KCa3.1-dependent uptake of the cytotoxic DNA-binding dye Hoechst 33258 into cancerous but not healthy cervical cells.

The poor and nonselective penetration of current chemotherapeutics across the plasma membranes of cancer cells, which is necessary for the targeted disruption of the intracellular machinery, remains a major pharmaceutical challenge. In several cell types, including mast cells and macrophages, exposure to extracellular ATP is known to stimulate passive entry of large and otherwise membrane impermeable cationic dyes, which is usually attributed to conduction through ionotropic P2X receptors. Here, we report that elevations in cytosolic Ca2+ stimulate the rapid uptake and nuclear accumulation of a DNA-binding fluorescent cation, Hoechst 33258 (H33258), in cervical cancer cells. The H33258 uptake was dependent on activation of intermediate conductance Ca2+-activated K+ channels (KCa3.1), and direct stimulation of the channel with the activators SKA 31 and DCEBIO was sufficient to induce cellular uptake of H33258 directly. In contrast to the results from cancerous cervical cells, KCa3.1-dependent H33258 uptake was rarely observed in epithelial cells derived from the ectocervix and transformation zone of healthy cervical tissue. Furthermore, whole-cell patch clamp experiments and assessment of membrane potential using the slow voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid)trimethine oxonol revealed a significant difference in functional KCa3.1 activity between cancerous and healthy cervical epithelial cells, which correlated strongly with the incidence of KCa3.1-dependent H33258 uptake. Finally, we show that activation of KCa3.1 channels caused a modest but significant sensitization of cancer cells to the growth suppressant effects of H33258, lending plausibility to the idea of using KCa3.1 channel activators to enhance cell penetration of small cationic toxins into cancer cells expressing these channels.

Ivermectin-induced cell death of cervical cancer cells in vitro a consequence of precipitate formation in culture media.

The anthelmintic ivermectin has been reported to possess anticancer and antiviral efficacy. However, the effective concentrations reported in vitro are near the predicted aqueous solubility limit for this hydrophobic drug. We observed that ivermectin-induced cell death in two cervical cancer cell lines correlated with the formation of solid ivermectin aggregates in both serum-free and serum-supplemented culture media. Filtration of ivermectin particles >0.2 µm abolished these cytolytic effects in both cell lines. An inhibitory effect on cell proliferation persisted for filtered solutions, but only for ivermectin concentrations higher than reported to be clinically attainable in humans. In addition to the importance of distinguishing between free and bound drug in solution, our data emphasize the importance of acknowledging the likely solubility limit of hydrophobic drugs when assessing their in vitro cytotoxicity.

Ca2+ flux through splice variants of the ATP-gated ionotropic receptor P2X7 is regulated by its cytoplasmic N terminus.

Activation of ionotropic P2X receptors increases free intracellular Ca2+ ([Ca2+] i ) by initiating a transmembrane cation flux. We studied the "a" and "k" splice variants of the rat purinergic P2X7 receptor (rP2X7aR and rP2X7kR) to exhibit a significant difference in Ca2+ flux through this channel. This difference is surprising because the variants share absolute sequence identity in the area of the pore that defines ionic selectivity. Here, we used patch-clamp fluorometry and chimeric receptors to show that the fraction of the total current carried by Ca2+ is a function of the primary sequence of the cytoplasmic N terminus. Using scanning mutagenesis, we identified five sites within the N terminus that respond to mutagenesis with a decrease in fractional calcium current and an increase in permeability to the polyatomic cation, N-methyl-d-glucamine (NMDG+), relative to Na+ (PNMDG/PNa). We tested the hypothesis that these sites line the permeation pathway by measuring the ability of thiol-reactive MTSET+ to alter the current of cysteine-substituted variants, but we detected no effect. Finally, we studied the homologous sites of the rat P2X2 receptor (rP2X2R) and observed that substitutions at Glu17 significantly reduced the fractional calcium current. Taken together, our results suggest that a change in the structure of the N terminus alters the ability of an intra-pore Ca2+ selectivity filter to discriminate among permeating cations. These results are noteworthy for two reasons: they identify a previously unknown outcome of mutagenesis of the N-terminal domain, and they suggest caution when assigning structure to function for truncated P2X receptors that lack a part of the N terminus.

Applications for Mass Spectrometry in the Study of Ion Channel Structure and Function.

