pulse type measurement systems when embedded into an artificial lipid bilayer. A single binding event causes a change in the flow of millions of ions across the membrane per second that is readily measured as a change in current with excellent signal-to-noise ratio. To date, ion channel-based biosensors have been limited to well-behaved proteins. Most demonstrations of using ion channels as sensors have been limited to proteins that remain in the open, conducting state, unless occupied by an analyte of interest. Furthermore, these proteins are nonspecific, requiring chemical, biochemical, or genetic manipulations to impart chemical specificity. Here, we report on the use of the pore-forming abilities of heat shock cognate 70 (Hsc70) to quantify a specific analyte. Hsc70 reconstitutes into phospholipid membranes and opens to form multiple conductance states specifically in the presence of ATP. We introduce the measurement of "charge flux" to characterize the ATP-regulated multiconductance nature of Hsc70, which enables sensitive quantification of ATP (100 µM-4 mM). We believe that monitoring protein-induced charge flux across a bilayer membrane represents a universal method for quantitatively monitoring ion-channel activity. This measurement has the potential to broaden the library of usable proteins in the development of nanopore-based biosensors.

Cationic polymers inhibit the conductance of lysenin channels.

The pore-forming toxin lysenin self-assembles large and stable conductance channels in natural and artificial lipid membranes. The lysenin channels exhibit unique regulation capabilities, which open unexplored possibilities to control the transport of ions and molecules through artificial and natural lipid membranes. Our investigations demonstrate that the positively charged polymers polyethyleneimine and chitosan inhibit the conducting properties of lysenin channels inserted into planar lipid membranes. The preservation of the inhibitory effect following addition of charged polymers on either side of the supporting membrane suggests the presence of multiple binding sites within the channel's structure and a multistep inhibition mechanism that involves binding and trapping. Complete blockage of the binding sites with divalent cations prevents further inhibition in conductance induced by the addition of cationic polymers and supports the hypothesis that the binding sites are identical for both multivalent metal cations and charged polymers. The investigation at the single-channel level has shown distinct complete blockages of each of the inserted channels. These findings reveal key structural characteristics which may provide insight into lysenin's functionality while opening innovative approaches for the development of applications such as transient cell permeabilization and advanced drug delivery systems.

[Transport of large organic ions through syringomycin channels in the membranes containing dipole modifiers].

The effect of the membrane dipole potential (Phid) on a conductance and a steady-state number of functioning channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes made from phosphocholine and bathed in 0.4 M solution of sodium salts of aspartate, gluconate and chloride was shown. The magnitude of Phid was varied with the introduction to membrane bathing solutions of phloretin, which reduces the Phid, and RH 421, increasing the Phid. It was established that in all studied systems the increase in the membrane dipole potential cause a decrease in the steady-state number of open channels. In the systems containing sodium salts of aspartate (Asp) or gluconate (Glc), changes in the number of functioning channels are in an order of magnitude smaller than in systems containing sodium chloride. At the same time, the conductance (g) of single SRE-channels on the membranes bathed in NaCI solution increases with the increase in Phid, and in the systems containing NaAsp or NaGlc the conductance of single channels does not depend on the Phid. The latter is due to the lack of cation/anion selectivity of the SRE-channels in these systems. The different channel-forming activity of SRE in the experimental systems is defined by the gating charge of the channel and the partition coefficient of the dipole modifiers between the lipid and aqueous phases.

Characterization of a membrane pore-forming protein from Entamoeba histolytica.

We describe the partial purification and characterization of a pore-forming material (PEM) from Entamoeba histolytica. The formation of ion channels by PFM was examined in three systems. (a) PFM depolarizes J774 macrophages and mouse spleen lymphocytes as measured by [3H]TPP+ uptake. (b) PFM induces rapid monovalent cation flux across the membrane of phosphatidylcholine-cholesterol vesicles. (c) PFM confers a voltage-dependent conductance to artificial planar bilayers, which is resolved as a summation of opening of individually conducting steps of 67 pS in 0.1 M KCl. Monomers of PFM are functional; however, a preferential aggregation occurs in the planar bilayer. Activity is pronase, trypsin, and heat sensitive and is stable between pH 5-8. PFM is not secreted by unstimulated amoebae but after exposure to the calcium ionophore A23187, concanavalin A, and E. coli lipopolysaccharide, 5-10% of the total cell content of PFM is released into the medium within 5-10 min. High-performance gel filtration results in an approximately 1,000-fold purification of PFM and gives an Mr of 30,000. This protein may play a role in the cytotoxicity mediated by E. histolytica.

Functional differences between two classes of sodium channels in developing rat skeletal muscle.