Ion channels are intrinsic membrane proteins that form gated ion permeable pores across biological membranes. Depending on the type, ion channels exhibit sensitivities to a diverse range of stimuli including changes in membrane potential, binding by diffusible ligands, changes in temperature and direct mechanical force. The purpose of these proteins is to facilitate the passive diffusion of ions down their respective electrochemical gradients into and out of the cell, and between intracellular compartments. In doing so, ion channels can affect transmembrane potentials and regulate the intracellular homeostasis of the important second messenger, Ca2+, modulating a multitude of cell signaling systems in the process. The ion channels of the plasma membrane are of particular clinical interest due to their regulation of cell excitability and cytosolic Ca2+ levels, and the fact that they are particularly amenable to manipulation by exogenously applied drugs and toxins. A critical step in improving the pharmacopeia of chemicals available that influence the activity of ion channels is understanding how their three-dimensional structure relates to their function. Historically, elucidation of the structure of membrane proteins has been slow relative to that for soluble proteins, due to limitations inherent in the most widely used methods, in particular X-ray crystallography. Over the course of the last decade, starting with significant advances in X-ray crystallography followed by the more recent, and profound, surge in the use of single particle cryo-electron microscopy (cryo-EM), a slew of high resolution ion channel structures have been resolved. Overshadowed during this period have been the equally marked advances in mass spectrometry, pushing this method to the fore as an important complimentary approach to studying the structure and function of ion channels. In addition to revealing the subtle conformational changes in ion channel structure that accompany gating and permeation, mass spectrometry is already being used effectively for identifying tissue-specific posttranslational modifications and mRNA splice variants. Furthermore, the use of mass spectrometry for high throughput proteomics analysis, which has proven so successful for soluble proteins, is already providing valuable insight into the functional interactions of ion channels within the context of the macromolecular signaling complexes that they inhabit in vivo. In this chapter, the potential for mass spectrometry as a complementary approach to the study of ion channel structure and function will be reviewed with examples of its application.

Quantifying Ca2+ current and permeability in ATP-gated P2X7 receptors.

ATP-gated P2X7 receptors are prominently expressed in inflammatory cells and play a key role in the immune response. A major consequence of receptor activation is the regulated influx of Ca(2+) through the self-contained cation non-selective channel. Although the physiological importance of the resulting rise in intracellular Ca(2+) is universally acknowledged, the biophysics of the Ca(2+) flux responsible for the effects are poorly understood, largely because traditional methods of measuring Ca(2+) permeability are difficult to apply to P2X7 receptors. Here we use an alternative approach, called dye-overload patch-clamp photometry, to quantify the agonist-gated Ca(2+) flux of recombinant P2X7 receptors of dog, guinea pig, human, monkey, mouse, rat, and zebrafish. We find that the magnitude of the Ca(2+) component of the ATP-gated current depends on the species of origin, the splice variant, and the concentration of the purinergic agonist. We also measured a significant contribution of Ca(2+) to the agonist-gated current of the native P2X7Rs of mouse and human immune cells. Our results provide cross-species quantitative measures of the Ca(2+) current of the P2X7 receptor for the first time, and suggest that the cytoplasmic N terminus plays a meaningful role in regulating the flow of Ca(2+) through the channel.

Measurement of relative Ca²âº permeability during sustained activation of TRPV1 receptors.

Some cation permeable ligand-gated ion channels, including the capsaicin-sensitive TRPV1, have been reported to exhibit a time-dependent increase in permeability to large inorganic cations during sustained activation, a phenomenon termed "pore dilation." TRPV1 conducts substantial Ca(2+) entry, and it has been suggested that this channel undergoes a time-dependent change in Ca(2+) permeability relative to Na(+) (P Ca/P Na) that parallels pore dilation. However, our experiments employing whole cell patch clamp photometry and single channel recordings to directly measure relative Ca(2+) current in TRPV1 expressing HEK293 cells show that relative Ca(2+) influx remains constant for the duration of capsaicin-evoked channel activation. Further, we present evidence from patch clamp photometry experiments suggesting that sustained activation of Ca(2+) permeable ion channels in the voltage-clamp configuration leads to rapid saturation of the pipette Ca(2+) chelator, and that subsequent observed shifts in the current reversal potentials in the presence of extracellular Ca(2+) are likely due to intracellular accumulation of this ion and a movement of the Ca(2+) equilibrium potential (E Ca) towards zero. Finally, using an adapted reversal potential-based protocol in which cells are only exposed to Ca(2+) after sustained capsaicin exposure in the absence of added extracellular Ca(2+), we demonstrate that the calculated P Ca/P Na is unaffected by duration of TRPV1 activation. In conclusion, we find no evidence in support of a time-dependent change in P Ca/P Na for TRPV1. Our data further urges caution in estimating relative Ca(2+) permeability using reversal potentials, as there is a limited time window in which the cytosolic Ca(2+) chelator included in the patch pipette can prevent localised elevations in cytosolic free Ca(2+) and thus allow for an accurate estimate of this important channel permeability parameter.

Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability.

Ionic "vital dyes" are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5'-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4',6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions.

Roles of the lateral fenestration residues of the P2X4 receptor that contribute to the channel function and the deactivation effect of ivermectin.

P2X receptors are cation-permeable ion channels gated by extracellular adenosine triphosphate (ATP). Available crystallographic data suggest that ATP-binding ectodomain is connected to the transmembrane pore domain by three structurally conserved linker regions, which additionally frame the lateral fenestrations through which permeating ions enter the channel pore. The role of these linker regions in relaying the conformational change evoked by ATP binding of the ectodomain to the pore-forming transmembrane domain has not been investigated systematically. Using P2X4R as our model, we employed alanine and serine replacement mutagenesis to determine how the side chain structure of these linker regions influences gating. The mutants Y54A/S, F198A/S, and W259A/S all trafficked normally to the plasma membrane of transfected HEK293 cells but were poorly responsive to ATP. Nevertheless, the function of the F198A/S mutants could be recovered by pretreatment with the known positive allosteric modulator of P2X4R, ivermectin (IVM), although the IVM sensitivity of this mutant was significantly impaired relative to wild type. The functional mutants Y195A/S, F200A/S, and F330A/S exhibited ATP sensitivities identical to wild type, consistent with these side chains playing no role in ATP binding. However, Y195A/S, F200A/S, and F330A/S all displayed markedly changed sensitivity to the specific effects of IVM on current deactivation, suggesting that these positions influence allosteric modulation of gating. Taken together, our data indicate that conserved amino acids within the regions linking the ectodomain with the pore-forming transmembrane domain meaningfully contribute to signal transduction and channel gating in P2X receptors.

Involvement of ectodomain Leu 214 in ATP binding and channel desensitization of the P2X4 receptor.

P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains.

Photopolymerized cross-linked thiol-ene polyanhydrides: erosion, release, and toxicity studies.

Several critical aspects of cross-linked polyanhydrides made using thiol-ene polymerization are reported, in particular the erosion, release, and solution properties, along with their cytotoxicity toward fibroblast cells. The monomers used to synthesize these polyanhydrides were 4-pentenoic anhydride and pentaerythritol tetrakis(3-mercaptopropionate). Techniques used to evaluate the erosion mechanism indicate a complex situation in which several phenomena, such as hydrolysis rates, local pH, water diffusion, and solubility, may be influencing the erosion process. The mass loss profile, the release rate of a hydrophilic dye, the rate of hydrolysis of the polyanhydride, the hydrolysis product solubility as a function of pH, average pK(a) and its cytotoxicity toward fibroblast cells were all determined. The solubility of the degradation product is low at pH values less than 6-7, and the average pKa was determined to be ~5.3. The cytotoxicity of the polymer and the degradation product was found to be low, with cell viabilities of >97% for the various samples studied at concentrations of ~1000-1500 ppm. These important parameters help determine the potential of the thiol-ene polyanhydrides in various biomedical applications. These polyanhydrides can be used as a delivery vehicle, and although the release profile qualitatively followed the mass loss profile for a hydrophilic dye, the release rate appears to be by both diffusion and mass loss mechanisms.

Applications for mass spectrometry in the study of ion channel structure and function.

Ion channels are intrinsic membrane proteins that form gated ion-permeable pores across biological membranes. Depending on the type, ion channels exhibit sensitivities to a diverse range of stimuli including changes in membrane potential, binding by diffusible ligands, changes in temperature and direct mechanical force. The purpose of these proteins is to facilitate the passive diffusion of ions down their respective electrochemical gradients into and out of the cell, and between intracellular compartments. In doing so, ion channels can affect transmembrane potentials and regulate the intracellular homeostasis of the important second messenger, Ca(2+). The ion channels of the plasma membrane are of particular clinical interest due to their regulation of cell excitability and cytosolic Ca(2+) levels, and the fact that they are most amenable to manipulation by exogenously applied drugs and toxins. A critical step in improving the pharmacopeia of chemicals available that influence the activity of ion channels is understanding how their three-dimensional structure imparts function. Here, progress has been slow relative to that for soluble protein structures in large part due to the limitations of applying conventional structure determination methods, such as X-ray crystallography, nuclear magnetic resonance imaging, and mass spectrometry, to membrane proteins. Although still an underutilized technique in the assessment of membrane protein structure, recent advances have pushed mass spectrometry to the fore as an important complementary approach to studying the structure and function of ion channels. In addition to revealing the subtle conformational changes in ion channel structure that accompany gating and permeation, mass spectrometry is already being used effectively for identifying tissue-specific posttranslational modifications and mRNA splice variants. Furthermore, the use of mass spectrometry for high-throughput proteomics analysis, which has proven so successful for soluble proteins, is already providing valuable insight into the functional interactions of ion channels within the context of the macromolecular-signaling complexes that they inhabit in vivo. In this chapter, the potential for mass spectrometry as a complementary approach to the study of ion channel structure and function will be reviewed with examples of its application.

Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors).

P2X receptors are trimeric cation channels with widespread roles in health and disease. The recent crystal structure of a P2X4 receptor provides a 3D view of their topology and architecture. A key unresolved issue is how ions gain access to the pore, because the structure reveals two different pathways within the extracellular domain. One of these is the central pathway spanning the entire length of the extracellular domain and covering a distance of ≈70 Å. The second consists of three lateral portals, adjacent to the membrane and connected to the transmembrane pore by short tunnels. Here, we demonstrate the preferential use of the lateral portals. Owing to their favorable diameters and equivalent spacing, the lateral portals split the task of ion supply threefold and minimize an ion's diffusive path before it succumbs to transmembrane electrochemical gradients.

Allosteric modulation of Ca2+ flux in ligand-gated cation channel (P2X4) by actions on lateral portals.

Human P2X receptors are a family of seven ATP-gated ion channels that transport Na(+), K(+), and Ca(2+) across cell surface membranes. The P2X4 receptor is unique among family members in its sensitivity to the macrocyclic lactone, ivermectin, which allosterically modulates both ion conduction and channel gating. In this paper we show that removing the fixed negative charge of a single acidic amino acid (Glu(51)) in the lateral entrance to the transmembrane pore markedly attenuates the effect of ivermectin on Ca(2+) current and channel gating. Ca(2+) entry through P2X4 receptors is known to trigger downstream signaling pathways in microglia. Our experiments show that the lateral portals could present a novel target for drugs in the treatment of microglia-associated disease including neuropathic pain.

Calcium-dependent decrease in the single-channel conductance of TRPV1.

TRPV1 is a Ca(2+) permeable cation channel gated by multiple stimuli including noxious heat, capsaicin, protons, and extracellular cations. In this paper, we show that Ca(2+) causes a concentration and voltage-dependent decrease in the capsaicin-gated TRPV1 single-channel conductance. This Ca(2+)-dependent effect on conductance was strongest at membrane potentials between -60 and +20 mV, but was diminished at more hyperpolarised potentials. Using simultaneous recordings of membrane current and fura-2 fluorescence to measure the fractional Ca(2+) current of whole-cell currents evoked through wild-type and mutant TRPV1, we investigated a possible link between the mechanisms underlying Ca(2+) permeation and the Ca(2+)-dependent effect on conductance. Surprisingly, we found no evidence of a structural correlation, and observed that the substitution of amino acids known to regulate Ca(2+) permeability had little effect on the ability for Ca(2+) to decrease TRPV1 conductance. However, we did observe that the Ca(2+)-dependent effect on conductance was not diminished by negative hyperpolarisation for a mutant receptor with severely impaired Ca(2+) permeability, TRPV1-D646N/E648Q/E651Q. This would be consistent with the idea that Ca(2+) reduces conductance by interacting with an intra-pore binding site, and that negative hyperpolarization reduces occupancy of this site by speeding the exit of Ca(2+) into the cell. Taken together, our data show that in addition to directly and indirectly regulating channel gating, Ca(2+) also directly reduces the conductance of TRPV1. Surprisingly, the mechanism underlying this Ca(2+)-dependent effect on conductance is largely independent of mechanisms governing Ca(2+) permeability.

The CDK domain of p21 is a suppressor of IL-1beta-mediated inflammation in activated macrophages.

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21((WAF1/CIP1)), an established suppressor of cell cycle progression, is a inhibitor of IL-1beta synthesis in macrophages. Mice deficient in p21 (p21(-/-)) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL-1beta. Administration of IL-1 receptor antagonist reduces LPS-induced lethality in p21(-/-) mice. Analysis of isolated macrophages, which are one of the central producers of IL-1beta, reveals that deficiency for p21 led to more IL-1beta mRNA and pro-protein synthesis following TLR ligation. The increase in IL-1beta pro-protein is associated with elevated secretion of active IL-1beta by p21(-/-) macrophages. siRNA-mediated knockdown of p21 in human macrophages results in increased IL-1beta secretion as well. A peptide mapping strategy shows that the cyclin-dependent-kinase (CDK)-binding domain of p21 is sufficient to reduce the secretion of IL-1beta by p21(-/-) macrophages. These data suggest a novel role for p21 and specifically for the CDK-binding domain of p21((WAF1/CIP1)) in inhibiting inflammation.