Excitability is generated in developing skeletal muscle by the incorporation of sodium-selective ion channels into the surface membrane. Whole-cell and patch voltage-clamp recording from myotubes and their embryologic precursors, myoblasts, indicated that voltage-activated sodium current in myoblasts was more resistant to block by tetrodotoxin (TTX) than that in myotubes. Single-channel recording from both cell types showed two classes of sodium channels. One class had a lower single-channel conductance, activated at more hyperpolarized voltages, and was more resistant to TTX than the other. The proportion of TTX-resistant to TTX-sensitive sodium channels was higher in myoblasts than in myotubes. Thus, the difference in TTX sensitivity between myoblasts and myotubes can be explained by a difference in the proportion of the two classes of sodium channels. In addition, the lower conductance of TTX-resistant channels provides insight into the relationship between the TTX binding site and the external mouth of the sodium channel.

A histidine residue associated with the gate of the cyclic nucleotide-activated channels in rod photoreceptors.

Ion channels directly activated by the binding of cGMP mediate the electrical response to light in rod photoreceptors. Here, we identify a region of the channel associated with the activation gate using potentiation by intracellular Ni2+. Low concentrations of Ni2+ caused a dramatic increase in the response of rod channels expressed in Xenopus oocytes to both cGMP and cAMP. Whereas saturating cAMP normally activated less than 1% of the channels, Ni2+ increased the cAMP potency nearly 50-fold. Ni2+ did not produce potentiation in the related channel from the olfactory epithelium. We localized the Ni(2+)-binding site to a histidine residue in the putative intracellular mouth of the rod channel (H420). We propose a mechanism for potentiation in which Ni2+ binds to H420 primarily when the channel is open, stabilizing the open conformation. These experiments suggest that H420 is associated with the activation gate.

Planar patch clamp: advances in electrophysiology.

Ion channels have gained increased interest as therapeutic targets over recent years, since a growing number of human and animal diseases have been attributed to defects in ion channel function. Potassium channels are the largest and most diverse family of ion channels. Pharmaceutical agents such as Glibenclamide, an inhibitor of K(ATP) channel activity which promotes insulin release, have been successfully sold on the market for many years. So far, only a small group of the known ion channels have been addressed as potential drug targets. The functional testing of drugs on these ion channels has always been the bottleneck in the development of these types of pharmaceutical compounds.New generations of automated patch clamp screening platforms allow a higher throughput for drug testing and widen this bottleneck. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole-cell configuration. Furthermore, the extracellular membrane remains accessible for compound application during the experiment.Internal perfusion can be used not only for patch clamp experiments with cell membranes, but also for those with artificial lipid bilayers. In this chapter we describe how internal perfusion can be applied to potassium channels expressed in Jurkat cells, and to Gramicidin channels reconstituted in a lipid bilayer.

Access of quaternary ammonium blockers to the internal pore of cyclic nucleotide-gated channels: implications for the location of the gate.

Cyclic nucleotide-gated (CNG) channels play important roles in the transduction of visual and olfactory information by sensing changes in the intracellular concentration of cyclic nucleotides. We have investigated the interactions between intracellularly applied quaternary ammonium (QA) ions and the alpha subunit of rod cyclic nucleotide-gated channels. We have used a family of alkyl-triethylammonium derivatives in which the length of one chain is altered. These QA derivatives blocked the permeation pathway of CNG channels in a concentration- and voltage-dependent manner. For QA compounds with tails longer than six methylene groups, increasing the length of the chain resulted in higher apparent affinities of approximately 1.2 RT per methylene group added, which is consistent with the presence of a hydrophobic pocket within the intracellular mouth of the channel that serves as part of the receptor binding site. At the single channel level, decyltriethyl ammonium (C10-TEA) ions did not change the unitary conductance but they did reduce the apparent mean open time, suggesting that the blocker binds to open channels. We provide four lines of evidence suggesting that QA ions can also bind to closed channels: (1) the extent of C10-TEA blockade at subsaturating [cGMP] was larger than at saturating agonist concentration, (2) under saturating concentrations of cGMP, cIMP, or cAMP, blockade levels were inversely correlated with the maximal probability of opening achieved by each agonist, (3) in the closed state, MTS reagents of comparable sizes to QA ions were able to modify V391C in the inner vestibule of the channel, and (4) in the closed state, C10-TEA was able to slow the Cd2+ inhibition observed in V391C channels. These results are in stark contrast to the well-established QA blockade mechanism in Kv channels, where these compounds can only access the inner vestibule in the open state because the gate that opens and closes the channel is located cytoplasmically with respect to the binding site of QA ions. Therefore, in the context of Kv channels, our observations suggest that the regions involved in opening and closing the permeation pathways in these two types of channels are different.

Calcium antagonist isradipine-induced calcium influx through nonselective cation channels in human gingival fibroblasts.

Isradipine raises the cytosolic Ca2+ concentration ([Ca2+]i) in human gingival fibroblasts by enhancing Ca2+ influx through the plasma membrane. To research the pathways through which Ca2+ enters the cells, we examined the interactive effects of isradipine and blockers or enhancers of nonselective cation channels (NSCCs) and Na+/Ca2+ exchangers (NCXs). Normal human gingival fibroblast Gin-1 cells were used. The [Ca2+]i was measured with the Ca2+-sensitive fluorescent dye fura-2/AM. Changes in the fluorescence intensity of fura-2 in the cells were recorded with a video-imaging analysis system. Ca2+ antagonists (nifedipine, verapamil, and diltiazem in the concentration range of 1 to 20 microM) other than isradipine also raised the [Ca2+]i. All of the NSCC inhibitors (SK&F 96365, GdCl3, HgCl2 and flufenamic acid), but none of the NCX inhibitors (KB-R 7943 and benzamil), significantly decreased the [Ca2+]i raised by isradipine (3 microM). Neither the Na+ ionophore monensin nor Na+/K+ ATPase inhibitor ouabain had any significant effect on the isradipine-induced [Ca2+]i rise. Taken together, our data indicate that Ca2+ entry through the NSCCs is involved in the isradipine-induced [Ca2+]i rise. The results obtained here play an important role in the development of drugs for etiologic therapy of gingival overgrowth.

Peptidomimetic Star Polymers for Targeting Biological Ion Channels.

Four end-functionalized star polymers that could attenuate the flow of ionic currents across biological ion channels were first de novo designed computationally, then synthesized and tested experimentally on mammalian K+ channels. The 4-arm ethylene glycol conjugate star polymers with lysine or a tripeptide attached to the end of each arm were specifically designed to mimic the action of scorpion toxins on K+ channels. Molecular dynamics simulations showed that the lysine side chain of the polymers physically occludes the pore of Kv1.3, a target for immuno-suppression therapy. Two of the compounds tested were potent inhibitors of Kv1.3. The dissociation constants of these two compounds were computed to be 0.1 µM and 0.7 µM, respectively, within 3-fold to the values derived from subsequent experiments. These results demonstrate the power of computational methods in molecular design and the potential of star polymers as a new infinitely modifiable platform for ion channel drug discovery.

Mechanism of blockage of amphotericin B channels in a lipid bilayer.

A number of organic compounds (non-electrolytes, tetraalkylammonia, etc.) with a molecular size of 6--8 angstrom decrease the conductance of ionic channels formed in the lipid bilayer by a polyene antibiotic amphotericin B. It is suggested that these compounds, upon entering the channel, block the passage of inorganic ions. The extent of conductance blockage by organic ions depends on the membrane potential and electrolyte concentration. In the presence of ionic blockers, for instance tetraethylammonium, amphotericin B-containing membranes assume some properties characteristic of excitable membranes, i.e. the current-voltage characteristic acquires the negative resistance region, and in response to a potential step activation followed by inactivation of conductance is observed. It is shown that the potential dependence of the blockage is due to interaction inside the channel of the blocker ion with penetrating ions, by a mechanism similar to that described by Armstrong ((1979) Q. Rev. Biophys. 7, 179--210) for blockage of squid axon potassium channels by ammonium derivatives.

The blockage of the electrical conductance in a pore-containing membrane by the n-alkanes.

1. In monooelein bilayers made highly conducting by the addition of a fixed amount of o-pyromellitylgramicidin, the membrane conductance has been shown to be strongly dependent on the chain length of the n-alkane with which the membrane is in equilibrium. Thus for n-hexadecane, the conductance is larger by approx. 10(4) times than it is for n-octane. This result is independent of whether the polypeptide is introduced via the aqueous or lipid phases. 2. The observed conductance variations have been accounted for in terms of a mechanism (outlined in earlier publications) which is based on the thickness and tension changes produced in bilayers by the adsorption of n-alkanes. Essentially quantitative agreement between theory and experiment is found.

Cholesterol markedly reduces ion permeability induced by membrane-bound amphotericin B.

It is widely accepted that amphotericin B (AmB) together with sterol makes a mixed molecular assemblage in phospholipid membrane. By adding AmB to lipids prior to preparation of large unilamellar vesicles (LUV), we directly measured the effect of cholesterol on assemblage formation by AmB without a step of drug's binding to phospholipid bilayers. Potassium ion flux assays based on 31P-nuclear magnetic resonance (NMR) clearly demonstrated that cholesterol markedly inhibits ion permeability induced by membrane-bound AmB. This could be accounted for by a membrane-thickening effect of cholesterol since AmB actions are known to be markedly affected by the thickness of membrane. Upon addition of AmB to an LUV suspension, the ion flux gradually increased with increasing molar ratios of cholesterol up to 20 mol%. These biphasic effects of cholesterol could be accounted for, at least in part, by the ordering effect of cholesterol.