Drug Delivery and Drug Efficacy from Amorphous Poly(thioether anhydrides).

The correlation between erosion and drug (lidocaine and 6-mercaptopurine, 6-MP) release from amorphous poly(thioether anhydrides), which are synthesized using radical-mediated thiol-ene polymerization, is reported. Cytotoxicity studies of the polymer toward human fibroblast human dermal fibroblasts adult, melanoma A-375, and breast cancer MCF-7 cells are conducted, and drug efficacy of a cancer and autoimmune disease drug (6-MP) when released from the poly(thioether anhydrides) is examined against two cancerous cell types (A-375 and MCF-7). Erosion and drug release studies reveal that lidocaine release is governed by network erosion whereas 6-MP is released by a combination of erosion and diffusion. The cytotoxicity studies show that all three cell types demonstrate high viability, thus cytocompatibility, to poly(thioether anhydrides). Toxicity to the material is dose dependent and comparable to other polyanhydride systems. The 6-MP cancer drug is shown to remain bioactive after encapsulation in the poly(thioether anhydride) matrix and the polymer does not appear to modify the efficacy of the drug.

Acidic amino acids impart enhanced Ca2+ permeability and flux in two members of the ATP-gated P2X receptor family.

P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca2+ permeability of the P2X2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca2+ currents (Pf%) of the rat and human P2X1 and P2X4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X1 receptor but had no effect on the lower Pf% of the P2X2 receptor, suggesting that ionized side chains regulate the Ca2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X1 and P2X4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X2 receptor. Taken together, the data suggest that COO- side chains provide an electrostatic force that interacts with Ca2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X1 and P2X4 receptors is conserved in the P2X3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His45 increased Pf% of the P2X3 channel, suggesting that this positive charge masks the facilitation of Ca2+ flux by the neighboring Glu46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca2+ permeability and flux of some P2X receptors.

Opioid elevation of intracellular free calcium: possible mechanisms and physiological relevance.

Opioid receptors are seven transmembrane domain Gi/G0 protein-coupled receptors, the activation of which stimulates a variety of intracellular signalling mechanisms including activation of inwardly rectifying potassium channels, and inhibition of both voltage-operated N-type Ca2+ channels and adenylyl cyclase activity. It is now apparent that like many other Gi/G0-coupled receptors, opioid receptor activation can significantly elevate intracellular free Ca2+ ([Ca2+]i), although the mechanism underlying this phenomenon is not well understood. In some cases opioid receptor activation alone appears to elevate [Ca2+]i, but in many cases it requires concomitant activation of Gq-coupled receptors, which themselves stimulate Ca2+ release from intracellular stores via the inositol phosphate pathway. Given the number of Ca2+-sensitive processes known to occur in cells, there are therefore a myriad of situations in which opioid receptor-mediated elevations of [Ca2+](i) may be important. Here, we review the literature documenting opioid receptor-mediated elevations of [Ca2+]i, discussing both the possible mechanisms underlying this phenomenon and its potential physiological relevance.

Fabrication of self-assembling nanofibers with optimal cell uptake and therapeutic delivery efficacy.

Effective strategies to fabricate finite organic nanoparticles and understanding their structure-dependent cell interaction is highly important for the development of long circulating nanocarriers in cancer therapy. In this contribution, we will capitalize on our recent development of finite supramolecular nanofibers based on the self-assembly of modularly designed cationic multidomain peptides (MDPs) and use them as a model system to investigate structure-dependent cell penetrating activity. MDPs self-assembled into nanofibers with high density of cationic charges at the fiber-solvent interface to interact with the cell membrane. However, despite the multivalent charge presentation, not all fibers led to high levels of membrane activity and cellular uptake. The flexibility of the cationic charge domains on self-assembled nanofibers plays a key role in effective membrane perturbation. Nanofibers were found to sacrifice their dimension, thermodynamic and kinetic stability for a more flexible charge domain in order to achieve effective membrane interaction. The increased membrane activity led to improved cell uptake of membrane-impermeable chemotherapeutics through membrane pore formation. In vitro cytotoxicity study showed co-administering of water-soluble doxorubicin with membrane-active peptide nanofibers dramatically reduced the IC50 by eight folds compared to drug alone. Through these detailed structure and activity studies, the acquired knowledge will provide important guidelines for the design of a variety of supramolecular cell penetrating nanomaterials not limited to peptide assembly which can be used to probe various complex biological processes.