Structure of planar membrane formed from liposomes.

Lipid vesicles with incorporated ion channels from polyene antibiotic amphotericin B were used to investigate structures of planar membranes formed by Shindler's techniques. A planar membrane assembled on the aperture in a lavsan film from two layers generated at the air-aqueous liposome suspension interface is not a simple bilayer but a bimolecular membrane containing numerous partly fused liposomes. A complete fusion of liposomal membranes with the planar bilayer is an unlikely event during membrane formation. A planar bimolecular lipid membrane without incorporated liposomes can be made by a method consisting of three stages: formation of a lipid layer on the air-water interface of a suspension containing liposomes, transfer of this layer along the surface of the solution into a chamber containing a solution without liposomes where a lipid monomolecular layer forms gradually (within about 20 min) at the air-water interface, assembling of the planar bilayer membrane from this monolayer. The knowledge of the planar membrane structure may be useful in experiments on incorporation of membrane proteins into a planar lipid bilayer.

Interactions of diphtheria toxin with lipid vesicles: determinants of ion channel formation.

Lipid vesicles have been utilized to study the interactions of diphtheria toxin (DT) with membranes. The assay for DT ion channel formation was fluorescence-detected membrane potential depolarization of vesicles in which valinomycin-induced potassium diffusion gradients had been generated. The following requirements for ion channel formation have been identified: (1) acid pH (less than 5); (2) trans-negative membrane potentials (35-fold increase in channel-forming activity from -6 mV to -59 mV); and (3) negatively charged phospholipid headgroups (about 100-fold more activity using vesicles formed from asolectin compared to soybean phosphatidylcholine). Concentration dependence plots of toxin activity showed a linear response with logarithmic slopes of nearly one for each lipid composition. These results show a close parallel to those obtained previously with planar lipid bilayers and thus provide guidelines for conditions which facilitate functional insertion of the toxin into vesicles.

Solid support membranes for ion channel arrays and sensors: application to rapid screening of pharmacological compounds.

The use of solid supported membranes (SSM) was investigated for reconstitution of ion channels and for potential application to screen pharmacological reagents affecting ion channel function. The voltage-gated Kv1.5 K+ channel was reconstituted on an SSM and a current was measured. This current was dependent on the presence of K+, but not Na+, indicating that the Kv1.5 K+ channel maintained cation specificity when reconstituted on SSM. Two pharmacological reagents applied to Kv1.5 K+ channels reconstituted on SSM had similar inhibitory effects as those measured using Kv1.5 in biological membranes. SSM-mounted ion channels were stable enough to be washed with buffer solution and reused many times, allowing solution exchange essential for pharmacological drug screening.

A quantitative explanation of the effects of some alcohols on gramicidin single-channel lifetime.

The effects of n-decanol, n-hexadecanol, n-octyl(oxyethylene)3 alcohol and cholesterol on gramicidin single-channel lifetime in planar lipid bilayers have been determined. The bilayers used were formed from a solution of monoolein in squalene. Measurements have also been made of the above compounds' effects on membrane thickness (as measured by electrical capacity and optical reflectance technique) and surface tension (as derived from bulk interfacial tension and bilayer-lens contact angle measurements). The reduction in single-channel lifetime caused by the n-alkanols may be accounted for quantitatively in terms of the effects of these compounds on bilayer thickness and surface tension. The n-octyl(oxyethylene)3 alcohol caused an increase in single-channel lifetime which is also consistent with the thickness/tension theory. The reduction in channel lifetime caused by cholesterol, however, was much larger than would be predicted from its effects on bilayer thickness and surface tension.

Approximate dimensions of membrane lesions produced by streptolysin S and streptolysin O.

Membrane lesions produced by the streptococcal membranolysins streptolysin S and streptolysin O were investigated. Escape of labeled marker molecules of various sizes from resealed sheep erythrocyte ghosts treated with the toxins for 30 min allowed estimation of the sizes of the primary channels formed. Streptolysin S formed lesions ranging in size up to 45 A in diameter, and even high toxin concentrations did not result in larger channels. The lesions produced by streptolysin O exceeded 128 A in diameter. Kinetics experiments demonstrated that the primary streptolysin O lesions were formed rapidly (1-2 min), but release of marker molecules from streptolysin S-treated vesicles began only after a 5-15-min lag period. Label release from large unilamellar liposomes treated with streptolysin S suggested that membrane fluidity does not affect the size of the streptolysin S lesions.